afab volume 4 issue 3
DESCRIPTION
This journal is a peer reviewed scientific forum for the latest advancements in bacteriology research on a wide range of topics including food safety, food microbiology, gut microbiology, biofuels, bioremediation, environmental microbiology, fermentation, probiotics, and veterinary microbiology.TRANSCRIPT
Volume 4, Issue 32014
ISSN: 2159-8967www.AFABjournal.com
158 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 159
Sooyoun Ahn University of Florida, USA
Walid Q. Alali University of Georgia, USA
Kenneth M. Bischoff NCAUR, USDA-ARS, USA
Debabrata Biswas University of Maryland, USA
Claudia S. Dunkley University of Georgia, USA
Michael Flythe USDA, Agricultural Research Service
Lawrence Goodridge McGill University, Canada
Leluo Guan University of Alberta, Canada
Joshua Gurtler ERRC, USDA-ARS, USA
Yong D. Hang Cornell University, USA
Armitra Jackson-Davis Alabama A&M University, USA
Divya Jaroni Oklahoma State University, USA
Weihong Jiang Shanghai Institute for Biol. Sciences, P.R. China
Michael Johnson University of Arkansas, USA
Timothy Kelly East Carolina University, USA
William R. Kenealy Mascoma Corporation, USA
Hae-Yeong Kim Kyung Hee University, South Korea
Woo-Kyun Kim University of Georgia, USA
M.B. Kirkham Kansas State University, USA
Todd Kostman University of Wisconsin, Oshkosh, USA
Y. M. Kwon University of Arkansas, USA
Maria Luz Sanz MuriasInstituto de Quimica Organic General, Spain
Byeng R. Min Tuskegee University in Tuskegee, AL
Melanie R. Mormile Missouri University of Science and Tech., USA
Rama Nannapaneni Mississippi State University, USA
Jack A. Neal, Jr. University of Houston, USA
Benedict Okeke Auburn University at Montgomery, USA
John Patterson Purdue University, USA
Toni Poole FFSRU, USDA-ARS, USA
Marcos Rostagno LBRU, USDA-ARS, USA
Roni Shapira Hebrew University of Jerusalem, Israel
Kalidas Shetty North Dakota State University, USA
EDITORIAL BOARD
160 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
EDITOR-IN-CHIEFSteven C. RickeUniversity of Arkansas, USA
EDITORSTodd R. CallawayFFSRU, USADA-ARS, USA
Philip G. CrandallUniversity of Arkansas, USA
Janet Donaldson Mississippi State University, USA
Ok-Kyung KooKorea Food Research Institute, South Korea
MANAGING and LAYOUT EDITOREllen J. Van LooGhent, Belgium
TECHNICAL EDITORJessica C. ShabaturaFayetteville, USA
ONLINE EDITION EDITORC.S. ShabaturaFayetteville, USA
ABOUT THIS PUBLICATION
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EDITORIAL STAFF
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 161
Batch Culture Characterization of Acetogenesis in Ruminal Contents: Influence of Aceto-
gen Inocula Concentration and Addition of 2-Bromoethanesulfonic AcidP. Boccazzi and J. A. Patterson
177
Survival of Salmonella enterica and Listeria monocytogenes in manure-based compost mix-tures at sublethal temperatures M.C. Erickson, C. Smith, X. Jiang, I.D. Flitcroft, and M.P. Doyle
224
The Effect of Phytochemical Tannins-Containing Diet on Rumen Fermentation Characteris-tics and Microbial Diversity Dynamics in Goats Using 16S rDNA Amplicon PyrosequencingB. R. Min, C. Wright, P. Ho, J.-S. Eun, N. Gurung, and R. Shange
195
Characterization of the Novel Enterobacter cloacae Strain JD6301 and a Genetically Modified Variant Capable of Producing Extracellular LipidsJ. R. Donaldson, S. Shields-Menard, J. M. Barnard, E. Revellame, J. I. Hall, A. Lawrence, J. G. Wilson, A. Lipzen, J. Martin, W. Schackwitz, T. Woyke, N. Shapiro, K. S. Biddle, W. E. Holmes, R. Hernandez, and W. T. French
212
ARTICLES
The Prevalence of E. coli O157:H7 in the Production of Organic Herbs and a Case Study of Organic Lemongrass Intended for Use in Blended TeaS. Zaman, Md. K. Alam, S. S. Ahmed, Md. N. Uddin, and Md. L. Bari
164
Instructions for Authors243
Introduction to Authors
The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.
TABLE OF CONTENTS
162 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Dr. Byeng R. Min appointed to AFAB editorial board
Agriculture, Food and Analytical Bacteriology is pleased to welcome Dr. Byeng R. Min to the editorial board. Dr. Byeng R. Min is an Animal nutritionist and rumen microbiolo-gist in the Animal Science Unit at Tuskegee University in Tuskegee, AL. He has been with Tuskegee University since 2009. Byeng earned degrees in Animal science from Kon-Kuk University (B.S.), South Korea, and Massey University (M.S. and Ph.D.), New Zealand, and gained experience with utilization of phytochemical tannin-containing forages by small ruminants during his studies at Massey University in New Zealand. Dr. Min conducts re-search on sheep, goats, and cattle, focusing on providing technology to enable small to mid-size farmers to maximize profits and sustainability. His primary interest in rumen and intestinal microbial diversity as well as alternative control of food borne pathogens and gastro-intestinal parasites. Dr. Min is author and/or co-author of over 52 refereed jour-nal articles, numerous technical/report papers, proceedings papers, and abstracts, and 2 book chapters.
NEW EDITORIAL STAFF
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 163
164 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
www.afabjournal.comCopyright © 2014
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Tea blended with different herbs bring a world of flavors, aromas and colors and is usually made with
dried tea leaves, or blended with other dried herbs and involves pouring boiling water over the leaves, let-
ting them steep for few minutes followed by consumption. This study was done to evaluate the insights of
potential microbial contamination of organic herbs production at the farm, after harvest, washing, before or
after drying and packaging of dried herbs sample. Organic compost, water quality, worker hygiene status
and overall food safety management systems were also evaluated to identify additional factors affecting
microbiological contamination. In addition, effect of pouring hot water over contaminated dried leaves in
a cup of tea was observed. The study was designed in such a way that reflects the actual tea preparation at
home. Presence of higher numbers of generic E. coli and pathogenic E. coli O157:H7 was observed in dried
tea, herbs and /or lemongrass samples, and blended tea mix lemongrass samples. However, no Salmonella
was detected in any of the samples tested. When hot water was added into dried lemongrass or blended
tea mix lemongrass samples in a cup of tea and held for 30, 60, 90, 120 or 180 seconds with or without a lid,
no generic E. coli and pathogenic E. coli O157:H7 was observed in the prepared cup of tea in 30 seconds or
above the holding time in selective medium. The bacteria might be severely injured by hot water treatment
and did not appear on the selective plates. To confirm whether the bacteria were inactivated or injured, an
enrichment study was done. Neither generic E. coli nor any pathogenic E. coli O157:H7 were detected in
the prepared tea in the cup. The hot water temperature was recorded as 82˚C when added in the cup and
after 60 seconds the temperature decreased to 78˚ C; further reduced to 73˚C after 3 minutes of holding
and at the end of 5 minutes the temperature reached 64˚ C. In addition, the natural microflora was reduced
to less than 100 CFU/ml. This finding suggested that addition of hot water (80˚C) in tea leaves resulted in
complete elimination of pathogens and thus the present tea making practice could provide safe tea for
drinking even though the tea leaves were contaminated. However, for sanitary reasons E. coli should be
eliminated from the organic products prior to consumption.
Keywords: Organic herbs, E. coli O157:H7, organic lemongrass, case study and blended tea
Correspondence: Md. Latiful Bari, [email protected]: 8801971560560 Fax: 8802-8615583
The Prevalence of E. coli O157:H7 in the Production of Organic Herbs and a Case Study of Organic Lemongrass Intended for Use in Blended Tea
S. Zaman1, Md. K. Alam2, S. S. Ahmed4, Md. N. Uddin3, and Md. L. Bari1
1Center for advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh2Institute of Food and Radiation Biology, Bangladesh Atomic Energy Commission, Savar, Dhaka, Bangladesh
3Bangladesh Agricultural Research Institute, Gazipur 1701, Bangladesh4Kazi & Kazi Tea Estate Ltd. University of Liberal Arts Bangladesh (ULAB), Dhanmondi, Dhaka-1209, Bangladesh
Agric. Food Anal. Bacteriol. 4: 164-176, 2014
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 165
INTRODUCTION
Lemongrass (Cymbopogan flexuosus, family:
Poaceae) is an aromatic plant which grows in many
parts of tropical and sub-tropical South East Asia
and Africa. Most of the species of lemon grass are
native to South Asia, South-East Asia and Australia
(USDA, 2008). Lemongrass naturally grows in tropi-
cal areas and can resist the heat of the sun. Fresh cut
lemongrass often lasts for several days and can be
preserved in fresh water for several months without
losing any flavor or nutritional properties. However,
these lemongrasses are dried easily, readily available
worldwide and can be used to make tea.
Lemongrass is usually ingested as an infusion made
by pouring boiling water on fresh or dried leaves and
is one of the most widely used traditional plants in
South American folk medicine (Blumenthal, 1998). It
is used as an antispasmodic, antiemetic, and anal-
gesic, as well as for the management of nervous and
gastrointestinal (GI) disorders and the treatment of
fevers (Leung, 1980). In India it is commonly used as
an antitussive, antirheumatic, and antiseptic. In Chi-
nese medicine, lemongrass is used in the treatment
of headaches, stomach aches, abdominal pain, and
rheumatic pain (Girón et al., 1991). Lemongrass is an
important part of Southeast Asian cuisine, especially
as a flavoring in Thai food. Lemongrass is used in
Cuban folk medicine for hypertension and as an anti-
inflammatory (Lewinsohn et al., 1998). It is also used
in Brazilian folk medicine in a tea called abafado as a
sedative, and for gastrointestinal problems and fever
(Martínez-de la Puente et al., 2009). Lemongrass and
closely related species are popularly used as insect re-
pellents (Wong et al., 2005; Tawatsin et al., 2001). They
may be found in sprays, candles, and other repellent
products. Various experimental studies support its use
as an insecticide or insect repellant. Lemongrass has
been shown to have antifungal properties in labora-
tory studies particularly against Candida species (Can-
dida albicans, Candida glabrata, Candida krusei, Can-
dida parapsilosis, and Candida tropicalis) (Warnke et
al., 2009). In a preliminary study, lemongrass infusion
had beneficial effects for the treatment of oral candi-
diasis in patients with HIV/AIDS (Wright et al., 2009).
These fresh herbs and leafy greens are potential
transmission sources of enteropathogens. In a recent
report from WHO/FAO on microbiological hazards in
fresh fruits and vegetables (FAO/WHO 2008) it was
stated that leafy green vegetables (including fresh
herbs) “currently presented the greatest concern in
terms of microbiological hazards.” This is because
these products are grown and exported in large
volumes, and they have been associated with many
foodborne disease outbreaks affecting considerable
numbers of people. Additionally, the production
chain for leafy greens is highly complex. The micro-
flora on these vegetables at harvest reflects the envi-
ronment in which they are grown, if the temperature
and humidity is relatively high then the occurrence of
enteropathogenic bacteria in this environment might
be considerable. During cultivation, use of contami-
nated water for irrigation, application of biocides,
and refreshing or washing of harvested crops, are po-
tential sources of contamination. Contamination from
contact with fresh manure used as fertilizer cannot be
excluded. Heavy rainfall may also lead to fecal con-
tamination from the environment. Direct sunshine will
most likely have a disinfection effect, but if the plants
are irrigated until harvest and the production hygiene
during harvest and post-harvest is inadequate, there
is a relatively high likelihood that the fresh herbs and
leafy greens may be fecally contaminated. These
fresh herbs and leafy greens and their products have
been found to be contaminated with pathogenic
bacteria such as Staphylococcus aureus, Escherichia
coli, Salmonella enterica serovar Typhi, Shigella spp,
Bacillus spp. amongst others that represent serious
public health hazards (Abadias et al., 2008; Esimone
et al., 2003; Oyetayo, 2008; Abba et al., 2009; Adel-
eye et al., 2005). Some of these pathogenic bacteria
originate from soil and adhere to parts of plants (Lau
et al., 2003) while most of them are being introduced
into leafy products through processes of harvesting,
drying, storage and manufacturing because of the
unhygienic practices of the product handlers (Lau et
al., 2003; Espen et al., 2008). In 2005, the Norwegian
Food Safety Authority (Mattilsynet) conducted an ad
hoc survey of 162 fresh herbs and green or leafy veg-
etables products, from South East Asia, and found
166 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
that 28% were contaminated with Salmonella, and
35% with E. coli at greater than 100 CFU/gram. This
resulted in a general import prohibition of such prod-
ucts from South East Asia, and now the EU accord-
ingly requires certificate of analysis for Salmonella
and E. coli before export (Olaimat and Holley, 2012).
The objectives of this present study were to evalu-
ate microbial contamination of organic herb pro-
duction at the farm level, and a case study of food
safety management in organic lemongrass produc-
tion intended for blended tea. Organic compost,
water quality, worker hygiene status and overall food
safety management systems were also evaluated to
identify the potential factors affecting microbiologi-
cal contamination. In addition, effect of pouring hot
water over contaminated dried leaves in a cup of tea
was observed. The study was designed in such a way
that reflects the actual tea preparation at home.
MATERIALS AND METHODS
Sample collection
Herb samples include, lemongrass, mint, neem
and jasmine were obtained from an organic farm
in Northern Bangladesh between May 15 and July
30, 2013. All samples were transported to the Food
Analysis and Research Laboratory, Center for Ad-
vanced Research in Sciences (CARS) at the Univer-
sity of Dhaka using a cool box at the earliest con-
venience for processing and further analysis. All the
microbial analysis was carried out according to the
standard methods described in United States Food
and Drug Administration (US-FDA) Bacteriological
Analytical Manual.
Selected critical sampling locations (CSLs)
The critical sampling locations were selected
based on the production scheme presented in Fig-
ure 1a and other sources of microbiological contam-
ination as identified in literature reviews (Ilic et al.,
2012; Vidal et al., 2004) i.e., soil, water, manure, food
contact surfaces, or food handlers. For dried organic
lemongrass production, 12 CSLs were selected (Fig-
ure 1b) including the lemongrass crop.
Figure 1a. Schematic flow diagram of lemongrass production chain (Farm to table).
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 167
Total aerobic count and total coliform count
Twenty five (25) g of each sample were homog-
enized in 225 mL of saline water (0.85% NaCl). Deci-
mal dilutions were prepared upto 10-6 mL and ap-
propriate dilutions were spread plated on Tryptic
soy agar (Oxoid Ltd., Hampshire, England) and in-
cubated at 35˚C for 24 hr for total aerobic bacterial
counts and on MacConkey agar (Oxoid Ltd., Hamp-
shire, England) with incubation at 35˚C and 42˚C for
24 hours for total coliform count. Total aerobic count
indicates the quality and shelf life of the products
and total coliform count indicates the unhygienic
condition of the food preparation surfaces.
Escherichia coli, fecal coliform bacteria
Twenty five (25) g of each sample were homog-
enized in 225 mL Enterobacteria enrichment broth-
Mossel pre-enrichment medium (Oxoid Ltd., Hamp-
shire, England) and incubated at 35˚C for 20 hours.
One mL of pre-enriched cultures were mixed with
nine mL of 2x EC medium (Nissui Co., Ltd., Tokyo,
Japan) and incubated at 35˚C for 20 hours. To con-
firm the presence of fecal coliforms, one loopful of
the culture was inoculated into 10 mL 1x EC medium
with Durham fermentation tubes and incubated at
42˚C for 20 hours. Gas production in the tube indi-
cates the presence of fecal coliforms. To isolate E.
coli, one loopful of gas producing 1x EC culture
broth was streaked on EMB agar plates (Nissui Co.,
Ltd., Tokyo, Japan) and the developed typical colo-
nies were then confirmed using biochemical charac-
terization (IMViC) and API 20E kit (bioMérieux, Dur-
ham, NC, USA). Presence of E. coli or fecal coliform
bacteria was used as an indicator that the food is
potentially contaminated with fecal material.
Escherichia coli O157, O111, O26
Twenty five (25) g of each samples were homog-
enized in 225 mL mEC medium (Nissui Co., Ltd.,
Tokyo, Japan) and incubated at 42˚C for 20 hours.
The enriched cultures were streaked on Sorbitol
MacConkey agar (Oxoid Ltd., Hampshire, England)
supplemented with Cefixime and potassium tellu-
rite admendments (Fluka, Sigma-Aldrich, Banglore,
India) and characteristic colonies were subjected to
biochemical tests (IMViC). Biochemically confirmed
isolates were screened using Rainbow agar (Biolog,
France) and CHROM agar (Kanto Co. Ltd., Kyoto, Ja-
pan). The colonies which gave the characteristic col-
or were serotyped using O157, O111 and O26 spe-
cific antisera. The isolates were subsequently tested
for the presence of stx1 and stx2 by NH-Immuno-
Figure 1b. Identification of selected Critical Sampling Locations (CSLs) in the production chain of dried lemongrass.S0: At the field; S1: weeks before harvest; S2: harvest and washing; S3: drying/sorting/grinding/packaging;
168 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
chromato VT1/2 and by polymerase chain reaction
(PCR) assay using primer 5’-CAGTTAATGTGGTG-
GCGAAGG-3’ and 5’-CACCAGACAAATGTAACC-
GCTC-3’ for stx1 and 5’-ATCCTATTCCCGGGAGTT-
TACG-3’ and 5’-GCGTCATCGTATACACAGGAGC-3’
for stx2, respectively (Vidal et al., 2004).
Salmonella spp.
Twenty five (25) g of each sample were homog-
enized in 225 mL of buffered peptone water (Merck,
Darmstadt, Germany) and incubated at 35˚C for 20
hours. One mL pre-enrichment cultures was mixed
with nine mL of Hanja Tetrathionate Broth (Eiken
Chemical Co. Ltd., Tokyo, Japan) and incubated at
35˚C for 20 hrs and nine mL of Rappaport-Vassiliadis
Broth (Eiken Chemical Co. Ltd., Tokyo, Japan) and
incubated at 42˚C for 20 hrs. The broth the culture
broths were subsequently streaked onto DHL and
MLCB and characteristic colony were characterized
with biochemical tests (TSI and LIM). Biochemically
confirmed isolates were re-confirmed using Salmo-
nella LA latex agglutination test and API 20E kits.
Hot water treatment in tea-cup & En-richment study
One gram of dried or blended herbs samples
were added in a cup and 50 mL of hot water was
poured over the dried leaves. The cup was kept with
and without lid up to 5 minutes. In each 30 second
interval microbiological parameters were done as
described in the previous section on microbiological
medium and conditions. For the enrichment study,
one mL of hot water treated sample was added into
9 mL of Tryptic soy broth (TSB; Oxoid Ltd., Hamp-
shire, England) medium and incubated at 37˚C for
6 hrs and then spread on to the selective medium
of interest. If any bacteria survived or injured non-
selective TSB medium was used to help resuscitate
these cells and enable them to grow in selective mi-
crobiological medium.
Statistical Analysis
Three samples of each category were taken from
the same farm. Reported plate count data represent-
ed in tables are the log10 mean values ± standard
deviation of three individual trials, and each of these
values were obtained from duplicate samples. Data
were subjected to analysis of variance using the Mi-
crosoft Excel program (Redmond, Washington DC,
USA). Significant differences in plate count data
were established by the least-significant difference
(P < 0.05) at the 5% level of significance.
RESULTS AND DISCUSSION
The search for healthy, safe, and sustainable food
production has increased the consumption of or-
ganic fresh produce. These products should be free
of pesticide residues and other synthetic substances
commonly used in conventional agriculture, such as
soluble fertilizers (Oliveira et al, 2012). At the same
time organic products have lower risks related to
chemical contamination; however, several investiga-
tions have raised concerns related to the microbio-
logical quality of these foods (Delaquis et al., 2007;
Itohan et al., 2011). Among organic fresh produce,
fresh and dried herbs stand out due to their flavors,
aromas, colors and continual availability in the mar-
ket as well as acceptability regardless of age or eco-
nomic group of the human population worldwide
(Esimone et al., 2003).
Thirteen categories of herbs and tea including
black tea, blend tea, neem blend herbs, neem tea,
mint (fresh and dry), jasmine (fresh & dry), lemon-
grass and lemongrass blend tea were analyzed for
total aerobic population (TAB), total coliform popu-
lation (TCC) and presence of E. coli, E. coli O157:H7
and Salmonella spp. Table 1 presents the results of
the distribution of natural aerobic population, co-
liform population and presence of E. coli, E. coli
O157:H7 and Salmonella spp in different fresh and
dry herbs; water and manure soil. Higher aerobic
bacterial counts were recorded as 6.9 log CFU/g
in liker base tea samples and the lowest aerobic
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 169
Table 1. Distribution of natural aerobic population, coliform population and presence of E. coli, E. coli O157:H7 and Salmonella spp in different fresh and dry herbs; water and manure soila
Herbs and tea Sample
Total Viable count
(log CFU/g)
Total Coliform count
(log CFU/g) )
Total E.coli Count
(log CFU/g)
E.coli O157:H7 counts
(log CFU/g)
Presence of Salmonella
Spp.pH
Black tea (Normal)
5.9 ± 0.08 6.9 ± 0.11 4.7± 0.14 3.8 ± 0.06 ND 4.84
Blend tea 6.4 ± 0.11 5.9± 0.11 4.5 ±0.11 4.5 ± 0.12 ND 5.01
Black tea (Original) 6.0± 0.14 6.0± 0.34 5.1±0.11 4.6± 0.11 ND 5.40
Neem blend herbs
4.1 ± 0.15 ND* ND ND ND 4.98
Neem Tea 3.9 ± 0.12 ND ND ND ND 5.04
Black tea (Premium) 5.4 ± 0.14 4.1 ± 0.21 4.0 ± 0.12 3.2 ± 0.11 ND 5.10
liker base 6.9 ± 0.22 6.2 ± 0.22 5.7 ± 0.13 5.2 ± 0.15 ND 7.13
Lemongrass 5.9 ± 0.11 5.8 ± 0.15 5.8 ± 0.23 4.8 ± 0.13 ND 4.60
Lemongrass blended tea 5.5 ± 0.24 5.0 ± 0.09 4.7 ± 0.19 4.4 ± 0.11 3.7 ± 0.07 6.00
Jasmine fresh 5.7 ± 0.11 5.3 ± 0.12 5.1 ± 0.11 4.0 ± 0.12 1.0 ± 0.09 5.94
Jasmine dried 5.4 ± 0.13 5.2 ± 0.17 5.0 ± 0.11 3.9 ± 0.15 1.0 ± 0.12 5.94
Mint fresh 4.5 ± 0.12 4.4 ± 0.19 4.2 ± 0.12 3.2 ± 0.11 1.3 ± 0.14 5.99
Mint dried 4.4 ± 0.14 3.5 ± 0.23 3.4 ± 0.11 2.4 ± 0.12 ND 5.94
Tap water 3.5 ±0.13 2.0 ± 0.14 1.8 ± 0.11 1.7± 0.13 ND* 6.60
Tank water 6.0 ±0.13 4.7 ± 0.09 3.9 ± 0.11 3.1 ± 0.11 ND 7.50
Ground water 3.8 ± 0.13 3.4 ± 0.07 3.0 ± 0.11 2.1 ± 0.12 ND 6.50
Manure soil 6.0 ± 0.14 5.8 ± 0.09 5.0 ± 0.11 4.7 ± 0.11 ND 7.80
*ND=Not detected; aResults are expressed in mean± standard deviation of three replicate samples, which are being calculated from duplicate plates.
170 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
counts were observed as 3.9 log CFU/g in neem tea
samples (Table 1). Among the herb and tea sample
tested, neem tea and neem blended herbs were de-
termined to be microbiologically safe, because no
coliform, fecal coliforms, E. coli, or Salmonella were
recovered throughout the study. In contrast, jasmine,
mint and tea blend with lemongrass were deter-
mined to be contaminated as the presence of E. coli
O157:H7 and Salmonella was observed. The supply
water used for irrigation, wash/rinse purposes, and
compost used as fertilizer of soil were also analyzed.
The water and composted manure was found to be
heavily contaminated with enteric bacterial patho-
gens (Table 1). The total coliforms, E. coli and E. coli
O157:H7 populations were enumerated as 5.0 log
CFU/ml, 4.7 CFU/ml and 4.2 CFU/ml, respectively.
Salmonella spp. was not detected in the manure
sample tested (Table 1). In this study, water for irriga-
tion and washing/rinse purpose was contaminated
with E. coli O157H7, therefore it was concluded that
there was a risk of contamination of final products.
Foodborne outbreaks involving green vegetables
contaminated by water have been reported in sever-
al studies around the world (Beuchat, 1996; Moyne,
et al., 2011). Pathogenic bacteria such as E. coli
O157:H7 are most often associated with outbreaks
of waterborne diseases, resulting from inadequate
treatment of water used for irrigation and washing
of fresh produce (Levantesi et al., 2012; Beraldo and
Filho, 2011; Fischer-Arndt et al., 2010). Therefore,
specific control measures should be developed in
order to prevent final product contamination.
Table 2. Distribution of natural aerobic population, coliform population and presence of E. coli, E. coli O157:H7 and Salmonella spp at different steps of dried lemongrass productiona.
Lemongrass pro-duction & process-
ing steps
Total aerobic counts
(log CFU/g)
Total Coli-form counts (log CFU/g)
Total E. coli counts
(log CFU/g)
E. coli O157:H7 counts
(log CFU/g)
Presence of Salmonella
Spp.pH
At Harvest 5.9 ± 0.08 5.8± 0.18 5.8± 0.18 4.8 ± 0.16 ND 4.6
Cleaning & No washing
5.7 ± 0.12 5.4 ± 0.08 4.7 ± 0.08 3.7 ± 0.10 ND 5.2
Cleaning & fresh water wash
5.0 ± 0.11 4.3± 0.16 4.3 ± 0.10 3.6± 0.20 ND 5.1
Cleaning with fresh hot water
5.2 ± 0.14 4.5 ± 0.08 3.8± 0.19 3.5 ± 0.17 ND 5.0
After dry heat at 900C for 20 min
4.4 ± 0.12 2.9 ± 0.12 2.8 ± 0.15 2.7 ± 0.12 ND 5.2
After grinding at room temperature
4.2 ± 0.13 4.0 ± 0.09 3.7 ± 0.18 3.7 ± 0.16 ND 5.4
After sorting at room temperature
5.6 ± 0.11 3.5± 0.20 3.3 ± 0.11 3.1 ± 0.11 ND 5.3
After final streaming 4.4 ± 0.10 4.3 ± 0.08 2.3 ± 0.10 2.3 ± 0.12 ND 5.4
*ND=Not detected; aResults are expressed in mean ± standard deviation of three replicate samples, which are being calculated from duplicate plates
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 171
However when composted manure was analyzed,
the presence of higher numbers of coliforms (5.8 log
CFU/g), E. coli (5.0 log CFU/g), and E. coli O157:H7
(4.7 log CFU/g), were observed. Salmonella spp. was
not detected in the compost samples (Table 1). Nu-
merous published reports have indicated that the
composting time and temperature of manure could
effectively reduce microorganisms like E. coli, E. coli
O157:H7, and Salmonella, which were routinely de-
tected in fresh compost (James, 2006; MAFF, 2000;
Millner, 2003; Johannessen, 2005). However, the or-
ganic fertilizer samples analyzed in the present study
were above the detection limit (3.0 log MPN/g), indi-
cating that the control of manure was not adequate.
From the same farm, lemongrass production prac-
tices were taken as a case study to determine the point
of E. coli O157:H7 contamination and to take correc-
tive measure in eliminating the risk. The average aero-
bic bacterial counts, coliform counts, E. coli and E. coli
O157: H7 counts were recorded as 5.9 log CFU /g., 5.8
log CFU /g, 5.8 log CFU/g and 4.8 log CFU /g, respec-
tively after harvest. However, Salmonella spp. was not
detected in the lemongrass sample (Table 2). After har-
vest, the lemongrass sample was washed with water,
in the hope of being able to remove the debris and to
reduce the microbial load. Washing with tap water re-
duced the microbial load by 0.5-1.0 log CFU/g of bac-
teria. The lemongrass sample was subsequently dried
in a fluid bed dryer for 20 minutes at a temperature
recorded as 90˚C. After the drying process was com-
pleted, the microbial load was decreased substantially
but not eliminated completely. Thereafter, grinding,
sorting and packaging were done at the commercial
settings. The contamination remaining was still evident
after packaging and in the finished product. Numer-
ous research reports have indicated that dry heating
at 90˚C for 20 minutes is sufficient to eliminate the
pathogen (Bari et al., 2009), however, in this study,
pathogens were not eliminated completely. This find-
ing suggested that heating temperature or the contact
Table 3. Recovery of natural aerobic population, coliform population and presence of E. coli, E. coli O157:H7 and Salmonella spp after corrective measures in processing and production of dried lemongrassa.
Lemongrass pro-duction & pro-cessing steps
Total aero-bic count
(log CFU/g)
Total Coli-form count (log CFU/g)
Total E. coli Count
(log CFU/g)
E.coli O157:H7 counts
(log CFU/g)
Presence of Salmo-nella Spp.
pH
Positive / No of sample tested
Control 5.9 ± 0.08 5.8 ± 0.18 5.8 ± 0.18 4.8 ± 0.16 ND 4.6
Lemongrass (After corrective measures 1)
2.7 ± 0.18 ND* ND ND ND 5.7 0/3
Lemongrass (After corrective measures 2)
2.5 ± 0.12 ND ND ND ND 5.8 0/3
Mint (After corrective measures 1)
2.7 ± 0.14 ND ND ND ND 6.0 0/3
Mint (After corrective measures 2)
2.9 ± 0.11 1.3 ± 0.09 1.0 ± 0.11 ND ND 5.6 1/3
*ND=Not detected; aResults are expressed in mean± standard deviation of three replicate samples, which are being calculated from duplicate plates.
172 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
time may not be adequate to inactivate pathogens in
lemongrass samples. After that an investigation of ac-
tual temperature and time inside the fluid bed dryer
was conducted and it was discovered that the actual
temperature at contact point was not homogeneous
for 90˚C, and the contact time was only a few seconds
because uniform conditions were not achieved by
passing air through the lemongrass layer under con-
trolled velocity conditions to create a fluidized state.
Therefore, when the sample comes in contact with
heat for few seconds, some bacteria may become in-
jured and could resuscitate in between the cycles and
in following steps, therefore, survive and subsequently
be detected in the final products. Therefore, corrective
actions of dryer temperature were undertaken and af-
ter these corrective measures, the same samples were
dried in the same machine, analyzed and the results
are presented in Table 3. It was found that drying at 90
˚C for 20 min in an oven was enough to eliminate the
pathogens even though the sample was contaminated
initially (Table 3). To prevent further contamination, the
workers was trained for personal hygiene and GAP, and
hand gloves, mask, hairnet, apron, hand washing soap/
sanitizers, and a single used towel was provided, along
with cleaning of utensils, machinery, and transport ve-
hicles was conducted using steam. After these steps
were taken, one batch of lemongrass was processed,
dried and analyzed for pathogens. Neither generic E.
coli nor any pathogenic E. coli O157:H7 were detect-
ed in the in the sample and the total viable bacteria
and coliform population counts were found to be less
than 100 CFU/ml, which is below the permissible limit
(Table 3). These findings again showed that good hy-
giene practices are necessary for reducing foodborne
pathogen contamination in the product.
For the consumer, a common strategy to avoid
foodborne disease is heating or cooking of potential
risk products before consumption. However, this ap-
proach is not appropriate for the majority of fresh herbs
and leafy greens that are mainly consumed raw, or
added to food after the heat-treatment. For example,
tea is usually made with dried tea leaves, or blended
with other dried herbs and pouring the boiling water
over the leaves and letting the combination remain for
a few minutes and then consumed. This general prac-
tice is consistent all over the world. If the herbs/tea
leaves were contaminated with pathogens, whether or
not hot water can reduce the risk of pathogen inges-
tion is a critical consideration. To solve this approach,
an experiment was designed to determine the effec-
tiveness of pouring hot water onto dried herbs/leaves
in a cup for eliminating the risk of pathogen exposure.
The results were presented in Table 4. Three differ-
ent contaminated tea samples include black, blend
and lemongrass tea were analyzed. One gram of each
sample was placed in a tea-cup and 50 ml of hot wa-
ter was added to each cup individually, either covered
with a lid or without a lid, and held up to 5 min. At
each 30 second time interval, microbiological popula-
tion counts were enumerated and recorded. The hot
water temperature was recorded as 82˚C when initially
added in the cup and after 60 seconds the tempera-
ture was reduced to 78˚C; further reduced to 73˚C after
3 minutes holding time and at the end of 5 minutes the
temperature decreased to 64˚C. It was determined that
the initial viable bacterial counts were 5.4 log CFU/g,
coliform counts were 4.1 log CFU/g, E. coli counts were
4.0 log CFU/g and E. coli O157:H7 counts were 3.2 log
CFU/g in the blended tea samples, respectively (Table
4). After 30 seconds of treatment with hot water with-
out a lid, a 2.0 log CFU/g reduction of viable bacterial
counts was observed for the blended tea samples. The
coliform bacteria, E. coli and E. coli O157:H7 counts
were reduced to non-detectable levels within 30 sec-
onds of hot water treatment despite the higher patho-
gen contamination levels in the initial samples. Similar
experimental results were observed in black tea, and
the lemongrass sample. The bacteria might be injured
or severely injured when hot water was added in the
cup and thus may not be able to grow in selective mi-
crobiological medium. To solve this issue, an enrich-
ment study was done. No coliform, E. coli and E. coli
O157:H7 were detected in the enrichment study after
30 seconds and above this holding time (Table 4). This
finding suggested that the addition of hot water (82˚C)
in the tea leaves resulted in the reduction of pathogens
below detection limits of the current study and thus the
present tea making practice is potentially capable of
providing safe tea for drinking even though the tea
leaves were initially contaminated.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 173
Tab
le 4
. Eff
ecti
vene
ss o
f p
our
ing
ho
t w
ater
ove
r co
ntam
inat
ed d
ried
(ble
nd t
ea, b
lack
tea
and
lem
ong
rass
sam
ple
s)
leav
es in
a c
up o
f te
a at
diff
eren
t ho
ldin
g t
ime.
Sam
ple
typ
eH
ot
wat
er
trea
tmen
t ti
me
(Sec
)
Rec
ove
ry o
f m
icro
org
anis
ms
(log
CFU
/g)a
Aft
er E
nric
hmen
t
Tota
l aer
o-
bic
co
unt
Tota
l Co
li-fo
rm c
oun
t E
. co
liE
. co
li O
157:
H7
Salm
one
lla
Spp
.P
rese
nce
of
Co
li-fo
rm
Pre
senc
e o
f E
.co
liP
rese
nce
of
E. c
oli
O15
7:H
7
Pre
senc
e o
f Sa
lmo
nella
Sp
p.
Ble
nded
Tea
Sa
mp
les
Co
ntro
l5.
4 ±
0.0
84.
1 ±
0.1
04.
0 ±
0.1
23.
2 ±
0.1
1N
D-
--
-
303.
5 ±
0.1
8<
1.0*
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
603.
2 ±
0.1
2<
1.0
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
903.
0 ±
0.1
1<
1.0
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
120
3.0
± 0
.14
<1.
0<
1.0
<1.
0N
DN
ilN
ilN
ilN
il
150
2.9
± 0
.09
<1.
0<
1.0
<1.
0N
DN
ilN
ilN
ilN
il
180
3.2
± 0
.09
<1.
0<
1.0
<1.
0N
DN
ilN
ilN
ilN
il
5 m
in3.
1± 0
.08
<1.
0<
1.0
<1.
0N
DN
ilN
ilN
ilN
il
Bla
ck T
ea
Sam
ple
sC
ont
rol
5.9
± 0
.11
6.9
± 0
.12
4.7
± 0
.11
3.8
± 0
.16
ND
--
--
301.
3 ±
0.1
4<
1.0
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
601.
3 ±
0.1
3<
1.0
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
901.
0 ±
0.0
9<
1.0
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
120
1.0
± 0
.07
<1.
0<
1.0
<1.
0N
DN
ilN
ilN
ilN
il
150
-<
1.0
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
180
-<
1.0
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
5 m
in-
<1.
0<
1.0
<1.
0N
DN
ilN
ilN
ilN
il
lem
ong
rass
Sa
mp
les
Co
ntro
l6.
0 ±
0.1
56.
0 ±
0.1
25.
1 ±
0.1
14.
6 ±
0.1
4N
D-
--
-
303.
1 ±
0.1
6<
1.0
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
603.
0 ±
0.1
2<
1.0
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
903.
0 ±
0.1
1<
1.0
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
120
3.0
± 0
.07
<1.
0<
1.0
<1.
0N
DN
ilN
ilN
ilN
il
150
-<
1.0
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
180
-<
1.0
<1.
0<
1.0
ND
Nil
Nil
Nil
Nil
5 m
in-
<1.
0<
1.0
<1.
0N
DN
ilN
ilN
ilN
il
<1.
0*=
Les
s th
an d
etec
tion
limit;
ND
= N
ot
Det
ecte
d, N
il= A
bse
nt; a R
esul
ts a
re e
xpre
ssed
in m
ean±
sta
ndar
d d
evia
tion
of t
hree
rep
licat
e sa
mp
les,
whi
ch
are
bei
ng c
alcu
late
d fr
om
dup
licat
e p
late
s.
174 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
CONCLUSIONS
In the present study it can be concluded that
the organic fertilizer and the water used for irriga-
tion and washing are critical sources of microbial
contamination that need to be controlled in the
production chain of organic produce. The contam-
ination of manures also highlighted the need for
a fertilizer control program in order to control the
composting time and avoid the addition of fresh
manure to the composted manure. Regarding
the issues of irrigation and wash water, the results
demonstrated the importance of using water from
safe sources. It is also essential to emphasize the
need for awareness and training to food handlers
because even though organic vegetables may
not be perceived as being chemically contami-
nated; nonetheless, they could very well be con-
taminated with pathogens and, for that reason,
sanitization procedures should be developed to
avoid foodborne illnesses. The use of a risk-based
sampling plan in combination with corrective
measures, personal hygiene and good agricultural
practices (GAP) allowed us to produce safe organ-
ic herbs. This case study provides an overview of
the organic farms’ status in northern Bangladesh,
where good hygiene practice and GAP were intro-
duced as a part of this study.
ACKNOWLEDGEMENTS
The authors would like to thank Mr. Harun-ur
Rashid and Mr. Abul Kalam Azad for the labora-
tory assistance required to complete this task. The
authors would also like to thank the United Nations
University, Tokyo, Japan (UNU-ISP) for financial sup-
port (FY 2013-2014) in this work.
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www.afabjournal.comCopyright © 2014
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Interspecies H2 transfer is a syntrophic interaction between H2-producing and H2-consuming organisms,
that plays an important role in regulating ruminal fermentation as well as other ecosystems. Any decrease
in hydrogen concentration, due to interspecies hydrogen transfer can influence volatile fatty acid fermenta-
tion patterns of many ruminal microorganisms. In the rumen, methanogens consume hydrogen to gener-
ate energy and thus serve as a hydrogen sink. However energy is lost due to eructation of methane which
can not be used by the ruminant animal. Alternative hydrogen consuming organisms, such as acetogens,
could be an attractive alternative hydrogen sink in rumen ecosystems because they generate actetate
from hydrogen and carbon dioxide, which can be used by the host animal. However, this would require
inhibiting methanogenic activity. Therefore, batch cultures were used to study acetogenesis as a functional
alternative to methanogenesis in the rumen in the presence of a methanognesis inhibitor. In batch culture
experiments, acetogen strains G1.5a, G2.4a, G3.2a, A10, and 3H were able to reduce H2 concentrations in
ruminal contents in the presence of bromoethanesulfonic acid, an inhibitor of methanogenesis. Batch cul-
ture studies indicated that acetogens could function as an alternative electron sink to methanogens under
some conditions.
Keywords: Acetogen, Acetogenesis, H2, Methane, Ruminal
INTRODUCTION
The productivity of ruminant domestic animals
is influenced, to a large extent, by the efficiency of
microbial fermentation of feedstuffs in the rumen.
During rumen fermentation, complex carbohydrates
Correspondence: John Patterson, [email protected]: +1-765-494-4826 Fax: +1-765-494-9347
(e.g., cellulose) are degraded to monomeric carbo-
hydrates (e.g., glucose) which are primarily ferment-
ed to pyruvate via the Embden-Meyerhof-Parnas
pathway (Ricke et al., 1996; Weimer, 1992; Weimer
et al., 2009). Pyruvate is subsequently metabolized
to volatile fatty acids (VFA; acetate, propionate, and
butyrate), CO2, H2, and microbial cells. While fer-
mentation acids provide 60 to 80% of the daily me-
tabolizable energy intake of ruminants (Annison and
Batch Culture Characterization of Acetogenesis in Ruminal Contents: Influence of Acetogen Inocula Concentration
and Addition of 2-Bromoethanesulfonic Acid
P. Boccazzi 1,2 and J. A. Patterson1
1 Department of Animal Sciences, Purdue University. West Lafayette, IN 479072 Current address: 147 Kelton St., Allston, MA 02134
Agric. Food Anal. Bacteriol. 4: 177-194, 2014
178 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Armstrong, 1970), microbial cells provide an impor-
tant source of amino acids, vitamins, and cofactors
(Hungate, 1966).
Interspecies H2 transfer is a syntrophic interaction
between H2-producing and H2-consuming organisms
that plays an important role in regulating ruminal
fermentation as well as fermentations in other an-
aerobic ecosystems (McInerney et al., 2011). Hydro-
gen produced by fermentative microorganisms is
consumed by H2 utilizing microorganisms, namely,
methanogens, sulfidogens, and acetogens (Lupton
and Zeikus, 1984; Drake, 1992, 1994; Saengkerdsub
and Ricke, 2014). The decrease in H2 concentration,
due to interspecies H2 transfer, influences the VFA
fermentation patterns of many ruminal microorgan-
isms because hydrogen is an end product inhibi-
tor of the hydrogenase enzyme (Wolin and Miller,
1983; Ricke et al., 1996; Weimer et al., 2009). When
H2 concentrations are high, pyruvate is utilized as
a reducing equivalent acceptor and more reduced
fermentation products (e.g., propionate, lactate,
and ethanol) are produced (Wolin and Miller, 1983).
When H2 concentrations are low, there is an increase
in acetate and ATP production that could be con-
verted into an increase in overall microbial cell yields
(Wolin and Miller, 1983).
The energy present in methane escapes the ru-
men through eructation and is lost to the animal.
Because energy lost as methane has been estimated
to be 2.4 to 7.4% of the gross energy intake (Branine
and Johnson 1990) or 10 to 15% of the apparent di-
gestible energy of the diet of ruminants (Blaxter and
Clapperton, 1965), there has been an interest to spe-
cifically inhibit methanogenesis to enhance animal
productivity. Direct inhibition of methanogenesis,
however, also results in loss of energy in the form
of H2, and reduction in production of microbial pro-
teins (Chalupa, 1980).
Maintaining the beneficial effects of interspecies
H2 transfer while minimizing loss of energy as meth-
ane could enhance energy provided to ruminants by
22% (Schaefer, D., personal communication). How-
ever, an alternative electron sink is required to trap
electrons into a form utilizable by the animal if meth-
anogens are to be directly inhibited. To date the
major method used to manipulate rumen fermen-
tation has been the use of ionophore antibiotics
such as monensin and lasalocid. These compounds
improve the efficiency of animal production by de-
creasing methane production and increasing ruminal
propionate concentration by 15%. Methane produc-
tion decreases primarily because monensin inhibits
H2 producing microorganisms, therefore decreasing
the amount of H2 available for methanogenesis.
Chemolithoautotrophic acetogenic bacteria
achieve reductive acetogenesis, utilizing CO2 and H2
as their sole carbon and energy source, respectively,
fixing CO2 into acetate (Ragsdale, 1991). Acetogen-
esis has been demonstrated to be the predominant
fate of H2 in some humans, swine, xylophagus ter-
mites, cockroaches and rats (Breznak and Blum, 1991;
Lajoie et al., 1988). Replacing methanogenesis with
acetogenesis in the rumen may have potential in de-
creasing energy losses in ruminants. Peptostrepto-
coccus productus (Bryant et al., 1958), Eubacterium
limosum (Genthner, 1981), and Acetitomaculum ru-
minis (Greening and Leedle, 1989) are chemolitho-
autotrophic acetogenic bacteria that have been iso-
lated from the bovine rumen. However they are not
considered the primary H2 consuming organisms in
this environment, since their numbers are consistent-
ly lower than methanogens.
Factors dictating whether acetogenesis or metha-
nogenesis will predominate in anaerobic environ-
ments are not well understood. Breznak and Kane
(1990) suggested several possible factors that may
influence the competitiveness of acetogens with
methanogens. One factor is that methanogenesis
has a higher energy yield than acetogenesis (Breznak
and Blum, 1991).
Another important factor is that methanogens
have a higher affinity for H2 than acetogens. The
normal rumen hydrogen concentration is between
10-5 and 10-6 atm (Czerkawski and Breckenridge,
1971; Robinson et al., 1981). Ruminal methanogens
have an affinity for H2 between 1 and 4x10-6 atm
(Greening et al., 1989). Different acetogenic isolates
have been shown to have affinities for H2 between
10-4 and 10-5 atm (Greening et al., 1989; LeVan etal.,
1998). In general, methanogens have been found
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 179
to have H2 thresholds 10 to 40 fold lower than ace-
togens (Greening et al., 1989; Breznak and Blum,
1991). However, in our laboratory, acetogens with
H2 thresholds only 2 to 4 fold higher than those of
methanogens were isolated from ruminal contents
using a hydrogen limited continuous culture system
(Boccazzi and Patterson, 2011).
Methane contributes roughly 25% of the global
green house warming and is considered the second
most important “greenhouse gas” after CO2 (Ty-
ler, 1991). Atmospheric CH4 is presently increasing
by 1% per year and it has reached a concentration
unprecedented in the past 160,000 years (Pearman
and Fraser, 1988). Ruminal and other gastrointesti-
nal fermentations account for some 14% of the total
CH4 emissions amounting to 70 to 100 Tg per year
(EPA, 1993, Moss, et al., 2000). Limiting CH4 emis-
sions from livestock and livestock waste while main-
taining interspecies H2 transfer would improve ru-
minant productivity and at the same time would be
beneficial for the environment. Certainly acetogens
offer that possibility, but rumen ecosystem condi-
tions would need to be designed that favor not only
their presence but their acetogenic activities. One
approach is to administer a methane inhibitor such
as BES (Immig et al. 1996), but before this can at-
tempted in a practical application in vitro screening
needs to be done to confirm that this will generally
be effective and which acetogens are the best candi-
dates. Batch culture growth experiments, while not
necessarily representative of the rumen from a pas-
sage rate and rumen turnover standpoint, still offer
a means to rapidly screen multiple bacterial isolate
responses and have been used for a wide range of
physiological studies on rumen bacteria includ-
ing rumen acetogens (Russell and Baldwin, 1978;
Schaefer et al., 1980; Ricke and Schaefer, 1991, 1996;
Ricke et al., 1994; Jiang et al., 2012; Pinder and Pat-
terson, 2012, 2013). The objective of this study was
to conduct batch culture screening experiments to
determine the feasibility of a functional replacement
of methanogenesis with reductive acetogenesis in
ruminal contents in the presence of a methanogen
inhibitor.
MATERIALS AND METHODS
Source of Organisms
Acetobacterium woodii (ATCC 29683) was ob-
tained from the American Type Culture Collection
(Rockville, MD). Acetogenic bacterial strains 3H,
G1.5a, G1.5e, G2.4a, G3.2a and A10 were isolated
and characterized in our lab and reported previously
(Boccazzi and Patterson, 2011, 2013; Pinder and Pat-
terson, 2011, 2012, 2013; Jiang et al., 2012).
Media and Growth Conditions
Growth and H2 threshold experiments were con-
ducted with a basal rumen fluid based acetogen
medium (Table 1) or with Mac-20 medium containing
casein hydrolysate and no rumen fluid (Table 1). Both
media were prepared as described in Table 1 with the
anaerobic techniques of Hungate (1966) as modified
by Bryant (1972) and Balch and Wolfe (1976). The
Mac-20 medium was only used with cultures of Ace-
tobacterium woodii. The prepared medium was dis-
pensed anaerobically into 60 ml serum bottles (West
Company, Phoenixville, PA) in an anaerobic glove
box (Coy Laboratories, Ann Arbor, MI) containing a
H2:CO2 (5:95) gas phase. Serum tubes and bottles
were sealed with butyl rubber serum stoppers and
aluminum seals (Bellco Inc., Vineland, NJ). All stock
solutions utilized to formulate media were prepared
anaerobically by boiling and cooling distilled water
under a CO2 gas phase and sterilized either by auto-
claving or by injecting the solution through a 0.2 μm
filter (Nalgene, Nalge Company, Rochster, NY).
For chemolithoautotrophic growth in broth me-
dium, bacterial cultures were grown in serum bottles
closed with butyl rubber stoppers and aluminum seals.
After medium sterilization, cooling and inoculation,
the bottles were flushed for 30 sec with an appropri-
ate gas mixture by inserting both a sterile gassing and
a release needle through the serum stoppers and then
bottles were pressurized to 200 kPa by removing the
release needle. Oxygen traces were removed from
gas mixtures by passing the gas through a reduced
180 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Table 1. Media compositiona
Acetogen Medium
(amounts per liter)
MAC-20 Mediumb
(amounts per liter)
Rumen Fluid 50.0 ml ---
Mineral 1c 40.0 ml 40.0 ml
Mineral 2d 40.0 ml 40.0 ml
Additional Trace Min. Sol.e 10.0 ml 10.0 ml
Wolfe’s Trace Min. Sol.f 10.0 ml 10.0 ml
Vitamin Solutiong 10.0 ml 10.0 ml
Na2CO3 4.0000 g 4.0000 g
Yeast Extract 0.5400 g 2.0000 g
Casein Hydrolysate --- 1.0000 g
Betaine --- 1.0000 g
NH4Cl 0.5400 g 0.5400 g
Cysteine.HCl 0.5000 g 0.5000 g
Resazurin solution 0.0010 g 0.0010 g
Hemin solution 0.0001 g 0.0001 g
a All components, except Na2CO3 and cysteine. HCl were dissolved in distilled water and brought to a vol-ume to 1000 ml. These were mixed thoroughly and the pH was adjusted to 7.0 with 1 M NaOH followed by gentle heating to a boil for 1 min. Na2CO3 was added and the solution was cooled rapidly to 25°C under 100% CO2. Cysteine.HCl was added, mixed thoroughly and autoclaved anaerobically for 12 min at 121°C and 15 psi
b Modification of AC-19 medium by Breznak et al. (1988)
c Mineral 1 (g/liter): 6.00 K2HPO4
d Mineral 2 (g/liter): 12.00 NaCl, 6.00 K2HPO4, 6.00 (NH4)2SO4, 2.45 MgSO4.7H20, 1.60 CaCl2.2H2O
e Additional Trace Mineral Solution (g/Liter): 0.10 NiCl2.6H2O, 0.01 H2SeO3
f Wolfe’s Trace Mineral Solution (g/liter): 3.00 Mg SO4.7H20, 1.00 NaCl, 0.50 MnSO4.H20, 0.10 CoCl2.6H20, 0.10 FeSO4.7H20, 0.10 CaCl2.2H20, 0.18 CoSO4.6H20, 0.19 ZnSO4.7H20, 0.02 AlK(SO4)2.12H20, 0.01 CuSO4.5H20, 0.01Na2MoO4.2H20
g Vitamin Solution (g/liter): 0.10 pyridoxine.HCl, 0.056 ascorbic acid, 0.05 choline chloride, 0.05 thiamine.HCl, 0.05 D,L-6,8-thioctic acid, 0.05b riboflavin, 0.05 D-calcium panthotenic acid, 0.05 p-amino benzoic acid, 0.05 niacinamide, 0.05 nicotinic acid, 0.05 pyridoxal.HCl, 0.05 pyridoxamine, 0.05 myo-inositol, 0.02 biotin, 0.02 folic acid, 0.001 cynocobalamin
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 181
copper column. Pressurized bottles, unless otherwise
specified, were incubated on their side on a rotatory
shaker (New Brunswick Scientific Co. Inc., Model M52)
operating at 200 rpm. For growth on solid medium,
60 mm disposable petri plates were incubated in an
anaerobic growth vessel (made by the Agricultural and
Biological Systems Department. Purdue University, IN)
able to withstand high gas pressures. Prior to incuba-
tion the container was flushed for 2 min and then pres-
surized to 110.34 kPa with gas mixtures specified in
the text for each experiment. More recently we have
used a less expensive approach using one gallon glass
screw cap storage jars with aluminum lids, the jars were
flushed with CO2 and the environment reduced with
BBL Gaspak Plus Anaerobic System Envelopes with
Catalyst (H2 + CO2) (Becton Dickinson & Co, Sparks,
MD), once the lids were closed, they were sealed with
plastacine modeling clay (Figure 1). These vessels will
not withstand pressurization, but that could be offset
with inserting swagelok fittings and a septa into the lid
and flushing with fresh gas on a routine basis.
General experimental procedure
Serum bottles (60 ml) were anaerobically filled
with 0.35 g alfalfa, 6 ml ADS (Table 2), 4 ml ruminal
contents, 4 ml of an acetogen culture (inoculum) or
acetogen medium (control), and 1 ml stock solution
of 2-bromoethanesolfonic acid (BES, Sigma Aldrich)
was added to provide a final concentration of 5
mM. Control cultures received 1 ml sterile anaero-
bic water. The alfalfa was dried at 60°C and ground
through a 1 mm screen. Ruminal contents were col-
lected from a Holstein Friesian dairy cow, fed a 57:43
concentrate:forage diet, prior to morning feeding
and immersed in ice during transportation to the lab,
where the rumen contents were filtered through a
double layer of cheesecloth under a stream of CO2
and anaerobically inoculated into the serum bottles.
Serum bottles were anaerobically sealed and incu-
bated in a rotatory shaker at 37°C operating at 200
rpm.
Figure 1. (A) Anaerobic incubation vessels made from one gallon glass screw cap storage jars with aluminum lids, the jars were flushed with CO2 and the environment reduced with BBL Gas-pak Plus Anaerobic System Envelopes with Catalyst (H2 + CO2) (Becton Dickinson & Co, Sparks, MD), once the lids were closed, they were sealed with plastacine modeling clay. These vessels will not withstand pressurization, but that could be offset with inserting swagelok fittings for gas chromatography and a septa into the lid and flushing with fresh gas on a routine basis (B).
A B
182 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Acetogenic bacteria inoculum prepara-tion
Enumeration of acetogen strains to prepare inoc-
ulum was done by growing cultures in acetogen me-
dium plus 5 mM glucose under 200 kPa of a H2:CO2
(80:20) gas mixture for 36 h at 37°C. Cultures were
then brought into a glove box where they were seri-
ally diluted to 10-8 with ADS (Table 2). Each dilution
was plated (25 μl) on acetogen medium containing
2% (w/v) agar (Bacto-Agar, Difco, Fisher) and 5 mM
glucose in triplicate 60 mm petri plates. Plates were
incubated, at 37°C for 3 days, in an anaerobic growth
vessel pressurized to 16 psi with a H2:CO2 (80:20) gas
mixture. The bacterial concentration was calculated
by counting colony-forming units (CFU) per ml of
culture. To make the acetogen inoculum for experi-
ments, the acetogens were grown under the same
growth conditions that were used for enumeration
and diluted to the appropriate concentration. Cul-
tures were centrifuged anaerobically at 8,000 rpm for
15 minutes and resuspended in acetogen medium
to reach the CFU/ml desired. The titer for each
acetogen strain used for methanogen replacement
studies was determined before each experiment.
Effect of bromoethane sulfonic acid on H2 and CH4 production in ruminal contents
To test BES as a methanogenesis inhibitor in
ruminal contents with or without the addition of
acetogenic bacteria, duplicate serum bottles (60 ml)
were anaerobically filled with 0.15 g alfalfa, 6 ml of
fresh ruminal contents, 4 ml of acetogen medium
(Table 1), 4 ml of an acetogen culture, 1 ml of a BES.
BES was added as sterile stock solution to the ace-
togen medium to reach final concentrations ranging
between 0.0 to 10 mM, respectively. Both the 5mM
and 10 mM BES effectively inhibited methanogen-
esis and only the 10 mM data is shown. Because 5
mM BES was as effective as 10 mM BES, the lower
concentration was used in all other experiments.
Controls that did not receive a methanogen inhibitor
received an equal volume of acetogen medium in-
stead. Acetogenic bacteria utilized were A. woodii,
and strains A10 and G3.2a. Acetogen inocula were
prepared as described previously to give a final con-
centration of 5x108 CFU/ml. Duplicate serum bottles
for each treatment were anaerobically sealed, and
incubated in a rotatory shaker at 37°C operating at
200 rpm. Headspace gas volume, and H2 and CH4
concentrations were measured at 0, 12, and 36 h of
incubation.
Table 2. Anaerobic dilution solution (ADS) compositiona
Component (amounts per liter)
Mineral 1b 75.0 ml
Mineral 2c 75.0 ml
Cysteine.HCl 0.5000 g
Resazurin solution 0.0010 g
a All components, except cysteine.HCl were added to distilled water and the volume brought to 1000 ml. These were mixed thoroughly and the pH adjusted to 7.0 with 1 M NaOH. These were gently heated and brought to a boil for 1 min. Cysteine.HCl under 100% CO2 was mixed thoroughly and autoclaved anaerobi-cally for 12 min at 121°C and 15 psib Mineral 1 (g/liter): 6.00 K2HPO4
c Mineral 2 (g/liter): 12.00 NaCl, 6.00 KH2PO4, 6.00(NH4)2SO4, 2.45 MgSO4.7H20, 1.60 CaCl2.2H2O
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 183
Effect of acetogenic bacteria on H2 uti-lization in ruminal contents
In an initial experiment using the acetogen strain
3H to determine efficiency of H2 utilization, either 3H
or acetogen medium was added to serum bottles
containing ruminal contents as described above.
Strain 3H was added to the experimental medium
to reach the initial concentration of 5x108 CFU/ml.
Cultures were incubated at 37°C in a rotatory shaker,
operating at 200 rpm, for 72 h. Headspace gas vol-
ume and H2 concentrations were measured from a
triplicate set of serum bottles for each treatment at
0, 12, 24, 36, 48, 60, and 72 h. Volatile fatty acid pro-
files of cultures were also determined at 12, 48 and
72 h by gas chromatography.
Similar procedures were used to determine ef-
ficiency of acetogen strains G1.5a, G1.5e, G2.4a,
and G3.2a except 40 mM of MES (final concentra-
tion) was added for additional buffering. A control
treatment receiving sterile anaerobic water instead
of BES was also added. Final concentrations of ace-
togens were 5x108 CFU/ml. Headspace gas volume
and H2 and CH4 concentrations were measured from
duplicate serum bottles for each treatment at 0, 12,
24, and 72 h.
The experimental procedure was slightly modi-
fied to determine the effect of acetogen dose on H2
concentrations. The following three modifications
were made: 0.15 g alfalfa was used instead of 0.35
g, 40 and 3 mM (final concentrations) of MES and
K2CO3, respectively, were added to the experimen-
tal medium, and a control treatment received sterile
anaerobic water instead of BES. In separate experi-
ments, the acetogen strain G2.4a was added to pro-
vide final concentrations of 5x107 and 1x108 CFU/ml.
Headspace gas volume and H2 and CH4 concentra-
tions were measured from a duplicate set of serum
bottles for each treatment at 0, 12, 24, and 72 h. The
acetogen strain G3.2a was added to provide final
concentration of 5x108, 1x109, and 5x109 CFU/ml.
Headspace gas volume and H2 and CH4 concentra-
tions were measured from duplicate serum bottles
for each treatment at 0, 12, and 36 h.
Analytical Methods
Bacterial growth: optical density was measured
at 660nm using a Spectronic 70 spectrophotometer
(Bausch and Lomb, Rochester, NY). VFA analysis:
volatile fatty acid concentrations were measured
by gas-liquid chromatography (GLC; Holdeman et
al., 1977). At sampling time, samples were acidi-
fied by adding 20% (v/v) of meta-phosphoric acid
(25% w/v) and then frozen. Samples to be analyzed
were thawed, centrifuged at 26,892 x g for 5 min,
and the supernatant was analyzed. A 0.92 meter
long column, packed with SP1220 (Supelco, Belle-
fonte, PA, USA), was used in a Hewlett Packard 5890
GLC equipped with a flame ionization detector.
Oven temperature was 130°C (isothermal), injector
temperature was 170°C, detector temperature was
180°C, the carrier gas was N2 flowing at a rate of 30
ml per minute.
Gas Analysis: for the measurements of hydrogen
and methane concentrations, gas samples were
analyzed using a Varian 3700 Gas Chromatograph
equipped with a thermal conductivity detector, and
a 1.83 meter silica gel column (Supelco). Tempera-
tures of the injector, oven, and detector were room
temperature, 130°C, and 120°C respectively. The
carrier gas was N2 flowing at a rate of 30 ml per min-
ute. The volume of gas injected for standards and
samples was 0.5 ml. The GC was standardized with
5 different concentrations of H2 (400 to 25,000 ppm)
and CH4 (900 to 32,000 ppm). A regression line was
obtained from the output values of the standard con-
centrations. The regression line was then utilized to
calculate H2 and CH4 concentrations in experimen-
tal samples. All gas mixtures were purchased from
Airco (Indianapolis, IN).
184 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Figure 2. Effect of 10 mM of BES and acetogen strains Acetobacterium woodii (Aw), A10 and G3.2a on CH4 production (Fig. A) and H2 utilization (Fig. B) by ruminal contents (C). Duplicate serum bottles (60 ml), containing 6 ml of ruminal contents, received 0.15 g of alfalfa and were incubated at 37°C. Cultures were incubated in duplicate and one standard deviation is depicted with error bars.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 185
RESULTS
Functional Replacement of Methano-genesis with Acetogenesis in Ruminal Contents
The effect of 0.0 to 10 mM of BES on methano-
genesis and H2 utilization was determined in batch
culture and data for 10 mM BES is shown in Figure
2A and B. Methanogenesis was totally inhibited in
all treatments that received 5 and 10 mM BES, but
not with lower doses of BES (data not shown). After
12 h of incubation, cultures receiving the acetogens
G3.2a and A10 had lower hydrogen concentrations
than either A. woodii or the control plus BES cultures
(Figure 5B). Hydrogen concentrations for cultures
receiving A10 and G3.2a were below 18 μmoles by
36 h (Figure 2B). Hydrogen concentrations for cul-
tures receiving A. woodii were similar to the control
plus BES cultures at 12 h, but H2 concentrations did
not decline as rapidly over time (Figure 2B), suggest-
ing that A. woodii is not as effective at utilizing H2 as
the other isolates.
The efficacy of acetogen strain 3H to utilize H2 was
determined by adding strain 3H to ruminal contents
containing 5 mM BES to inhibit methanogenesis.
After 12 and 24 h of incubation, the H2 concentra-
tion of the 3H treatment was about half of that of the
control treatment (Figure 3). However, after 36 h of
incubation the control treatment had similar H2 con-
centrations as the 3H treatment (Figure 3). While the
3H cultures had the highest acetate concentration
after 12 h of incubation, by 72 h, acetate concentra-
tions were similar for both treatments (Figure 3).
An additional experiment was conducted to de-
termine how well 4 acetogen isolates could replace
methanogens as a hydrogen sink in ruminal con-
tents. Methane was produced only in the control
Figure 3. Acetate production (bars) and H2 utilization (lines) by ruminal contents with (3H) or without (C) the addition of 5x108 CFU/ml (final concentration) of the acetogen strain 3H. Metha-nogenesis was inhibited by 5 mM BES in all treatments. Cultures were incubated in triplicate and one standard deviation is depicted with error bars.
186 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Figure 4. CH4 production (Fig. A) and H2 utilization (Fig. B) by ruminal contents after the addi-tion of 0.35 g of alfalfa and with or without (C) the addition of 5x108 CFU/ml (final concentration) of acetogenic isolates G1.5a, G1.5e, G2.4a and G3.2a. Methanogens were inhibited by 5 mM BES in all treatments but C. Cultures were incubated in duplicate and one standard deviation is depicted with error bars.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 187
Figure 5. Effect of inoculum size of the acetogen G2.4a (a= 5x107 and b= 1x108 CFU/ml, final concentration) on CH4 production (Fig. A) and H2 utilization (Fig. B) in ruminal contents (C) after the addition of 0.35 g of alfalfa and in the presence (C) or absence (C – BES) of BES (5 mM) as an inhibitor of methanogenesis. Cultures were incubated in duplicate and one standard deviation is depicted with error bars.
188 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Figure 6. Effect of inoculum size of the acetogen G3.2a (a=5x108, b=1x109 and c=5x109 CFU/ml, final concentration) on CH4 production (Fig. A) and H2 utilization (Fig. B) in ruminal contents (C) after the addition of 0.35 g of alfalfa and in presence (C) or absence (C - BES) of BES (5 mM) as an inhibitor of methanogenesis. Cultures were incubated in duplicate and one standard devia-tion is depicted with error bars.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 189
culture where no BES was added (Figure 4A). The
acetogen isolate G2.4a had the lowest H2 concentra-
tion, followed by G3.2a, G1.5a, and G1.5e after 12 h
of incubation (Figure 4B). After 72 h of incubation,
of the acetogen tested, G1.5e, and G2.4a had the
highest (183.2 μmoles) and lowest (93.8 μmoles) H2
concentrations, respectively, and strain G1.5a had a
slightly lower H2 concentration than G3.2a (Figure 4
B). Although, the control plus BES culture had the
highest H2 concentration (332.2 μmoles) at all times,
H2 concentrations for this treatment decreased be-
tween 24 and 72 h of incubation (Figure 4B), indi-
cating that ruminal contents have some capability to
utilize H2.
Dose response experiments were performed to
determine the optimal acetogen dose for the func-
tional replacement of methanogenesis in ruminal
contents. Methane production was inhibited in
all cultures receiving BES, and remained below 20
μmoles for up to 72 h (Figure 5A). Doubling the dose
of the acetogen G2.4a, from 5x107 to 1x108 CFU/ml,
increased H2 utilization (Figure 5B). Hydrogen con-
centrations for cultures containing G2.4a were lower
than the control without BES at 12 h, but not after
24 h. Cultures containing the highest dose of G2.4a
had a H2 concentration approximately fourfold lower
than the control plus BES (Figure 4B) after 12 h of in-
cubation. The positive effect of increasing acetogen
numbers persisted even after 72 h of incubation (Fig-
ure 4 and 5). However, H2 concentrations of control
plus BES cultures declined to a level similar to that of
the highest dose of G2.4a added after 72 h.
Similar experiments were conducted with aceto-
gen isolate G3.2a with dosages increasing between
5x108 to 5x109 CFU/ml. Methane production oc-
curred only in the control cultures in which BES was
not added (Figure 6A). Hydrogen concentrations for
all the levels of G3.2a remained below 15 μmoles
for 36 h (Figure 6B). Hydrogen concentrations for
control, and control plus BES cultures increased to
148 and 88 μmoles, respectively at 12 h, and then
declined to below 15 μmoles after 36 h. These data
show that the lowest level of G3.2a used, which was
5 fold higher than in the previous study, was suffi-
cient to decrease H2 concentrations effectively.
DISCUSSION
Methane accumulated over time during in vitro
fermentations of alfalfa hay inoculated with ruminal
contents (Fig 2, 4, 5, 6). The addition of 5-10 mM
BES completely inhibited methane production in
these fermentations, with a resultant accumulation
of H2 as would be expected. Additions of less than
5 mM BES did not completely inhibit methane pro-
duction (data not shown). Inoculating BES treated
rumen content cultures with 5 x 108 cfu of acetogenic
bacteria reduced H2 concentrations compared to the
control cultures (Fig. 2) with isolate G3.2a reducing
H2 concentrations most rapidly. Isolate A10 also
significantly decreased H2 concentrations at 12 hr,
compared to the 5mM BES control and Acetobac-
terium woodii. H2 concentration decreased by 40%
in the controls, indicating that there is some ability
to utilize H2 in ruminal contents. However, H2 con-
centrations in the acetogen inoculated cultures were
significantly lower than that of the control cultures.
Thus, acetogens used in this study have the ability to
reduce H2 concentrations during rapid fermentation
and isolate G3.2a has a greater hydrogen utilization
potential than isolate A10 or Acetobacterium woodii
(Fig 2). Acetogenic isolate 3H significantly reduced
H2 concentrations over the first 24 hours of incuba-
tion compared to the BES treated control culture and
also produced more acetate during this time (Fig 3).
Additon of 5 x 108 cfu /ml of isolates G1.5e, G3.2a,
G1.5a and G2.4a showed a similar rapid reduction
of H2 concentration over the first 24 hr, with isolate
G2.4a having the greatest reduction in H2 concen-
tration (Fig. 4).
Two dose level experiments were conducted to
determine the effect of the number of acetogens on
H2 concentrations under slightly different conditions.
Only 0.15 g ground alfalfa hay was added to the in-
cubation bottles and 40 mM MES was added to pro-
vide additional buffering. In the first dose level ex-
periment, isolate G2.4a was inoculated at 5 x 107 cfu/
ml and 1 x 108 cfu. Under these conditions, H2 con-
centrations were significantly lower for the higher
concentration of acetogen addition at all time points
(Fig. 5). In the second dose level experiment isolate
190 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
G3.2a was added inoculated at 5 x 108 cfu and 1 x 109
cfu/ml. In this experiment the higher dose of Isolate
G3.2a completely prevented any accumulation of H2
in the culture bottles, wherease the typical accumu-
lation and then decrease in H2 concentrations was
observed with the lower dose of acetogen (Fig 6).
These experiments demonstrate that acetogens
can utilize H2 produced by rumen microorganisms
during fermentation of substrates during in vitro in-
cubations where methanogenensis has been inhib-
ited by the addition of a methanogen inhibitor, BES.
Thus, these acetogens can utilize H2 in the presence
of other substrates that may be available during fer-
mentation, although Pinder and Patterson (2012)
have shown diauxic growth of isolate A10 in the pres-
ence of glucose in pure culture. The data also show
that different isolates have different capacities for uti-
lizing H2 during fermentation. Acetogens can reduce
H2 concentrations in relatively low numbers (5 x 107
to 1 x 109 cfu/ml), although H2 utilization is greater
with the higher number of acetogens present.
Hydrogen thresholds have been argued to be one
possible reason that methanogens outcompete ace-
togens in the rumen. Methanogens have lower H2
thresholds than acetogens. The acetogens we iso-
lated using H2 limited continuous culture have lower
hydrogen thresholds than most acetogens that have
been isolated. Acetobacterium woodii, isolates
G1.5a, G1.5e, G2.4a, G 3.2a, A10 and 3H have hy-
drogen thresholds of 1007, 800, 635, 908, 960, 209
and 951ppm H2, respectively in our system (Boccazzi
and Patterson 2011, 2013) whereas a methanogenic
isolate had a H2 threshold of 91 ppm in our system
(unpublished data). The reduction of H2 produced
during in vitro incubations of alfalfa hay did not di-
rectly correlate with H2 thresholds within the range
of H2 threshold differences of the acetogens used in
this study, although Acetomaculum ruminis, which
had the highest H2 threshold, also was less able to
reduce H2 concentrations in the in vitro incubations.
The differences between pure culture H2 threshold
measurements and ability to reduce H2 concentra-
tions during incubation may be because H2 threshold
measurements estimate the lowest level of H2 the or-
ganism can utilize over time with no other nutrients
available, whereas the mixed culture incubations
measure how much and how rapidly the acetogens
can utilize H2 as it is being produced. Thus, other
factors such as ability to utilize multiple substrates,
resistance to low pH, growth rate and maximum rate
of H2 utilization may be more important for acetogen
utilization of H2 in ruminal conditions.
This data demonstrates that ruminal acetogens
can reduce H2 concentrations in a mixed ruminal
fermentation when methanogenesis is inhibited and
that different acetogenic isolates have different ca-
pabilities for H2 utilization in batch culture. Thus,
there is potential for acetogens to effectively utilize
H2 for interspecies H2 transfer to increase efficiency
of ruminal fermentation, trap more of the feedstuff
energy into acetate in the absence of methanogen-
esis. Feasibility of utilizing acetogens increase effi-
ciency of ruminant fermentation is dependent upon
identifying safe, cost effective methods to inhibit
methanogenesis.
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www.afabjournal.comCopyright © 2014
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Two grazing experiments were performed to 1) investigate the effects of supplementing condensed
tannins-containing pine bark powder on average daily gain, ruminal fermentation, and rumen microbial
diversity dynamics (Experiment 1), and 2) to quantify the influence of different sources of extracted tannins
supplementations on ruminal fermentation and rumen microbial diversity changes of goats grazing fresh
forages (Experiment 2). In experiment 1, 20 Kiko-Boer cross male goats (Capra hircus; initial body weight=
39.7 ± 2.55 kg) were randomly assigned to 2 experimental diets (alfalfa pellet vs. pine bark powder). Alfalfa
pellet (no tannin as a control) or pine bark powder (11% condensed tannins) was supplemented at 0.5%
body weight for targeted total dry matter intake of 1.2% body weight. The remaining dry matter intake
of each diet was obtained from grazing for 55 days. In experiment 2, 12 Kiko-Boer cross goats were used
to measure average daily gain, ruminal fermentation, and gut microbial population in the rumen of goats
grazing bermudagrass. The animals were randomly assigned to 3 experimental diets: 1) no tannins (con-
trol), 2) chestnut extract at 100 g/d, and 3) quebracho tannin extract at 100 g/day. In experiment 1, aver-
age daily gain and rumen fermentation status as measure of volatile fatty acids production were similar
between diets. Bacterial population in pine bark powder-supplemented group was greater for Bacteroides
(20.5 vs. 33.2%) and Firmicutes (67.2 vs. 57.3%) phylum compared with control group, respectively. In experi-
ment 2, average daily gain was greatest (P < 0.05) for chestnut tannins extract (278.6 g/d) than quebracho
tannins extract (150 g/d) and the control (42.9 g/d). Goats grazing bermudagrass pasture with chestnut tan-
nins extract had greater (P < 0.05) concentrations of acetate, propionate, butyrate, and total volatile fatty
acids compared to those in quebracho tannins extract and control. Bacterial population in chestnut tannins
extract-supplemented group was greatest for Bacteroides (51.5, 52.9, and 35.3%) phylum compared with
quebracho tannin extract and control group, respectively. Current study shows that tannins from plants can
exhibit a positive or negative effect both on rumen fermentation and on rumen microflora, and it is possible
that this effect is depending on sources of tannins or tannin-containing diet.
Keywords: Goats, gut microbial diversity, plant tannins, pyrosequencing
Correspondence: B.R. Min, [email protected]: +1-334-524-7670 Fax: +1-334-727-8552
The Effect of Phytochemical Tannins-Containing Diet on Rumen Fermentation Characteristics and Microbial Diversity Dynamics in Goats
Using 16S rDNA Amplicon Pyrosequencing
B. R. Min1, C. Wright1, P. Ho2, J.-S. Eun3, N. Gurung1, and R. Shange1
1Tuskegee University, Tuskegee, AL, USA
2Montgomery Blair High School, Silver Spring, MD, USA3Utah State University, Logan, UT, USA
Agric. Food Anal. Bacteriol. 4: 195-211, 2014
196 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
INTRODUCTION
The microbial populations of the rumen, par-
ticularly bacteria and archaea (methanogens), have
been extensively studied (Coleman, 1975; Williams
and Coleman, 1988; Fernando et al., 2010). Much is
known about the ruminal bacterial populations, but
our understanding of interactions between ruminal
bacteria and sources of plant tannins in vivo is limit-
ed, and very little data exist on the effects of sources
of plant tannins or tannin-containing diet on rumen
microbiome diversity in goats. Tannins are usually
classified either hydrolyzable tannins (HT) or con-
densed tannins (CT; proanthocyanidins) based on
their molecular structure. (Min et al., 2003). Our un-
derstanding of interactions between rumen bacte-
ria and HT or CT in the rumen is still in its infancy.
Tannins have traditionally been considered antinu-
tritional but it is now known that their beneficial or
antinutritional properties depend upon their chemi-
cal structure and dosage (Min et al., 2003). Structural
and chemical dissimilarities between HT (chestnut
tannins extract) and CT (Quebracho tannins extract)
may offer an explanation for differences in their bio-
logical effects and, therefore, results obtained using
a particular type of tannins cannot be applied to oth-
ers. In our study, we utilized a combination of CT and
HT instead of CT alone to examine if some phenolic
metabolites deriving from HT degradation in the ru-
men may affect the rumen fermentation and micro-
biome diversity, giving it added value.
Recent studies have demonstrated that chestnut
tannins have been shown to have positive effects
on silage quality in round bale silages, in particular
reducing non protein nitrogen (NPN) in the lowest
wilting level (Tabacco et al., 2006). Improved fer-
mentability of soya meal nitrogen in the rumen has
also been reported by Mathieu and Jouany (1993).
Studies by Gonzalez et al. (2002) on in vitro ammonia
release and dry matter degradation of soybean meal
comparing three different types of tannins (quebra-
cho, acacia and chestnut) demonstrated that chest-
nut tannins are more efficient in protecting soybean
meal from in vitro degradation by rumen bacteria.
This has been confirmed by the findings that supple-
mentation of tannins in heifers grazing winter wheat
reduced the rate of gas and biofilm production with
chestnut tannin being more efficacious than mimosa
tannins, but some selected rumen bacterial species
such as Prevotella ruminicola and strains of both
Fibrobacter succinogens and Ruminococcus flave-
faciens populations were decreased with chestnut
and mimosa tannins supplemented animals (Min et
al., 2012a). The implication of sources of plant tan-
nins (Chestnut vs. Quebracho tannins extracts or CT-
containing pine bark powder) likely being associated
with specific rumen microorganisms led us to study
the effect of two contrasting plant tannins on rumen
microbial diversity associated with animal perfor-
mance. The primary hypothesis of the in vivo grazing
research was that different sources of tannins supple-
mentation would selectively reduce rumen microbial
diversity and as a result would increase average daily
gain in meat goats. Thus, our objective was to in-
vestigate concurrent changes in ruminal bacterial
diversity and animal performance in goat response
to plant tannins using a modern pyrosequencing ap-
proach.
MATERIALS AND METHODS
Care and handling of all experimental animals
were conducted under protocols approved by the
Tuskegee University Institutional Animal Care and
Use Committee.
Experimental Animals and Diets
In Exp. 1, 20 Kiko-Boer cross male goats (Capra
hircus; initial body weight (BW) = 39.7 ± 2.55 kg)
were randomly assigned to 2 experimental diets (al-
falfa pellet vs. PB powder). Alfalfa pellet (no CT as a
control) or PB (11% CT) was supplemented at 0.5%
BW for targeted total dry matter intake (DMI) of 1.2%
BW. The remainder DMI of each diet was obtained
from grazing for 55 days (Table 1). Animals were fed
once a day at 0900 h and had free access to wa-
ter and trace mineral salt blocks grazing on winter
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 197
pea and rye grass dominant forages. Animal body
weight (BW) were measured before and after experi-
ment completion. Animals were drenched orally with
Cydectin (1 ml/10 kg BW) when fecal egg count was
over 1000 egg per gram of feces. Individual rumen
fluid samples (50 ml) were collected on day 55 af-
ter slaughter at the end of the experiment for rumen
volatile fatty acids (10 ml) and microbial diversity (40
ml) analyses. Rumen fluid samples from ten animals
per treatment were then pooled to three samples
sizes within treatment for bacterial analysis.
In Exp. 2, 12 Kiko-Boer cross male goats were
used to measure average daily gain (ADG), ruminal
fermentation, and rumen microbial population in the
rumen of goats grazing bermudagrass (Cynodon
dactylon) dominant pasture. The animals were ran-
domly assigned to 3 experimental diets: 1) no tan-
nins (control), 2) chestnut HT-extract at 100 g/d (CTE),
and 3) quebracho CT-extract at 100 g/d (QCTE). Ex-
perimental diets were gradually fed to animals in a
stepwise increasing fashion, and at the end of week
2, all animals were fed whole, pre-assigned experi-
mental diets (Table 1). Rumen fluid was collected via
stomach tube, fitted with a small cylindrical strainer,
before the morning feeding, into 50 mL serum vials
that were filled to capacity, capped immediately and
stored at -20°C until analysis later that day.
Chemical Analysis
Feed and forage samples were collected daily
during the collection period, dried at 60°C for 48 h,
ground to pass a 1 mm screen (standard model 4; Ar-
thur H. Thomas Co., Swedesboro, NJ), and stored
for subsequent analyses. Daily portions of ground
samples were composited for each animal and ana-
lyzed for DM, crude protein (CP), acid detergent lig-
nin, ether extract, and ash according to the methods
described by AOAC (AOAC, 1998). Nitrogen for diet
sample was determined using a Kjeldahl N, and CP
was calculated by multiplying N by 6.25. The neutral
detergent fiber (NDF) and acid detergent fiber (ADF)
concentrations were sequentially determined using
Table 1. Chemical compositions (%) of the pine bark powder, alfalfa pellet, winter forage (ryegrass and pea) and bermuda grass.
Experimental diet
Winter forage Alfalfa pellet BermudagrassPine bark
powderSD
Ingredients
Dry matter 91.9 91.8 92.1 92.5 0.33
Crude protein 19.0 22.0 16.9 9.1 6.17
Acid detergent fiber 32.4 37.3 30.4 49.6 9.60
Neutral detergent fiber
41.5 48.7 40.3 50 9.57
SD = standard deviation
198 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
an ANKOM200/220 Fiber Analyzer (ANKOM Technol-
ogy, Macedon, NY). Sodium sulfate heat stable amy-
lases (Sigma Aldrich Co., St. Louis, Mo) were used in
the procedure for NDF determination and pretreat-
ment with heat stable amylase (Type XI-A from Ba-
cillus subtilis; Sigma-Aldrich Corporation, St. Louis,
MO). Acetone (70%) extractable CT in grain mixes
were determined using a butanol-HCL colorimetric
procedure (Min et al., 2012). For volatile fatty acids
analysis, 5 mL of rumen fluid was diluted with 1 ml
of 3 M meta-phosphoric acids, and samples were
analyzed using a method described by Williams et
al., (2011). Volatile fatty acids were analyzed via gas
chromatography (Agilent 6890N, Santa Clara, CA,
USA) with a 007 series bonded phase fused silica
capillary column (25 m × 0.25 mm × 0.25 μm) and
a flame ionizing detector with the following param-
eters: 1 μL injection, injector temperature = 240 °C,
oven temperature = 80 °C for 1 min, ramp to 120 °C
hold for 5 min, ramp to 165 °C hold for 2 min, detec-
tor temperature = 260 °C.
DNA Extraction
Genomic bacterial DNA was isolated from 1 ml of
rumen samples according to the method described
in the QIAamp DNA Mini Kit (QIAGEN Inc., 27220
Turn berry Lane, Suite 200 Valencia CA). Extracted
DNA (2 μL) was quantified using a Nanodrop ND-
1000 spectrophotometer (Nyxor Biotech, Paris,
France) and run on 0.8% agarose gel with 0.5 M Tris-
Borate-EDTA (TBE) buffer. The samples were then
transported to the Research and Testing Laborato-
ry (Lubbock, TX) for PCR optimization and pyrose-
quencing analysis. Bacterial tag-encoded FLX ampli-
con pyrosequencing (bTEFAP) PCR was carried out
according to procedure described previously (Min et
al., 2012).
bTEFAP Sequencing PCR
The bTEFAP and data processing were performed
as described previously (Dowd et al., 2008). All DNA
samples were adjusted to 100 ng/μL. A 100 ng (1 μL)
aliquot of each sample’s DNA was used for a 50 μL
PCR reaction. The 16S universal eubacterial primers
530F (5’-GTG CCA GCM GCN GCG G) and 1100R
(5’-GGG TTN CGN TCG TTG) were used for amplify-
ing the 600 bp region of 16S rRNA genes. HotStar
Taq Plus Master Mix Kit (Qiagen, Valencia, CA) was
used for PCR under the following conditions: 94°C
for 3 min followed by 32 cycles of 94°C for 30 sec;
60°C for 40 sec and 72°C for 1 min; and a final elonga-
tion step at 72°C for 5 min. The resultant individual
sample after parsing the tags into individual FASTA
files was assembled using CAP3. The resulting tenta-
tive consensus FASTA (A database search tool used
to compare a nucleotide or peptide sequence to a
sequence database) for each sample was then evalu-
ated using BLASTn (Altschul et al., 1990) against a
custom database derived from the RDP-II database
(Cole et al., 2005) and GenBank website athttp://
www.ncbi.nlm.nih.gov. The sequences contained
within the curated 16S database were both >1200 bp
and considered as high quality based upon RDP-II
standards.
Data Processing and Statistical Analysis
Statistical analyses were performed using the
SPSS package (SPSS Inc., v 17.0, Chicago, IL). Pack-
age of NCSS (NCSS, 2007, v 7.1.2, Kaysville, UT)
was used for cluster analysis through which double
dendrograms were generated through use of the
Manhattan distance method with no scaling, and
the unweighted pair technique. Quality trimmed
sequences were provided with the sequencing ser-
vices by the Research and Testing Laboratory (Lub-
bock, TX; (Dowd et al., 2008). Tags which did not
have 100% homology to the original sample tag
designation were not included in data analysis. Se-
quences which were less than 250 bp after quality
trimming were not also considered. The resulting se-
quences were then evaluated using the classify.seqs
algorithm (Bayesian method) in MOTHUR against a
database derived from the Greengenes set using a
bootstrap cutoff of 65%.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 199
Relative abundance data are presented as per-
centages/proportions, but prior to subjection to
general linear model (GLM; SAS Inst., Cary, NC)),
they were transformed using the arcsine function
for normal distribution prior to analysis. In addition,
quantification of major rumen bacterial phylum,
classes and species populations was analyzed by the
GLM procedure of the SAS in a completely random-
ized design with the factors examined being sources
of tannins supplementation in the diets. Results are
reported as least square means.
RESULTS AND DISCUSSION
Regardless of numerous studies (Pitta et al., 2010;
Callaway et al., 2010; Hristov et al., 2012) demon-
strating the role of the gut microbial diversity in ru-
minants associated with different sources of forages
or dried distillers grains, the response of the micro-
biome to feeding various sources of phytochemical
tannins-containing diet remain largely unknown. The
most significant findings in the present study dem-
onstrates that when goats received tannins extracts
(chestnut and quebracho extracts) in the Firmicutes
phylum populations had significant (P < 0.01) de-
creased, while Bacteroidetes populations were
Table 2. Effects of condensed tannin-containing pine bark (PB) powder and different sources of tannins extracts supplementation on the animal body weight (BW) changes and average daily gain (ADG) in meat goats grazing fresh forages
Initial BW (kg) Final BW (kg) ADG (g)
Exp.1 (n = 10/diet)1
PB powder 37.3 46.6 169.1
Control 36.5 46.7 185.5
SD 8.05 0.34 12.67
P-value 0.76 0.35 0.71
Exp.2 (n = 4/diet)2
Chestnut 32.7 36.6 278.6
Quebracho 33.5 35.6 150.0
Control 30.5 31.1 42.9
SD 5.88 6.98 20.02
P-value 0.85 0.26 0.05
1 Animals were grazed on winter pea and ryegrass pasture with or without PB powder supplementation dur-ing 55 days2 Animals were grazed on bermudagrass forage with or without tannin extracts supplementation during 14 days.
200 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
significantly increased. The bacterial distribution
showed that Firmicutes (56-57%) was the most domi-
nant phyla with mean relative abundance values
ranging from 56% in control to 67 % in PB diets. This
suggests that phytochemical tannins supplementa-
tion alters the microbiome and animal performance
on goats grazing fresh forage diets.
Diet Composition
Ingredients and chemical composition of experi-
mental diet (alfalfa pellet), PB, and bermudagrass
forage are presented in Table 1. Goats were provided
diets that met all animals’ requirements for growth
and gain according to National Research Council
(NRC, 2007). Total CT concentration in the PB, alfalfa
pellet, winter forages, and bermudagrass was 10.3,
0.03, 0.03, and 0.05% DM, respectively (Table 1). All
the experimental treatments provided similar nutri-
ent profiles, except CT and ADF that were higher in
PB, but lower in CP compared to other diets. In our
previous study, addition of PB in goat diets improved
ADG and favorably modified ruminal fermentation
(Min et al., 2012b).
Animal Performance and Rumen Fer-mentation
The animal performance and ruminal volatile fatty
acids concentrations in goats grazing fresh forages
in response to different sources of tannins supple-
mentation are shown in Tables 2 and 3, respectively.
In Exp. 1, Initial BW, final BW, ADG, total volatile fatty
acids, acetate: propionate ratios, and individual vol-
atile fatty acids concentration were similar between
PB powder and control alfalfa pellet diets (Tables 2
and 3). In Exp. 2, Initial BW and final BW were similar
among treatments (Table 2), but ADG was greatest
for CTE (275 g/d) than QCTE (145 g/d) and the con-
trol (41 g/d). Goats grazing bermudagrass pasture
with CTE had greater (P < 0.05) concentrations of
acetate, propionate, butyrate, caprionate, and to-
tal volatile fatty acids concentrations compared to
those in QCTE and control (Table 3). However, goats
grazing bermudagrass forage without tannins sup-
plementation increased (P < 0.05) concentrations of
iso-valerate and acetate: propionate ratio compared
to tannins supplemented groups.
A number of studies have demonstrated that ef-
fects of tannins on ruminal fermentation is dose
dependent, and a negative effect only occurs when
they are fed at high concentrations (Hervás et al.,
2003; Mueller-Harvey, 2006). In addition, previous
studies have reported that mimosa and chestnut
tannins supplementations were not affected animal
performance and rumen fermentation in steers fed
a high-grain diet (Krueger et al., 2010) or hay sup-
plementation with chestnut tannins spray (Zimmer
and Cordesse, 1996). However, Min et al. (2012a)
reported that heifers grazing on high quality (about
28% crude protein content) winter wheat forage and
supplemented with 1.5% tannins (DM basis) experi-
enced 82% fewer days of bloat, and had 6 and 17%
greater ADG for mimosa and chestnut tannins ex-
tracts, respectively, than animals receiving the con-
trol diet principally through reducing the rate of
rumen fermentation as well as modification of micro-
bial populations in the rumen of cattle. Our chestnut
tannins supplementation study shows a similar trend
to this. This suggests that plant tannins supplemen-
tation in high quality forage may have more impact
on mitigating rumen fermentation and improving
animal performance than low quality forages diets.
Relative Abundance of Bacterial Phyla
In this study, bacterial (Fig. 1a,b) community com-
position of the rumen fluids were examined at de-
scending levels of biological classification to deter-
mine the effect of PB powder (Exp. 1) or tannin extract
(Exp. 2) supplementation on community membership.
Detailed phylogenic analyses grouped the rumen bac-
teria associated bacterial sequences into 45 phyla (in-
cluding unknown). The relative abundances of the 19
most abundant phyla (>1%) are presented in Figure 1.
Interestingly, the gut of human and many other verte-
brae are mostly dominated by two groups of bacteria,
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 201
Bacteroidetes and Firmicutes (Backhed et al., 2004),
which is similar to the result obtained current study.
This finding agrees with the results within the cur-
rent goat study showing that Firmicutes (56-57%) and
Bacteroidetes (33-35%) were the dominant bacterial
phylum in the goat rumen fluid. With the addition of
Firmicutes, together these phyla constituted approxi-
mately 89 to 92% of the total gut population. A study
by Henderson et al. (2013) demonstrated an increase
in the abundance of the phylum Firmicutes correlated
with a decrease in the abundance of Bacteroidetes
in cow (r= -0.805) and sheep (r= -0.976), which also
shows similarity to the results obtained in the current
study. This has been confirmed by the findings that
Firmicutes, Bacteroidetes, Actinobacteria, and Pro-
teobacteria were reported to be dominant bacterial
phyla in the goat intestine (Min et al., 2014) and human
gut (Schloss et al., 2009). It has been shown that the
Bacteroidetes and Firmicutes phyla comprised 35%
of all sequences, followed by Proteobacteria (13% to
15%) and Fusobacterium (7% to 8%). The bacterial
distribution showed that Firmicutes (56-57%) was the
most dominant phylum with mean relative abundance
values ranging from 56% in control to 67 % in PB diets
(Fig. 1). However, goats that received CTE and QCTE
extract supplementation had significant decreases
(P<0.01) in Firmicutes populations, while Bacteroide-
tes populations were significantly increased (Fig. 1b).
Table 3. Effects of condensed tannin-containing pine bark (PB) powder and different sources of tannin extracts supplementation on the ruminal volatile fatty acids (VFA) concentration and ac-etate: propionate (A:P) ratios in meat goats grazing winter pea and ryegrass dominant forages.
Item1 C2 C3 Iso-C4 C4 Iso-C5 C5 C6Total VFA
A:P ratio
Exp.1 (n = 10/diet)
PB powder2 14.4 3.38 0.99 1.82 1.52 0.39 0.01 22.54 4.23
Control 15.0 3.04 0.93 2.0 1.47 0.41 0.06 23.67 4.05
SD 4.74 1.35 0.15 0.66 0.28 0.09 0.03 6.98 0.10
P-value 0.78 0.47 0.37 0.53 0.73 0.77 0.64 0.73 0.22
Exp.2 (n = 4/diet)2
Chestnut3 54.4 13.55 0.23 4.4 0.09 0.5 0.15 73.4 4.02
Quebracho 44.0 12.4 0.25 3.9 0.08 0.4 0.05 61.2 3.59
Control 49.1 10.5 0.37 3.6 0.31 0.4 0.03 64.4 4.69
SD 7.44 1.96 0.10 0.92 0.02 0.08 0.08 9.12 0.46
P-value 0.02 0.001 0.02 0.01 0.07 0.21 0.01 0.01 0.001
1 Volatile fatty acids (VFA): acetic (C2), propionic acid (C3), butyric (C4), valeric (C5), and caproic acid (C6)2 Animals were grazed on winter pea and ryegrass pasture with or without PB powder supplementation dur-ing 55 days. 3Animals were grazed on bermudagrass forage with or without tannin extracts supplementation during 14 days.
202 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Figure 1. Predominant bacterial phylum observed in rumen samples of healthy goats with or with-out tannin-containing diets supplementation based on pyrosequencing of the 16S rRNA gene. Goats with pine bark powder (A) had significantly decreases in Bacteroidetes, but increased Fir-micutes (Data expressed as % of total 16S rRNA sequences). Goats with tannins extracts (B) had significant (P < 0.01) decreases in Firmicutes. Bacteroidetes were significantly increased.
0
10
20
30
40
50
60
70
80
90
100
Control Pine bark powder
33.320.5
57.367.2
ParabasaliaProteobacteriaEuryarchaeotaStreptophytaTenericutesFirmicutesElusimicrobiaCyanobacteriaAcidobacteriaNitrospiraePlanctomycetesVerrucomicrobiaFibrobacteresLentisphaeraeSpirochaetesActinobacteriaSynergistetesChloroflexiBacteroidetes
A
B
0
10
20
30
40
50
60
70
80
90
100
Control Chestnut Quebracho
35.3
51.5 52.9
55.942.6 36.7
ParabasaliaProteobacteriaEuryarchaeotaStreptophytaTenericutesFirmicutesElusimicrobiaCyanobacteriaAcidobacteriaNitrospiraePlanctomycetesVerrucomicrobiaFibrobacteresLentisphaeraeSpirochaetesActinobacteriaSynergistetesChloroflexiBacteroidetes
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 203
The gastrointestinal microbiota performs a large
number of important roles that define the physiol-
ogy of the host, such as immune system maturation
(Mazmanian et al., 2005), the intestinal response to
epithelial cell injury (Rakoff-Nahoum et al. (2004),
xenobiotic (Wilson and Nichoson, 2009), and energy
metabolism (Turnbaugh et al., 2006). In most mam-
mals, the gastrointestinal microbiome is dominated
by four bacterial phyla that perform these tasks:
Firmicutes, Bacteroidetes, Actinobacteria and Pro-
teobacteria (Ley et al., 2007). The current study has
shown that the number of Firmicutes population was
notably greater than the number of Bacteroidetes in
PB fed animals compared to alfalfa supplemented
animals, while CTE and QCTE plant extracts supple-
mentation had the opposite trend (lower abundance
in Firmicutes and greater density in Bacteroidetes,
respectively) compared to control group in grazing
goats. The mechanism of action of tannin-resistant
bacteria in animals exposed to condensed tannins
is not known between two different dietary supple-
mentations.
Past research into the correlation between gut mi-
crobiota and diet had demonstrated a complex rela-
tionship between the population of the gut and fatty
acid absorption. Although exact mechanisms are not
yet known, it has been observed that obesity due to
a high fat or high polysaccharide diet correlates with
a decrease in the amount of Bacteroidetes and a
proportional increase in Firmicutes. This was shown
by Ley et al. (2005) in mouse models with obese and
normal genotypes, and was later supported by Ley
et al. (2007) in studies of human fecal matter. The
number of Firmicutes was notably higher than the
number of Bacteroidetes in obese mice, and vice
versa for the lean mice (Ley et al., 2005). Along with
increased fatty acid absorption, more energy was
also found to be efficiently obtained from diet in the
obese mice compared to the lean mice, illustrating
the connection between Firmicutes and improved
efficiency in energy harvesting (Turnbaugh et al.,
2006). The replacement bacteria are more efficient
at harvesting energy from food than the bacteria
they replaced, resulting in increased calorie intake
by the host (Turnbaugh et al., 2006), and ultimate-
ly, an increase in weight (Turnbaugh et al., 2006).
The Bacteroidetes spp, in particular Bacteroides
thetaiotaomicron, hydrolyzes otherwise indigest-
ible polysaccharides and accounts for 10% to 15%
of caloric requirement in humans (Xu et al. 2003).
Human colonocytes derive 50% to 70% of their en-
ergy from butyrate, which is derived from complex
carbohydrates metabolized by Firmicutes spp via
fermentation (Pryde et al., 2002). However, the cur-
rent study had opposite trends to human study in
that the both CTE and QCTE extract supplemented
groups have greater butyrate, iso-butyrate, acetate,
and propionate concentrations in the rumen, and
had higher Bacteroidetes population compared to
control group. It is unclear what factors in the setting
of average daily gain tip the scales in favor of the
Firmicutes over Bacteroidetes in ruminants. Perhaps
the Bacteroides possess may more tannins-resistant
mechanisms or more diverse enzymatic capabilities
(Odenyo and Osuji, 1998; Smith et al., 2003) that
more efficiently extract energy when a variety of
complex organic matter is available in goats. This hy-
pothesizes that the metabolic and energy extraction
functions in ruminants may be fundamentally due to
microbiota, such that all are affected by alterations in
nutritional state.
Diversity and Abundance of Rumen Bac-terial species
More than 332 bacterial species (including un-
known) were classified from the ruminal fluid of
the goats in this study. However, the relative abun-
dances of the 12 most abundant species (>1%) are
presented in Tables 4 and 5. Tannins are one of the
most abundantly available plant secondary metabo-
lites, and have positive or adverse effects on rumen
microbial populations, feed digestibility and animal
performance (Min et al., 2003). The bacterial spe-
cies distribution showed that Ruminococcaceae spp.
(12-15%) and Prevotella spp. (21-40%) were the most
dominant species with mean relative abundance val-
ues ranging from 42 to 55% in control group with-
out tannins supplementation (Table 4). In Exp. 1,
204 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Prevotella spp. was decreased (P < 0.05) in PB diet,
while Butyrivibrio spp. (P < 0.05) and Ruminococ-
caceae spp. (P = 0.08) were increased compared to
control diet. However, bacterial populations in CTE-
supplemented group in Exp. 2 were significantly in-
creased for Prevotella spp. (40.4, 33, and 32%) com-
pared with QCTE and control groups, respectively.
It has been shown that the gastrointestinal micro-
bial population was dominated by Prevotella (18.2%
of total population) in the rumen and Clostridium
(19.7% of total population) in the feces of cattle (Cal-
laway et al., 2010). Consequently, analysis of human
microbiota-associated rat feces using molecular ap-
proach has revealed that the Bacteroides/Prevotella
and Faecalibacterium species are dominant in both
humans and rats post-transfection (Licht et al., 2007).
These findings also agree with the results of a
metabolic finger print study of a rat fed CT extract
from Acacia angustissima. Condensed tannin ex-
tracts from A. angustissima altered fecal bacterial
populations in the gastrointestinal tract, resulting in
a shift in the predominant bacteria towards tannin-
resistant gram-negative Enterobacteriaceae and
Bacteroidetes (Smith et al., 2003). Presence of bac-
teria able to tolerate elevated levels of condensed
tannins in the rumen of animals fed forages high in
tannins has been reported by Nelson et al. (1995).
Different groups of microbes have different toler-
ance to tannin. Rumen fungi, proteolytic bacteria
and protozoa are more resistant to tannin as com-
pared to other microbes (McSweeny et al., 2001).
McSweeny et al. (1999) observed that in the animals
Table 4. Effects of condensed tannin-containing pine bark (PB) powder supplementation (n = 10/diet) on the rumen bacterial species population diversity (%) in meat goats grazing fresh forages1
Bacterial species Pine bark Control SD P-value
Roseburia spp. 1.96 1.62 2.34 0.83
Ruminococcus spp. 1.03 1.89 0.85 0.31
Paraprevotella spp. 0.54 0.67 0.50 0.71
Succiniclasticum spp. 3.18 5.58 4.42 0.51
Prevotellaceae spp. 1.81 1.15 1.61 0.59
Victivallis spp. 0.38 0.58 0.41 0.56
Ruminococcaceae spp. 18.78 15.2 4.99 0.08
Prevotella spp. 11.46 21.4 3.19 0.05
Butyrivibrio spp. 13.56 5.08 3.76 0.05
Blautia spp. 4.92 4.76 5.30 0.97
Desulfovibrio spp. 0.24 0.28 0.72 0.89
Saccharofermentans spp. 4.37 6.97 3.44 0.54
1Animals were grazed on winter pea and ryegrass pasture with or without PB powder supplementation dur-ing 55 days
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 205
fed on tannin rich Calliandra calothyrsus, the popu-
lation of Ruminococcus spp. and Fibrobacter spp.
was reduced considerably. Min et al. (2002) reported
that a decrease of 0.5-0.1 log in proteolytic ruminal
bacteria Clostridium proteoclasticum, Streptococ-
cus bovis, Eubacterium spp. and Butyrivibrio fibri-
solvenes when CTs from Lotus corniculatus (3.2 %
CT/kg DM) were fed to sheep. Recently, Min et al.,
(2014) reported that Stenotrophomonas koreensis
was the most dominant bacterial species with mean
relative abundance values ranging from 23.9% (con-
trol) to 9.9% (15% PB) and 17.2% (30% PB). The re-
maining bacterial species accounted for fewer than
10% of the relative abundance observed. Of these
groups, Flavobacterium gelidilacus and Myroides
odoratimimus were decreased with increasing di-
etary PB concentration. However, Bacteroides cap-
illosus, Clostridium orbiscindens, and Oscillospira
guilliermondii were linearly increased with increasing
PB concentration. This suggests that phytochemical
tannins supplementation alters microbial diversity
and thereby improves animal performance. The au-
thors observed significantly lower (P<0.05) Prevotella
bacteria populations in goats fed CTE extract was
increased of as compared to animals fed QCTE or
control diets.
For ease of presentation and interpretation, we
present prevalent bacterial genera (Figures 2 and 3)
observed in the community based on a cutoff value
of 0.9% of relative abundance for inclusion in a hier-
archal cluster analysis of individual animal microbial
diversity within and among diets in Figures 2 and 3.
Table 5. Effects of different sources of tannins extracts supplementation (n = 4/diet) on the rumen bacterial species population diversity (%) in meat goats grazing fresh forages1
Bacterial species Chestnut Quebracho Control SD P-value
Roseburia spp. 1.13 0.81 0.87 0.04 0.56
Ruminococcus spp. 2.28 1.94 2.57 0.20 0.41
Paraprevotella spp. 1.53 1.31 1.67 0.06 0.21
Succiniclasticum spp. 2.52 1.90 2.66 0.32 0.15
Prevotellaceae spp. 6.16 8.24 6.54 2.44 0.67
Victivallis spp. 1.21 0.37 1.37 0.56 0.27
Ruminococcaceae spp. 11.65 14.4 12.7 3.74 0.22
Prevotella spp. 40.37 32.8 31.76 12.77 0.05
Butyrivibrio spp. 2.36 1.62 1.93 0.28 0.38
Blautia spp. 4.15 6.91 6.95 5.14 0.29
Desulfovibrio spp. 1.13 0.69 1.12 0.12 0.71
Saccharofermentans spp. 5.2 1.45 2.30 3.98 0.88
1Animals were grazed on bermudagrass forage with or without tannin extracts supplementation during 14 days
206 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Figure 2 (Exp. 1). Heat-map double dendrogram of the 44 most abundant bacterial genera in the ru-men of various sources of tannin extracts supplementation from a common cohort of 20 meat goats. Clustering in the Y-direction is indicative of abundance, not phylogenetic similarity. RA = relative abundance; pine bark = sample no. 9, 10, and 11; Control = 12, 13, and 14. Rumen fluid samples from ten animals per treatment were pooled to three samples sizes within treatment for bacterial analysis.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 207
Figure 3 (Exp. 2). Heat-map double dendrogram of the 44 most abundant bacterial genera in the rumen of various sources of tannin extracts supplementation from a common cohort of 12 meat goats. Cluster-ing in the Y-direction is indicative of abundance, not phylogenetic similarity. RA = relative abundance; Chest nut = tag no. 1 and 2; Quebracho = 3 and 4; Control = 7 and 8. Rumen fluid samples from four animals per treatment were pooled to two samples sizes within treatment for bacterial analysis.
208 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Overall, animals clustered relatively well within diet
and animals. However, one of PB powder supple-
mented goats had more dissimilarity between treat-
ments. Similar trends were found in Exp. 2. Chestnut
diet of goats (sample # 1 and 2) clustered more closely
to the quebracho (# 3) supplementation, but one of
control animal (# 7) was relatively not clustered within
control diet (# 8). Goats that received PB powder in
Exp. 1 had greater relative community abundance of
Clostridia population compared to the control diet,
and the opposite was observed for Bacterodia pop-
ulation (Figure 2). In Exp. 2, chestnut and quebracho
tannin extracts supplemented groups had greater
relative abundance (%) of Bacteroidia compared to
the control diet. Lower abundance of Clostridia in
tannins extract groups compared to control diet, in-
dicated that tannins extracts supplementation may
have decreased the abundance of Clostridia popula-
tion in rumen of goats.
CONCLUSIONS
In conclusion, the current results show that tannin
can exert a positive or negative effect both on ru-
men fermentation and on rumen microflora, and it
is possible that this effect is depending on sources
of tannins or tannin-containing diet. Rumen micro-
bial population is very dynamic and tannin inclusion
impacts specific members of the microbial popula-
tion. There is also possible adaptation of ruminal mi-
crobiota to tannin and beneficial effect of tannin on
some class of rumen microbes has been observed.
However, there is need for detailed study involving
effect of varying concentration of tannins on rumen
bacteria, archaea and fungal diversity of goats in re-
sponse to ingestion of different sources of tannin-
containing diet.
ACKNOWLEDGEMENTS
This project was supported by USDA-NIFA, The
USDA-NIFA Evans-Allen Research Program and
Tuskegee University, George Washington Carver
Agricultural Research Station. Research and Testing
Laboratory (Lubbock, TX) for PCR optimization and
pyrosequencing analysis are also acknowledged.
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212 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
www.afabjournal.comCopyright © 2014
Agriculture, Food and Analytical Bacteriology
ABSTRACT
This work presents results from the phenotypic and genotypic characterization of a novel Enterobacter
cloacae strain (#JD6301) recently isolated from a mixed population of oleaginous microorganisms. Lipid
analysis of this strain indicated that JD6301 produces nearly 50% of its cellular weight as lipids. The yield
of fatty acid methyl esters for this microorganism was 76 μg/mL. Transmission electron microscopy obser-
vations showed inclusion bodies form within this isolate. To improve the recovery of these useful lipids
from this microorganism, a random mutagenesis assay was utilized to isolate an alternative form of this
bacterium capable of producing extracellular lipids. The extracellular fraction of the mutant strain JD8715
had a total fatty acid methyl esters yield of 86 μg/mL, which was similar to the intracellular yield of JD6301.
Furthermore, cell viability and microscopic analysis indicated that the presence of extracellular lipids was
not due to cell lysis. Comparative genome analysis of JD8715 against JD6301 revealed 24 single nucleotide
polymorphisms, of which 17 resulted in non-synonymous amino acid changes. Seven of these changes oc-
curred in genes related to membrane proteins. The application of oleaginous microorganisms capable of
producing extracellular lipids while still retaining cell viability represents a promising approach for provid-
ing energy required for biotechnological applications.
Keywords: Enterobacter cloacae, triacylglyceride, lipids, extracellular lipids, electron microscopy,
oleaginous, biofuels, biodiesel, JD6301, membrane transport
Correspondence: J. R. Donaldson, [email protected], Tel: +1-662-325-9547
Characterization of the Novel Enterobacter cloacae Strain JD6301 and a Genetically Modified Variant Capable of Producing Extracellular Lipids
J. R. Donaldson1*, S. Shields-Menard1, J. M. Barnard1, E. Revellame2, J. I. Hall3, A. Lawrence4, J. G. Wilson1, A. Lipzen5, J. Martin5, W. Schackwitz5, T. Woyke5, N. Shapiro5, K. S. Biddle1,
W. E. Holmes2, R. Hernandez2, and W. T. French3
1Department of Biological Sciences, Mississippi State University, Mississippi State, MS, USA. 2Department of Chemical Engineering, University of Louisiana Lafayette, Lafayette, LA, USA. 3Renewable Fuels and Chemicals Laboratory, Dave C. Swalm School of Chemical Engineering,
Mississippi State University, Mississippi State, MS, USA.4Institute for Imaging and Analytical Technologies, Mississippi State University, Mississippi State, MS, USA.
5 DOE Joint Genome Institute, Walnut Creek, CA, USA.
Agric. Food Anal. Bacteriol. 4: 212-223, 2014
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 213
INTRODUCTION
Lipids are important energy sources in both hu-
man health and also in biofuel production, such as
biodiesel (Ranganathan et al., 2008; Rosen and Spie-
gelman, 2006). Biodiesel is produced through the
transesterification/esterification of triacylglycerides
(TAGs) to yield fatty acid alkyl esters and commonly
exists as fatty acid methyl/ethyl esters, or FAMEs (Le-
stari et al., 2009). While plant and animal fat stores
are the most common source of TAGs for biofuel
production, certain prokaryotes have been identi-
fied to accumulate TAGs as a form of energy storage
(Alvarez and Steinbuchel, 2002; Holder et al., 2011).
These microorganisms, termed oleaginous microor-
ganisms, accumulate more than 20% of their biomass
as TAGs and include the bacteria Streptomyces, No-
cardia, Rhodococcus, Mycobacterium, Dietzia, and
Gordonia (Alvarez and Steinbuchel, 2002; Wynn and
Ratledge, 2005), as well as yeast and fungi, such as
Yarrowia lipolytica and Mortierella isabellina (Meng
et al., 2009).
Recently, the novel Enterobacter cloacae strain
JD6301 was isolated from a mixed culture contain-
ing oleaginous microorganisms and microorganisms
from a municipal wastewater treatment facility and
was sequenced (Wilson et al., 2014). The goal of this
study was to further analyze this novel isolate. En-
terobacter cloacae strain JD6301 was found capable
of producing large quantities of lipids through both
transmission electron micrograph observations and
lipid analyses. A variant form of this strain was con-
structed following a random mutagenesis that was
able to produce extracellular lipids. This strain was
further analyzed through genomic comparisons to
determine candidate gene mutations that resulted
in the observed phenotype. The ability of oleagi-
nous microorganisms to produce extracellular lipids
could lead to advancements in lipid biotechnology,
especially in the areas of lipid recovery and utiliza-
tion.
MATERIALS AND METHODS
Culture conditions
Frozen stocks of the Enterobacter cloacae
wild type (WT) strain JD6301 and resulting isogen-
ic mutant JD8715 were maintained at -80°C in 20%
glycerol. Frozen stocks were cultured on nutrient
agar and allowed to grow for 48 – 72 h at 30°C. For
lipid analysis of JD6301 and JD8715, bacteria were
cultured in mineral salts medium (MSM) supplement-
ed with 3% (w/v) sodium gluconate in baffled culture
flasks (Schlegel et al., 1961). The auxotrophic mutant
Saccharomyces cerevisiae strain KD115 (MATα ole1)
was purchased through the American Type Culture
Collection (Stukey et al., 1989) and was cultured in
YPOD media (1% yeast extracts, 2% bacto-peptone,
2% glucose, 1% brij 58, and 0.2% oleic acid) under
aerobic conditions for 24 – 48 h at 30°C in a shaking
incubator (180 rpm). For plates, 2% agar was added
to the YPOD medium.
Mutant construction
A mutant of JD6301 capable of producing extracel-
lular lipids was constructed as previously described
for S. cerevisiae with minor modifications (Nojima et
al., 1999). A 24 h culture of JD6301 in MSM supple-
mented with 3% sodium gluconate was treated for
3 h at 30°C with 3% ethyl methanesulfonate (EMS;
Acros Organics), which is a mutagenic chemical that
introduces predominately GC to AT base transitions
(Ingle and Drinkwater, 1989). The treated cells were
then plated onto nutrient agar and incubated for 48
h at 30°C. Colonies were then overlaid with 6 mL of
YPD (1% yeast extracts, 2% bacto-peptone, 1% agar,
and 2% glucose) agar containing 1x107 CFU/mL of S.
cerevisiae KD115 and 50 U of lipoprotein lipase (Sig-
ma, L9656). Cultures were incubated for an addition-
al 16 h at 30°C to allow for growth of the auxotrophic
KD115 strain. The presence of microcolony growth
of KD115 around the periphery of the Enterobacter
cloacae cells indicated the EMS introduced genetic
alterations that promoted the presence of extracel-
lular lipids, which were then available for utilization
by KD115. The potential mutants were transferred to
214 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
individual tubes with 2 mL of MSM with 3% sodium
gluconate and incubated in a shaking incubator for
24 h at 30°C, plated on nutrient agar, and incubated
for an additional 48 h. After incubation the mutants
were tested again through an overlay with KD115
and lipase-containing YPD agar. Of the 1,000 colo-
nies screened, the mutant JD8715 was selected for
further analysis.
Genomic resequencing and analysis
Resequencing of JD8715 was generated by the
Department of Energy Joint Genome Institute (JGI)
using the Illumina HiSeq 2000 platform as previous-
ly described (Wilson et al., 2014), which generated
178,495,750 reads. The final draft assembly con-
tained 53 contigs and 49 scaffolds, totaling 4.8 Mb;
draft assembly was deposited in NCBI (GenBank
accession JDWG00000000). Alignments between
the JD6301 reference genome (GenBank Accession
JDWH00000000) and JD8715 were performed with
Burrows-Wheeler Alignment (bio-bwa.sourceforge.
net; (Li and Durbin, 2009) and putative single nucle-
otide polymorphisms (SNPs) and small indels were
identified using samtools/mpileup/bcftools (Li et al.,
2009). This analysis resulted in the identification of 24
SNPs, which included 17 non-synonymous, 4 synony-
mous, and 3 non-coding variants (Table 1).
Growth responses and sugar consump-tion
JD6301 and JD8715 (25 mL) were grown overnight
at 30°C in a shaking incubator (200 rpm) in flasks
containing MSM supplemented with 13 g/L glu-
cose, diluted 1:100 in fresh media, and transferred
to a 96-well microtiter plate. Optical density (OD600)
measurements were recorded over 24 h in triplicate
using a Molecular Devices spectrophotometer Spec-
traMax Plus 384 plate reader at 30°C with intermit-
tent shaking.
For glucose consumption analysis, 1 mL of cells
was filtered through a 0.2 μm filter (Corning Life Sci-
ences, Amsterdam, The Netherlands). Glucose con-
centrations were determined using an Agilent 1100
High Performance Liquid Chromatography (HPLC;
Agilent Technologies, Inc., Santa Clara, CA) system.
The HPLC system was coupled to a Varian 385-LC
evaporative light scattering detector (ELSD; Varian
Inc., Palo Alto, CA) and a Restek Pinnacle II Amino
column (5 μm, 150 × 4.6 mm; Restek, Inc., Bellefonte,
PA). The temperature of the nebulizer in the ELSD
was set to 60°C and the drift tube was held at 80°C
with a nitrogen nebulization gas flow rate of 1.8 L/
min. The mobile phase consisted of acetonitrile and
water (83:17) with an injection volume of 2 μL. The
flow rate was 1 mL/min. Results represent the aver-
age of three independent replicates.
Transmission electron microscopy
Two mL aliquots of 24 h cultures cultured at 30°C
in a shaking incubator in MSM media supplemented
with 3% sodium gluconate were centrifuged (10,000
x g) for 2 min at 4°C, fixed in ½ strength Karnovsky’s
fixative in 0.1 M Na cacodylate buffer at pH 7.2, rinsed
with 0.1 M Na cacodylate buffer, and then post fixed
in buffered 2% osmium tetraoxide. Samples were
rinsed once more in buffer, en bloc stained with 2%
aqueous uranyl acetate, dehydrated in a graded eth-
anol series, and embedded in Spurr’s resin. Ultra-
thin sections were cut with a Reichert-Jung Ultracut
3 ultra-microtome and were stained with uranyl ac-
etate and lead citrate. Stained sections were viewed
on a JEOL JEM-100CXII TEM at 80KV. A minimum
of 50 cells from two independent replicates was ana-
lyzed by transmission electron microscopy.
Scanning electron microscopy
Both JD6301 and JD8715 strains were grown for
24 h at 30°C in MSM supplemented with 3% sodium
gluconate in a shaking incubator. Cells (1 mL) were
pelleted by centrifugation at 8,000 x g for 10 min and
washed with 1 mL of chloroform, pelleted again by
centrifuging for 10 min at 8,000 x g. Bacterial pellets
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 215
were fixed in 2.5% glutaraldehyde in 0.1 M cacodyl-
ate buffer, washed in 0.1 M cacodylate buffer, post-
fixed in 1% osmium tetraoxide in 0.1 M cacodylate
buffer, rewashed in distilled water, dehydrated in an
ethanol series, and finally dried in a hexamethyldisi-
lazane series as previously described (Merritt et al.,
2010). Samples were sputter coated with gold-pal-
ladium using a Polaron SEM coating system prior to
observation with a field emission scanning electron
microscope (JEOL JSM-6500F). A minimum of 50
cells from two independent replicates was analyzed.
Live/Dead BacLightTM bacterial viability
One μL (approximately 1x107 cells) of JD6301 and
JD8715 was collected at 24 h of growth and viabil-
ity was determined using the LIVE/DEAD BacLightTM
bacterial viability kit consisting of SYTO 9 and prop-
idium iodide (Invitrogen, Calsbad, CA). Staining
was performed in the dark for 15 min, after which
cells were analyzed by a BD FASCaliber flow cytom-
eter using Cell Quest software (BD Biosciences, San
Jose, CA). Analyses were performed on 4,500 to
5,000 cells (gated events) using instrument param-
eters previously described by others (Gunasekera et
al., 2003). Percent viability was determined by a two-
parameter comparison of green (live cells) and red
(dead cells) fluorescent emission for individual bac-
teria using the formula: [% live green-emitting cells/
(% dead red-emitting cells + % live green-emitting
cells)] x 100. Three independent replicates were per-
formed for each strain.
Lipid analysis
Cultures (20 mL) incubated at 30°C for 24 h were
centrifuged for 10 min at 6,000 x g, after which 10
mL of the supernatant was extracted for lipid analy-
sis; the remainder of the supernatant was discarded.
The resulting cell pellet was rinsed gently with 1 mL
of chloroform, centrifuged for an additional 5 min,
and the wash was combined with the 10 mL of super-
natant collected. Lipids were extracted from the col-
lected supernatant and cell pellet using a standard
Bligh and Dyer lipid extraction technique (Bligh and
Dyer, 1959).
Extracted lipids were derivatized using N-Methyl-
N-(trimethylsilyl)-trifluoroacetamide (MSTFA) follow-
ing ASTM D6584, which uses tricaprin as an inter-
nal standard at a concentration of 100 μg/mL. This
method also utilizes triolein, diolein and monoolein
as reference compounds. Briefly, tricaprin (12.5 μL),
MSTFA (25 μL), and pyridine (62.5 μL) were added
to the lipid extracts. Samples were vortexed and al-
lowed to react for at least 20 min, after which 900
μL of n-heptane was added. Samples were then fil-
tered through a 0.45 μm PTFE filter (SUN Sri, Rock-
wood, TN) and transferred to auto-sampler vials for
analysis on a Varian 3600 GC (Varian Inc., Palo Alto,
CA) equipped with a flame ionization detector (FID).
The GC column was a RTX®-65TG (15m × 0.25 mm
ID, with a 0.10 μm film thickness) and utilized a 2 m
x 0.53 mm Rxi® guard column (Restek, Bellefonte,
PA). Samples were analyzed using cool-on-column
injection with an initial injector temperature of 50°C
and a final injector temperature of 380°C, at a ramp
rate of 180°C/min. The GC oven temperature was
programmed at an initial temperature of 50°C, held
for 1 min, then ramped to 180°C at 15°C/min, then
to 230°C at 7°C/min, to 370°C at 20°C/min, and fi-
nally held for 11.20 min. The FID was retained at
380°C for the duration of the GC analysis. Analysis
was performed on three independent replicates of
each extraction for each strain.
FAMES analysis
Lipids extracted from JD6301 and JD8715 were
converted to FAMEs using 1.5 mL of 14% BF3 in
methanol at 65°C for 30 min, after which 5 mL of 5%
NaCl and 2% NaHCO3 in distilled water was added.
FAMEs were then extracted twice with 10 mL n-hex-
ane and recovered from the solvent at 45°C under
15 psi of N2 using a TurboVap LV (Caliper Sciences,
Hopkinton, MA). The solid residue was re-dissolved
in 1 mL toluene containing 100 μg/mL butylated hy-
droxytoluene and 200 μg/mL 1,3-dichlorobenzene.
216 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Table 1. Summary of SNPs identified.
SNP Ref-Mut
Scaffold/Pos Gene Function
Non-synonymous
G-A 2/8877 P698DRAFT_00653ABC-type branched-chain amino acid transport systems, periplasmic component
C-T 2/82327 P698DRAFT_00726 Paraquat-inducible protein B
C-T 2/99542 P698DRAFT_00743 Predicted membrane protein
G-A 2/250263 P698DRAFT_00887 Uncharacterized protein family (UPF0259)
G-A 4/321850 P698DRAFT_01861 Mg/Co/Ni transporter MgtE (contains CBS domain)
G-A 6/39934 P698DRAFT_02343 ATPase components of ABC transporters
G-A 6/182761 P698DRAFT_02476Phosphotransferase system IIC components, glu-cose/maltose/N-acetylglucosamine-specific
G-A 6/193118 P698DRAFT_02492 2-methylthioadenine synthetase
C-T 6/233659 P698DRAFT_02537Ribulose-5-phosphate 4-epimerase and related epimerases and aldolases
C-T 6/299293 P698DRAFT_02598 Cation transport ATPase
C-T 8/199438 P698DRAFT_03078 G:T/U mismatch-specific DNA glycosylase
T-C 9/90423 P698DRAFT_03214 Hemolysin activation/secretion protein
C-T 12/90473 P698DRAFT_03655 Flagellar hook-associated protein
G-A 13/4044 P698DRAFT_03706SAM-dependent methyltransferases related to tRNA (uracil-5-)-methyltransferase
C-T 14/8347 P698DRAFT_03815 Protein of unknown function (DUF968)
G-A 18/63736 P698DRAFT_041987,8-dihydro-6-hydroxymethylpterin-pyrophosphoki-nase
C-T 32/7612 P698DRAFT_04585 Phage-related minor tail protein
Synonymous
G-A 1/491858 P698DRAFT_00477 Transcriptional regulator
C-T 2/127810 P698DRAFT_00771 Alanine racemase
C-T 4/371589 P698DRAFT_01904 Glutamine synthetase
G-A 19/23709 P698DRAFT_04231 Sugar phosphate permease
Non-Coding
C-T 2/160082 - -
C-A 4/240806 - -
C-T 7/209964 - -
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 217
The FAMEs were analyzed using an Agilent 6890N
gas chromatograph equipped with a flame ioniza-
tion detector (GC-FID) and a fused silica column Sta-
bilwax-DA (30 m × 0.25 mm, film thickness 0.25 μm)
(Restek, Bellefonte, PA). The operating conditions
were as follows: initial oven temperature of 50°C for
2 min to a final oven temperature of 250°C with a rate
of increase of 10°C/min and was held at 250°C for
18 min with helium as the carrier gas at 1.5 mL/min
and 260°C detector temperature. Instrument cali-
bration was achieved using a 14-component FAMEs
standard mixture (C8 – C24) (Supelco, Bellefonte, PA)
containing saturated, mono-unsaturated and poly-
unsaturated fatty acids. Analysis was performed on
three independent replicates of each extraction for
each strain. Analysis of variance (ANOVA), followed
by a Tukey post-hoc range test (p < 0.05), was used
to analyze the significance of total biodiesel lipid
analysis.
RESULTS AND DISCUSSION
Lipid production by Enterobacter cloa-cae JD6301 and construction of the mu-tant JD8715
A novel strain of Enterobacter cloacae previously
isolated by our group (Wilson et al., 2014) was ana-
lyzed by transmission electron microscopy, where in-
clusion bodies were found to form in the cytoplasm
within 24 h of growth. By 48 h, it appeared that the
inclusion bodies had formed around nearly the en-
tire inner portion of the cell membrane (Fig. 1). As
this culture was in a mixed environment containing
oleaginous microorganisms, it was hypothesized
that these may be representative of lipid inclusion
bodies similar to what has been observed in other
oleaginous microorganisms (Alvarez et al., 1996; Al-
varez and Steinbuchel, 2002; Waltermann et al., 2005;
Waltermann and Steinbuchel, 2005). Lipids were iso-
lated by Bligh and Dyer extraction and based on the
dry weight of the cells, 50% of the cellular weight
was attributed to lipids, indicating that this is a novel
oleaginous isolate of Enterobacter cloacae.
A limitation to the usefulness of oleaginous mi-
croorganisms in industry is in recovery of the useful
end products (Grima et al., 2003). However, once
the lipids are outside of the cell, separation from the
aqueous solution is effortless due to the insolubil-
ity of lipids in water (Fischer et al., 2008). Therefore,
this bacterium was modified to produce extracellu-
lar lipids. This strain was treated with the carcinogen
EMS as previously described (Nojima et al., 1999)
and the mutant JD8715 was subsequently identified
Figure 1. Inclusion bodies form within the cytoplasm of JD6301. TEM images were acquired at 24 h and 48 h. A minimum of 50 cells was observed for each time point. Scale bars represent 1 μm
24 h 48 h
218 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
as producing extracellular lipids through a screen
for growth of an auxotrophic strain of S. cerevisiae.
The genome of JD8715 was resequenced using the
Illumina platform. A total of 52 scaffolds and 61 con-
tigs were generated; genome size was 4,769,233
bp and a total of 4,508 protein coding genes were
identified. The genomes of JD8715 and JD6301
were compared to identify locations of SNPs that
may have contributed to the observed phenotype.
A total of 24 SNPs were identified, with a majority
being G->A or C->T transitions (Table 1). Seventeen
of the identified SNPs resulted in a non-synonymous
substitution. A majority of these SNPs were linked to
membrane proteins, including ATP synthase compo-
nents, transporter proteins, and a hook associated
protein. It is possible that the resulting phenotype
was due to a combination of these SNPs. Individual
mutants need to be generated to determine which
mutation(s) is sufficient for the release of lipids.
Lipid and sugar analysis
The mutant selection procedures and micro-
scopic observations suggested that the JD8715
mutant had extracellular lipids. Quantification of
extracellular and intracellular lipids, extracted from
the supernatant and cell pellets respectively, from
JD6301 and JD8715 was performed using gas chro-
matography after 24 h of growth. Mass of the re-
Table 2. Yield of lipids produced relative to consumption of glucose.
Strain%Glucose Consumed (±StDev)
Cells: glucose consumed
Yield extracellular lipid: glucose
Yield cell mass: glucose
JD6301 99.51 (0.02) 4.62X1011 CFU/g 0.098g lipid/g glucose 0.0803g cells/g glucose
JD8715 85.86 (0.31) 1.56x1011 CFU/g 0.353g lipid/g glucose 0.0588g cells/g glucose
Table 3. Extracellular and intracellular lipid concentrations.
JD6301 (μg/ml, ±StDEV) JD8715 (μg/ml, ±StDEV)
Lipid intracella extracellb intracella extracellb
Monoglycerides 17.32 (2.85) 56.15 (0.19) 12.27* (0.81) 98.23* (3.40)
Diglycerides 8.27 (0.95) 16.03 (2.32) 10.65 (1.40) 18.52 (1.44)
Triglycerides 43.28 (0.01) 3.83 (0.21) 28.74 (0.22) 5.21*(1.41)
a Intracellular concentrations of lipids are based on 20mL cell pellets collected from 24hr cultures of JD6301 or JD8715. StDev represents ± standard deviations.
b Extracellular concentrations of lipids are based on 10mL of supernatant collected from 24hr cultures of JD6301 or JD8715. StDev represents ± standard deviations.
*Indicates significant change (p < 0.05) in concentration in JD8715 compared to JD6301.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 219
Figure 2. Total FAMEs and lipid profiles of JD6301 and JD8715. (A) Total mean FAMEs yield (± SD) of JD6301 (black) and JD8715 (white) of intracellular and extracellular fractions. (B) FAMEs profile (± SD) of JD6301 intracellular (black), JD6301 extracellular (blue), JD8715 intracellular (white), JD8715 supernatant (grey). Intracellular concentrations of lipids are based on 20 mL cell pellets collected from 24 h cultures of JD6301 or JD8715. Extracellular concentrations of lipids are based on 10 mL of supernatant collected from 24 h cultures. Means denoted by the same let-ter are not significantly different (p <0.05).
A
B
220 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Figure 3. Enterobacter sp. JD6301 and JD8715 mutant exhibit similar growth and viability. (A) Viable growth curves of JD6301 (▀) and JD8715 (□) represent the average of three independent replicates. (B) Presence of extracellular lipids is most likely not due to lysis of JD8715. JD6301 and JD8715 cells were treated with chloroform. Scale bars represent 1 μm.
A
B
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 221
sulting lipid extractions was determined (in lieu of
“media only” controls) to obtain the yield of lipid
production relative to glucose consumption (Table
2). JD6301 produced a greater cell mass yield per
gram of sugar consumed (0.0803 g/g sugar) than the
mutant JD8715 (0.0588 g/g sugar). However, JD8715
consumed less sugar (85.86% versus 99.51% for WT)
and also produced greater amounts of extracellu-
lar lipids (0.353 g lipid/ g sugar for JD8715 versus
0.098 g lipid/ g sugar for JD6301 WT; Table 2), sug-
gesting that more energy may have been directed
towards lipid production rather than cell replication.
This is further supported by the reduced cell mass
of JD8715 and therefore the greater production of
extracellular lipids compared to WT.
Lipid analyses indicated that the extracellular lip-
ids present in the JD8715 culture consisted of mono-
glycerides, diglycerides, and triglycerides and that
the extracellular quantity of mono- and triglycerides
was significantly different between the JD6301 and
JD8715 strains (Table 3). The mutant had a nearly
four-fold increase in extracellular lipids. However,
comparing the cell pellet (intracellular) lipid com-
position from JD8715 to JD6301 indicated that the
mutant had significantly less amounts of intracellular
monoglycerides and triacylglycerides. No signifi-
cant difference was observed in the concentration
of diacylglycerides present in the JD8715 cells as
compared to the WT intracellular lipid composition.
The predominant lipids identified in the extracellular
fraction were monoglycerides. It is possible that the
difference in the quantity of triglycerides between
the combined intracellular and extracellular frac-
tions of JD8715 (33.95 μg/mL) and the triglyceride
concentration identified in the WT (43.28 μg/mL) is
due a defect in TAGs formation.
The total FAMEs of JD8715 extracellular lipids
were not significantly different from the intracellu-
lar yield of WT (Fig. 2A). The extracellular yield from
JD8715 was significantly greater than the extracel-
lular yield of WT and the intracellular yield of JD8715
(p < 0.05). Furthermore, the FAMEs profiles between
WT and JD8715 was similar, suggesting a consisten-
cy between strains in regard to lipids produced (Fig.
2B). A large amount of total unknown FAMEs were
detected for JD8715 and WT, which could include
odd-numbered fatty acids.
Growth and viability of JD8715 and WT JD6301
To determine if the presence of extracellular lip-
ids was due to the JD8715 cultures containing more
lysed or dead cells as compared to JD6301, the per-
centages of live cells for both the mutant and WT
were determined using the LIVE/DEAD BacLightTM
bacterial viability kit (Invitrogen). These results indi-
cated that the JD8715 and JD6301 strains exhibited
similar viability at 24 h (77% versus 78%). The growth
of the JD8715 strain was compared to WT to deter-
mine whether the two strains grew similarly under
standard growth conditions. Both strains exhibited
similar growth patterns, indicating that the external
production of lipids did not affect the growth of the
mutant for at least the first 24 h of growth (Fig. 3A).
To further determine whether the increase in the
presence of extracellular lipids was due to cell lysis
occurring during the wash that precedes the ex-
traction procedure, cells were washed with chloro-
form and analyzed by scanning electron microscopy
(SEM). Results indicated that out of a minimum of 50
cells analyzed, 98% of WT and 96% of JD8715 cells
remained intact with no structural deformities fol-
lowing the chloroform wash (Fig. 3B), indicating that
the chloroform wash that precedes the lipid extrac-
tion procedure did not unintentionally lyse the cells,
therefore skewing the extracellular fraction.
CONCLUSIONS
To the author’s knowledge, this is the first report
of a strain of Enterobacter cloacae that is capable
of producing large quantities of lipids. Additionally,
this is the first report of a genetically altered form of
an oleaginous bacterium that is capable of produc-
ing extracellular lipids. The formation of lipid inclu-
sion bodies initiates at the cell membrane (Walter-
mann et al., 2005) proposing several potential areas
222 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
of mutation associated with membrane transport
and integrity resulted in the observed phenotype of
JD8715. The elucidation of this mechanism would
allow application of similar mutations to other oleag-
inous microorganisms for optimization of extracellu-
lar lipid production, providing a substantial advance-
ment to the biofuels industry.
ACKNOWLEDGEMENTS
We would like to thank John Brooks, Karen Coats,
Kendrick Currie, Linda MacFarland, Julie Newton,
John Stokes, Darrell Sparks, and Justin Thornton
for their assistance and helpful discussions with this
project. This project was funded by the Northeast
Mississippi Daily Journal Undergraduate Research
Award to JMB and by the Mississippi State University
Sustainable Energy Research Center funded through
the Department of Energy to JRD. The work con-
ducted by the DOE Joint Genome Institute is sup-
ported by the Office of Science of the DOE under
contract number DE-AC02-05CH11231.
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www.afabjournal.comCopyright © 2014
Agriculture, Food and Analytical Bacteriology
ABSTRACT
Aerobic composting of animal manures has been advocated as an effective management tool to inac-
tivate resident zoonotic pathogens where the time at lethal temperatures is used to determine the effec-
tiveness of the treatment. In the absence of meeting these process conditions, the relative contributions
of other physical factors on growth and persistence of zoonotic pathogens is vague and therefore the
required storage time necessary for elimination of pathogens cannot be adequately estimated. This study
explored the influence of sublethal temperatures, moisture levels, and light exposure on the survival of Sal-
monella and Listeria monocytogenes in compost mixtures that were prepared with three different sources
of manure (dairy cow, swine, and chicken). As ambient temperatures increased from 20°C to 40°C, persis-
tence of both pathogens decreased, which was likely due to the increased competitive activity of the more
dominant indigenous microflora. During storage at 30°C, evaporation of water from compost mixtures
occurred rapidly. Under those conditions, populations of L. monocytogenes declined in cow compost mix-
tures throughout a 4-week storage period, whereas Salmonella populations increased. In chicken compost
mixtures at 30°C, populations of both pathogens decreased only during the first week of storage, which
was likely due to the antimicrobial properties of ammonia initially present in chicken manure. When stored
at 20°C, L. monocytogenes populations decreased more rapidly when compost mixtures were exposed
to more intense light conditions whereas no discernible differences in Salmonella populations occurred in
swine or cow compost mixtures under the different light conditions. These results indicate that developing
safety guidelines for times to hold compost mixtures at sublethal temperatures, prior to land application,
will be challenging.
Keywords: Salmonella, Listeria monocytogenes, compost, dairy, swine, chicken, temperature,
moisture, light, manure
Correspondence: M.C. Erickson, [email protected]: +1 770-412-4742 Fax: +1 770-229-3216
Survival of Salmonella enterica and Listeria monocytogenes in manure-based compost mixtures at sublethal temperatures
M.C. Erickson1, C. Smith2, X. Jiang3, I.D. Flitcroft4, and M.P. Doyle1
1 Center for Food Safety and Department of Food Science and Technology, University of Georgia, Griffin, GA 2 Food Safety Net Services, Atlanta Laboratory, Covington, GA
3 Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, SC 4 Department of Crops and Soil Science, University of Georgia, Griffin, GA
Agric. Food Anal. Bacteriol. 4: 224-238, 2014
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 225
INTRODUCTION
Livestock and poultry production are major enter-
prises worldwide that in addition to the production
of food, waste by-products that include solid ma-
nure and manure slurries are also produced in large
quantities. For example, the Environmental Protec-
tion Agency (EPA) estimated that 1.1 billion tons of
manure was produced annually within the U.S. (US
EPA, 2013), with cattle contributing the greatest pro-
portion (83%), followed by swine (10%), and poultry
(7%). Land application of these wastes has been one
of the most cost effective approaches to dispose of
such large quantities of manure, with 5% of all crop-
land (15.8 million acres) in 2006 reported as having
been fertilized with livestock manure (MacDonald et
al., 2009). Zoonotic pathogens are sporadically resi-
dent within animal manure (Le Bouquin et al., 2010;
LeJeune et al., 2006; Lomonaco et al., 2009). Hence if
manure is applied to land, these pathogens can con-
taminate the soil, crops grown in those fields, and
waterways that collect runoff from the fields.
Aerobic composting of animal wastes can inac-
tivate zoonotic bacterial pathogens while creating
a stable amendment that improves soil quality and
fertility (Berry et al., 2013; Raviv, 2005). Heat gener-
ated from the metabolic activity of thermophilic mi-
croorganisms in manure piles that are self-insulating
is the primary mechanism for inactivating zoonotic
pathogens (Pell, 1997; Wichuk and McCartney, 2007).
Hence, process conditions for composting manures
in the U.S. are based on EPA’s regulations for com-
posting biosolids that includes either a minimum
temperature of 55°C for 3 days in aerated static piles
or in-vessel systems, or 55°C for 15 days in windrow
systems. Moreover, in the windrow systems, the ma-
terial must be turned a minimum of 5 times to en-
sure that all material is subjected to the necessary
thermal conditions (US EPA, 1999a). Composting at
40°C for 120 h or more, during which time the tem-
perature exceeds 55°C for 4 h, has also been desig-
nated by EPA in Appendix B of the 503 Regulations
as a process to significantly reduce pathogens (US
EPA, 1999b). Unfortunately, when these EPA criteria
are not met (Wichuk and McCartney, 2007), as could
occur during winter composting or if piles are not
turned to expose the surface material to sufficient
internal heat, the holding time of compost materials
to ensure pathogen inactivation is uncertain.
Compared to lethal heat exposure, the contribu-
tion of other physical factors (e.g., non-lethal tem-
peratures, light, and desiccation) to inactivation of
zoonotic pathogens in manure-based compost mix-
tures has not been elucidated because biological
(i.e., competition for nutrients) and chemical (e.g.,
ammonia, volatile acids or other antimicrobials) fac-
tors that affect pathogen inactivation are also likely
affected by the physical parameter. Such is the case
with soil systems in which increased temperatures,
despite being near the organism’s optimal growth
temperature, led to greater inactivation of Esche-
richia coli O157:H7 as a result of an accompanying
increase in competition by the dominant native mi-
crobial community (Semenov et al., 2007). Similarly,
the effect of moisture levels on the fate of pathogens
(Salmonella and E. coli O157:H7) or their surrogates
in soil systems has been dependent on the patho-
gen population levels relative to the levels of the
indigenous microbial community (Lang et al., 2007;
Ongeng et al., 2011) and likely play a similar role in
compost mixtures. Ammonia that is generated dur-
ing the composting process (Beck-Friis et al., 2003)
and has been shown to be an antimicrobial agent
toward Salmonella and Listeria monocytogenes in
chicken and cattle manure (Himathongkham and
Riemann, 1999; Park and Diez-Gonzalez, 2003) is also
affected by moisture levels, with drying of manure
accelerating the volatilization of ammonia (Gotaas,
1956) and inhibiting the conversion of nitrogenous
compounds to aqueous ammonia (Hutchison et al.,
2000). Considering the complex interactions that
moisture and temperature exert on the activity of in-
digenous microbial communities, it is of interest to
investigate the role of moisture levels on inactivation
of pathogens in compost mixtures that would likely
be populated with different indigenous microflora
from the different nitrogen feedstocks.
Another physical factor that has received little at-
tention for its involvement in inactivating pathogens
in manure-based compost mixtures is sunlight. Due
226 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
to its inability to penetrate compost mixtures, sun-
light would be lethal to pathogens primarily at the
surface of compost mixtures as has occurred at the
surfaces of natural waterways and lagoons (Davies
and Evison, 1991; Maynard et al., 1999). Hutchison
et al. (2005) postulated that lack of surface contami-
nation by Salmonella, Listeria, Campylobacter, or E.
coli O157:H7 in composted static pile wastes after
eight days was due to exposure of their surfaces to
sunlight; however, the experiment lacked a control
sample not exposed to sunlight. In contrast, Erick-
son et al. (2010) were able to detect both Salmonella
and Listeria on the surface of static piles comprised
of chicken litter and peanut hulls after composting
for 14 and 56 days in the summer and winter, respec-
tively. To gauge the potential impact of sunlight on
pathogens in compost more specifically, results of
a study on the survival of pathogens in beef cattle
fecal pats is presented here for comparison (Meays
et al., 2005). In that experiment, E. coli survival un-
der 4 different levels of solar exposure (controlled
by using a shade cloth) was determined. After 45
days, fecal pats under the 0% shade cloth had the
least surviving E. coli, followed by the 40%, 80%, and
100% treatments. A similar response in non-turned
composting systems could result in longer recom-
mended holding times for regions with a large num-
ber of overcast days compared to regions that are
dominated by sunny days.
The purpose of this study was to determine the in-
fluence of several physical factors (i.e., temperature,
level of light exposure, and moisture levels) on the
inactivation of Salmonella and L. monocytogenes in
compost mixtures that were stored in environmen-
tal chambers at temperatures ranging from 20°C to
40°C in amounts that would not be self-insulating.
To account for the potential confounding influence
of indigenous microflora on pathogen inactivation,
this variable was addressed indirectly by utilizing ma-
nure in compost mixture formulations from different
sources (dairy, chicken, and swine) that should have
different microbial compositions.
MATERIALS AND METHODS
Pathogen Strains and Preparation
Three strains of green-fluorescent protein (GFP)-
labeled Salmonella enterica serovar Enteritidis (ME-
18, H4639, and H3353) and one strain of GFP-labeled
S.enterica serovar Newport containing an ampicillin-
resistant marker were selected from the culture col-
lection at the University of Georgia, Center for Food
Safety (Griffin, GA). Five strains of GFP-labeled L.
monocytogenes containing an erythromycin-resistant
marker (12443, H7550, G3982, 101M, and F6845) were
also selected from the culture collection. Details on
the construction of these GFP strains has been de-
scribed by Ma et al. (2011) and they also reported
that the loss of the GFP-plasmid after 20 generations,
indicative of its stability, has ranged from 15 to 77%
and 8 to 52% for the Salmonella and L. monocyto-
genes strains, respectively.
Frozen stock cultures of each GFP-labeled Sal-
monella strain and GFP-labeled L. monocytogenes
strain were thawed and streaked onto tryptic soy agar
(Difco, Becton Dickinson, Sparks, MD) containing 100
μg/ml ampicillin (TSA-A) and brain heart infusion agar
(Becton Dickinson) containing 8 μg/ml erythromycin
(BHIA-E), respectively. Following incubation at 37°C
for 20 to 24 h, individual colonies from each plate
were subsequently streaked onto a second plate that
was incubated for another 20 to 24 h at 37°C. Individ-
ual Salmonella and L. monocytogenes colonies from
these plates were then inoculated into 100 ml of tryp-
tic soy broth (Becton Dickinson) containing 100 μg/ml
of ampicillin (TSB-A) and 100 ml brain heart infusion
broth (Becton Dickinson) containing 8 μg/ml erythro-
mycin (BHIB-E), respectively. Broths were incubated
at 37°C for 20 to 24 h with agitation (150 rpm) and
bacteria were subsequently harvested by centrifuga-
tion (4,050 x g, 15 min, 4°C) with cell pellets being
washed three times in 0.1% peptone water (Difco,
Becton Dickinson). Reconstitution of the individual
strains in 0.1% peptone water to an optical density
of 0.5 (approximately 109 CFU/ml) was made prior to
combining equal volumes of each strain to comprise
one four-strain mixture of Salmonella and one five-
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 227
strain mixture of L. monocytogenes. Salmonella and
L. monocytogenes populations were determined by
plating on TSA-A and modified Oxford agar (Acu-
media Manufacturers, Lansing, MI) containing 8 μg/
ml erythromycin (MOX-E), respectively. Salmonella
transformed colonies emitted bright green fluores-
cence when viewed at 365 nm under a handheld UV
light (Fotodyne Inc., Hartland, WI); however, visual-
ization of fluorescent L. monocytogenes transformed
colonies required use of a Leica MZ16 FA stereo fluo-
rescence microscope (Bannockburn, IL).
Compost Feedstocks and Chemical Analysis
Three sources of manure including dairy cow ma-
nure, swine manure, and broiler chicken litter were
used as the primary nitrogen source for compost
mixtures. These materials were collected from farms
located near Griffin, GA, and upon arrival at the labo-
ratory were frozen for at least 24 h to kill the majority
of insect eggs (Sherman et al., 2006). Carbon amend-
ments in compost formulations (i.e., wheat straw and
cottonseed meal) were purchased from a local feed
supply store. To improve the homogeneity of com-
post formulations, wheat straw was shredded using a
Flowtron Leaf Eater (Malden, MA) for lengths of 1 to
5 cm. Carbon, nitrogen, and moisture content analy-
ses were conducted on all raw ingredients used in
the compost mixtures (Erickson et al., 2010) to assist
in determining recipes for formulation of compost
mixtures.
Compost Mixture Formulation
Each type of manure was individually sprayed in
a 28-L sanitized bowl with either GFP-labeled Sal-
monella or both GFP-labeled-Salmonella and L.
monocytogenes to give initial cell populations of 3.3
to 7.5 log CFU/g. Carbon amendments and sterile
deionized water were then added to the inoculated
manure to comprise formulations having initial levels
of 30% or 60% moisture and initial carbon:nitrogen
(C:N) ratios of 20:1 to 40:1. Compost amendments
and inoculated manure were mixed thoroughly for
ca. 5 min in a Hobart mixer (model D320; ¾ h.p.) prior
to distributing the mixtures into containers for exper-
imental studies.
Experimental Design
Four studies were conducted that varied in their
experimental design. In the first experimental study
investigating the role of temperature on survival of
Salmonella in manure, no carbon amendment was
added to the manure source (dairy cow manure and
chicken litter) that were each obtained at two sepa-
rate times. Dairy cow manure (2 kg) or chicken litter
(1 kg) was sprayed with the Salmonella inoculum mix-
ture (20 ml or 10 ml of 7 log CFU/ml, respectively) to
obtain ca. 5 to 6 log CFU/g. Inoculated material (100
g) was placed into multiple square (12.7 cm2) Ziplock
plastic containers (S.C. Johnson & Sons, Racine, WI).
With the first batch of dairy cow manure, three con-
tainers were held at 25°C and another three were
held at 40°C. With the second batch of dairy cow ma-
nure, three containers were held at 35°C and another
three were held at 40°C. For inoculated chicken litter,
the first batch was stored in three separate contain-
ers only at 25°C whereas the second batch was filled
into three containers that were stored at 40°C only.
Samples were removed from each container initially
and after 3 days of storage for analysis of Salmonella
and mesophilic and thermophilic bacteria. Only one
replicate trial of this experimental design was con-
ducted.
The second experimental study investigated the
role of temperature on survival of enteric pathogens
in manure-based compost mixtures. Chicken litter
was collected at six separate times, with each col-
lection being used as an independent replicate trial.
Each batch of chicken litter was sprayed with both an
inoculum of Salmonella and an inoculum of L. mono-
cytogenes before blending with wheat straw, cotton-
seed meal and sterile deionized water to obtain mix-
tures having an initial carbon:nitrogen (C:N) ratio of
40:1 and 60% moisture content Initial pathogen pop-
228 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
ulations in three of the batches was targeted at a low
level (ca. 3.5 log CFU/g) while another three batches
had a target at a higher level (ca. 6.7 log CFU/g). Du-
plicate samples from each inoculated mixture were
obtained for pathogen and moisture content analy-
sis prior to distributing compost mixtures into two
uncovered translucent plastic cups (8.5 cm diameter
x 5 cm height, ca. 45 g/cup). Cups containing the
low level inoculum were placed in an environmental
chamber at 30°C with a 12-h light and 12-h dark cy-
cle whereas cups containing the high level inoculum
were placed in a dark environmental chamber set to
20°C. In the lighted chamber, light was supplied by
ten 400W Metal Halide MVR400/U bulbs (General
Electric, Cleveland, OH) and ten 400W high pressure
sodium lamps LU400/H/ECO, LUCALOS bulbs (Gen-
eral Electric). High pressure sodium lamps emit no
ultraviolet (UV) light and while the metal halide bulbs
emit a small band of long band UV light (ca. 375
nm), compost mixtures were not exposed as this UV
light was filtered out by diffusive panels separating
the lamps and chamber. The compost mixtures of
all batches were held for two weeks at the specified
temperature after which time the cups were removed
and mixtures assayed for surviving pathogens.
For the third experimental study, both dairy cow
manure and chicken litter were used as nitrogen
sources to determine the role of moisture content
on the survival of Salmonella and L. monocytogenes.
Manure or litter was initially sprayed with an inocu-
lum of Salmonella and an inoculum of L. monocyto-
genes and, then mixed with wheat straw, cottonseed
meal, and sterile deionized water to obtain cow and
chicken compost mixtures having an initial C:N ra-
tio of 20:1, initial moisture contents of either 30% or
60%, and initial pathogen populations of ca. 3 to 4
log CFU/g. The cow and chicken compost mixtures
were then distributed into small translucent cups (ca.
45 g/cup) used in study 2. The cups were stored un-
covered in an environmental chamber at 30°C with a
12-h light (602 μmol/m2/sec) and 12-h dark cycle for
up to 4 weeks. Two cups from each treatment were
removed initially and at weekly intervals and analyzed
for pathogen populations, moisture content, and pH.
In addition, half of the remaining cups were adjusted
back to their initial weights and original moisture
contents by spraying the sample with a light mist
of sterile deionized water. Two cups whose mois-
ture contents had been adjusted were also removed
from each of the four treatments (manure source
x target moisture content) at 2, 3, and 4 weeks for
analysis of pathogen populations, moisture content,
and pH. Three independent replicate trials in which
samples were not adjusted for moisture content were
conducted whereas two independent replicate trials
were conducted for samples adjusted for moisture
content. For each independent trial, the dairy cow
manure and chicken litter were collected at separate
times and were inoculated with a different batch of
pathogen inocula.
For the fourth experimental study, three different
sources of manure (dairy cow manure, chicken litter,
and swine manure) were inoculated and incorpo-
rated into compost mixtures to examine the effect
of light on survival of Salmonella and L. monocyto-
genes. Wheat straw and cottonseed meal served as
the carbon amendments and were mixed with the
Salmonella- and L. monocytogenes-inoculated ma-
nure sources and sterile deionized water to obtain
cow-, chicken-, and swine-compost mixtures having
an initial C:N ratio of 30:1, an initial moisture content
of 60%, and pathogen populations of ca. 6.7 to 7.5
log CFU/g. Duplicate samples from each of the three
contaminated compost mixtures were analyzed for
initial pathogen populations, moisture content, and
pH. The remainder of each compost mixture was
then distributed into the small translucent cups (ca.
45 g/cup) used in studies 2 and 3 described above.
The cups for each treatment group were then divided
into three groups. One group was placed in a dark en-
vironmental chamber, the second group was placed
in an environmental chamber where all bulbs were
turned on to simulate daily “bright” sunny conditions
(12 h at 524-573 μmol/m2/sec and 12 h in the dark),
and the third group was placed in an environmental
chamber where only half the bulbs were turned on
to simulate daily “cloudy” conditions (12 h at 289-
359 μmol/m2/sec and 12 h in the dark). All chambers
were at 20°C. Duplicate samples from each treat-
ment at five sample times over the course of 4 weeks,
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 229
12 weeks, and 18 weeks for swine, chicken, and cow
compost mixtures, respectively, were enumerated
for pathogens and assayed for moisture content and
pH. Using the above experimental design, three in-
dependent replicate trials were conducted with each
trial using manure collected at separate times and a
different batch of pathogen inoculum cultured for in-
oculation of the manure.
Microbiological and pH Analyses
Both direct plating and selective enrichment cul-
ture was used for detection of GFP-labeled Salmo-
nella and L. monocytogenes. In direct plating as-
says, compost sample (5 g) was added to 45 ml of
0.1% peptone water in a sterile Whirl-Pak bag and
mixed in a stomacher. Ten-fold serial dilutions of
this homogenate were made prior to spreading on
TSA-A or MOX-E plates for enumeration of GFP-
labeled Salmonella or L. monocytogenes, respec-
tively. Selective enrichment culture for Salmonella
and L. monocytogenes consisted of adding compost
sample (5 g) directly to 45 ml of TSB-A or BHIB-E,
respectively, and incubating at 37°C for 24 h. These
enriched samples were then streaked on TSA-A or
MOX-E plates to determine the presence or absence
of Salmonella or L. monocytogenes, respectively, at a
detection limit of 20 cells/100 g.
To determine initial levels of mesophilic and ther-
mophilic microbial populations in chicken and cow
manure, ten-fold serial dilutions of the stomached
homogenates were spread onto Difco plate count
agar (Becton Dickinson, Sparks, MD). Colonies of
mesophilic and thermophilic bacteria were counted
after overnight incubation at 30°C and 55°C, respec-
tively.
Measurement of pH was determined with an Acu-
met Basic pH meter (Fisher Scientific, Pittsburgh, PA)
on 5-g compost samples dispersed in 250 ml of de-
ionized water. Compost mixtures were analyzed for
moisture using the same procedure described for
moisture analysis of feedstuffs.
Statistical Analyses
Salmonella and L. monocytogenes populations in
samples for each independent trial were converted
to logarithmic values prior to determining differences
from initial population levels. Logarithmic pathogen
decreases, % moisture content, and pH values were
subjected to the general linear- and one-way analy-
sis of variance (ANOVA) test using the StatGraphics
Centurion XV software package (StatPoint, Inc., Hern-
don, VA). When statistical differences were observed
(P < 0.05) with the ANOVA test, sample means were
differentiated with the least significant difference test
(P = 0.05).
RESULTS AND DISCUSSION
Several studies have previously addressed the
survival of zoonotic pathogens in manure at ambi-
ent temperatures (Himathongkham et al., 1999a, b;
2000; Sinton et al., 2007); however, this type of study
was repeated in our preliminary study (first experi-
mental study) with locally-obtained manure to give
some baseline information on the fate of Salmonel-
la and other indigenous microflora in the absence
of a carbon amendment. Different responses were
observed for Salmonella and the indigenous micro-
flora depending on the manure source and storage
temperatures. Following a 3-day storage period,
no changes in populations of the indigenous mi-
croflora (mesophilic and thermophilic bacteria) oc-
curred when present in cow manure and stored at
25°C (Table 1). In contrast, the populations of both
mesophilic and thermophilic bacteria increased in
cow manure stored at 35°C or 40°C, but decreased
in chicken litter stored at 40°C for a similar time pe-
riod. Salmonella decreases occurred in both manure
sources when held at 40°C, but reductions were sub-
stantially greater in chicken litter than in cow manure.
Salmonella populations also decreased in chicken
litter held at 25°C, but increased in cow manure held
at 25°C or 35°C. Transient increases in Salmonella
population have been observed previously in cow
230 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
manure held at ambient temperatures (Himathong-
kham et al., 1999a; Sinton et al., 2007) and a minimal
water content of 80% was a prerequisite (Sinton et
al., 2007). Salmonella die-off in chicken manure has
also been documented and has been ascribed to
the generation of ammonia (Himathongkham et al.,
1999b; 2000) which is an antimicrobial (Himathong-
kham and Riemann, 1999). For the current study, it
is plausible that the production of ammonia by the
indigenous microflora could be stimulated by the
presence of bedding material that was included dur-
ing collection of the chicken manure.
For the second experimental set of studies,
temperature was the main variable of interest, but
chicken litter was mixed with the carbon amend-
ments, wheat straw and cottonseed meal, to create
compost mixtures prior to their storage. When com-
post mixtures were formulated to an initial moisture
content of 60% and an initial C:N ratio of 40:1, stor-
age for 2 weeks at 20°C in the dark led to L. mono-
cytogenes reductions of 0.97 ± 0.66 log CFU/g. In
contrast, Salmonella populations remained relatively
constant (increase of 0.16 ± 0.56 log CFU/g) over
the same time period. Storage of chicken compost
mixtures at 30°C for the same time interval but un-
der lighted conditions, however, resulted in popu-
lation decreases for both L. monocytogenes (1.87
± 1.22 log CFU/g loss) and Salmonella (0.89 ± 1.54
log CFU/g loss). This trend of increased pathogen
inactivation with increasing ambient temperatures
Table 1. Indigenous bacterial populations in chicken and cow manure and fate of Salmonella when stored for 3 days at temperatures between 25°C and 40°C1.
Chicken manure2 Cow manure3
Day 25°C 40°C 25°C 35°C 40°C
Mesophilic bacteria populations (log CFU/g)5
0 ND4 9.38 ± 0.13 a 9.22 ± 0.10 a 6.88 ± 0.41 b 6.88 ± 0.06 b
3 7.65 ± 0.14 5.86 ± 0.33 b 9.51 ± 0.16 a 8.88 ± 0.41 a 8.44 ± 0.64 a
Thermophilic bacteria populations (log CFU/g)5
0 ND 7.08 ± 0.02 a 8.36 ± 0.08 a 6.35 ± 0.01 b 6.35 ± 0.01 b
3 8.32 ± 0.25 5.86 ± 0.06 b 8.54 ± 0.17 a 8.46 ± 0.38 a 8.19 ± 0.83 a
Salmonella’s fate after 3 days of storage (Δ log CFU/g)6
↓7 1.21 ↓ 5.56 ↑8 1.88 ↑ 1.11 ↓ 0.89
1 Data collected from first experimental study.2 Mesophilic and thermophilic bacteria population levels are mean ± S.D., n = 3.3 Mesophilic and thermophilic bacteria population levels are mean ± S.D., n = 3 for 25°C and 35°C samples,
n = 6 for 40°C samples.4 Not determined.5 Values for this parameter within each column followed by a different letter are significantly different (P <
0.05). 6 Salmonella initial populations in manure samples ranged from 5 to 6 log CFU/g.7 Decrease in population.8 Increase in population.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 231
is similar to the patterns in soil of pathogen survival
previously documented (Lang et al., 2007; Semenov
et al., 2007). Based on those studies, the investiga-
tors suggested that increases in temperature, de-
spite being close to the pathogen’s optimal growth
temperature, increased the competitive activity of
the more dominant indigenous microflora which
adversely affected the pathogen’s survival (Lang et
al., 2007). This explanation may be the basis for the
decreased pathogen persistence we observed in
chicken compost mixtures as exposure temperatures
increased.
Initial moisture contents (30% or 60%) and weekly
adjustment of the moisture contents of chicken- and
cow compost mixtures were the two variables of in-
terest in our third experimental study in determining
their influence on L. monocytogenes and Salmonella
inactivation in compost mixtures stored for up to 4
weeks at 30°C. However, under these conditions,
populations of either pathogen were not affected
by the initial moisture contents nor did weekly ad-
ditions of water to return the compost mixtures to
their original moisture contents affect the reduction
of pathogens (P > 0.05). Moisture analysis of the
compost mixtures revealed that water was lost very
quickly from the samples stored in uncovered con-
tainers and equilibrated to approximately the same
percentage of moisture (9.7 ± 2.7%) regardless of the
initial moisture content or when weekly additions of
water were applied to the mixtures. These condi-
tions were therefore likely responsible for the fail-
ure of moisture content to have an effect on patho-
Table 2. Comparison of Salmonella and L. monocytogenes losses in chicken and cow manure-based compost mixtures1 stored for up to 4 weeks at 30°C2.
Cumulative pathogen reduction (log CFU/g)3, 4
L. monocytogenes5 Salmonella6
Week Chicken manure compost mixture
Cow manure compost mixture
Chicken manure compost mixture
Cow manure compost mixture
1 2.09 ± 0.67 a 0.59 ± 1.27 b 1.36 ± 1.62 a -0.35 ± 1.73 a-c
2 1.87 ± 1.22 a 0.60 ± 1.25 b 0.89 ± 1.54 ab -0.82 ± 1.44 bc
3 2.11 ± 0.93 a 1.15 ± 1.22 ab 1.11 ± 1.50 ab -1.31 ± 2.10 c
4 2.11 ± 0.93 a 2.21 ± 0.82 a 0.90 ± 1.64 ab -0.58 ± 1.56 bc
1 Compost mixtures were formulated with carbon amendments to give an initial carbon:nitrogen ratio of 20:1 and pathogen populations of ca. 3.5 log CFU/g.
2 Data collected from third experimental study.3 Pathogen data collected from treatments evaluating compost mixtures formulated to either an initial
moisture content of 30% or 60% and then either readjusted to the original moisture content on a weekly basis or left undisturbed were not significantly different. The data were therefore pooled prior to statistical analysis and displaying the data in this table by the manure source used in the compost mixture.
4 Pathogen reductions were calculated by subtracting the population level at each time period from the initial population level.
5 Values for this pathogen (mean ± S.D.), across both rows and columns, followed by a different letter are significantly different (P < 0.05).
6 Values for this pathogen (mean ± S.D.), across both rows and columns, followed by a different letter are significantly different (P < 0.05).
232 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
gen inactivation. Hence, the data were pooled to
determine the changes that occurred in pathogen
populations in the chicken and cow compost mix-
tures (Table 2). As with dairy manure in the absence
of a carbon amendment and held at 25°C or 35°C
(Table 1), the populations of Salmonella in the cow
compost mixtures increased from initial populations
after holding of the materials at 30°C and remained
at these higher levels over the 4-week trial (Table 2).
In contrast, L. monocytogenes populations declined
in cow compost mixtures after only one week of stor-
age at 30°C and additional significant decreases oc-
curred up to 4 weeks of storage (P < 0.05). Over all
time periods, there were significantly greater reduc-
tions in Salmonella and L. monocytogenes popula-
tions in the chicken compost mixtures compared to
the cow compost mixtures (P < 0.05). Interestingly,
the decreases in pathogen populations in the chick-
en compost mixtures occurred during the first week
of storage but not after additional storage (Table 2).
Monitoring the pH of the chicken- and cow ma-
nure-based compost mixtures during storage at
30°C for 4 weeks (third experimental study) revealed
that initially the chicken compost mixtures were ap-
proximately 0.5 pH units higher than the cow com-
post mixtures (Table 3). Although not determined in
this study, the ammonia present that has previously
been associated with higher pH values in chicken
manure (Himathongkham et al., 1999b; 2000) could
have been the principal factor responsible for the
die-off of pathogens in the chicken compost mix-
tures. It appears, however, that pH alone may not be
used as an indicator of a compost mixture’s capacity
to sustain viable pathogen populations. In the cow
compost mixtures having an initial moisture content
of 60%, the pH increased to values approximating
those detected in the chicken compost mixtures (Ta-
ble 3) yet Salmonella grew in those mixtures (Table
2). Based on these results, it is likely that Salmonella
is susceptible to ammonia that is present in chicken
Table 3. pH of chicken and cow manure-based compost mixtures1 following storage at 30°C for 4 weeks when initial moisture contents were either 30% or 60%2.
pH3
Chicken manure compost mixture Cow manure compost mixture
Week 30% moisture 60% moisture 30% moisture 60% moisture
0 7.25 ± 0.66 a-f 7.58 ± 1.22 a-d 6.84 ± 0.33 d-f 6.64 ± 0.34 f
1 7.06 ± 0.32 b-f 7.85 ± 0.66 a 6.80 ± 0.29 ef 7.46 ± 0.21 a-e
2 6.97 ± 0.34 c-f 7.63 ± 0.59 a-c 6.77 ± 0.22 ef 7.09 ± 0.10 b-f
3 6.99 ± 0.42 c-f 7.75 ± 0.34 ab 6.81 ± 0.16 ef 7.32 ± 0.29 a-f
4 6.87 ± 0.41 d-f 7.74 ± 0.24 ab 6.77 ± 0.08 ef 7.45 ± 0.22 a-e
1 Compost mixtures were formulated with carbon amendments to give an initial carbon:nitrogen ratio of 20:1.
2 Data collected from third experimental study.3 Values within the table (mean ± S.D.) followed by a different letter are significantly different (P < 0.05).
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 233
Tab
le 4
. Sal
mo
nella
and
L. m
ono
cyto
gen
es r
educ
tio
ns d
urin
g s
tora
ge
at 2
0°C
und
er d
iffer
ent
light
co
ndit
ions
in c
om
po
st m
ixtu
res
pre
par
ed w
ith
diff
eren
t m
anur
e so
urce
s1 .
Cum
ulat
ive
pat
hog
en re
duc
tions
(lo
g C
FU/g
) 2
L. m
ono
cyto
gen
es3
Salm
one
lla3
Man
ure
sour
ceW
eek
Dar
kC
loud
y4Su
nny5
Dar
kC
loud
ySu
nny
Chi
cken
20.
86 ±
0.0
1 e
1.37
± 0
.74
e1.
37 ±
0.6
1 e
0.32
± 0
.65
e0.
48 ±
0.2
6 e
1.01
± 0
.76
de
62.
70 ±
0.2
7 d
4.71
± 1
.01
c5.
83 ±
0.3
0 ab
1.10
± 1
.02
c-e
1.39
± 0
.63
b-e
3.07
± 1
.44
ab
84.
38 ±
1.0
8 c
5.83
± 0
.30
ab5.
83 ±
0.3
0 ab
1.07
± 0
.56
de
2.09
± 0
.38
a-e
3.41
± 1
.14
a
105.
01 ±
1.1
2 b
c5.
83 ±
0.3
0 ab
6.03
± 0
.13
a1.
89 ±
0.8
1 a-
e2.
11 ±
0.7
9 a-
e2.
90 ±
1.9
1 a-
c
125.
93 ±
0.2
4 ab
5.83
± 0
.30
ab5.
93 ±
0.2
8 ab
3.14
± 1
.92
ab2.
45 ±
0.7
2 a-
d2.
99 ±
1.5
3 ab
Dai
ry c
ow
20.
36 ±
0.8
1 d
1.07
± 1
.85
d2.
03 ±
2.1
2 cd
-0.3
1 ±
1.0
8 a
0.23
± 2
.04
a1.
08 ±
3.4
3 a
61.
04 ±
0.8
1 d
2.39
± 2
.90
cd2.
43 ±
1.6
1 cd
0.44
± 1
.75
a0.
58 ±
2.4
0 a
1.01
± 2
.45
a
101.
28 ±
0.2
6 d
2.87
± 2
.50
b-d
2.90
± 2
.73
b-d
0.71
± 0
.89
a1.
63 ±
2.9
4 a
1.75
± 2
.84
a
142.
25 ±
0.3
9 cd
4.09
± 1
.46
a-c
5.29
± 0
.70
ab0.
93 ±
1.9
6 a
1.78
± 2
.81
a1.
67 ±
2.0
6 a
184.
70 ±
1.7
1 a-
c4.
29 ±
1.2
7 a-
c6.
27 ±
1.0
2 a
1.61
± 2
.96
a1.
90 ±
2.7
2 a
2.09
± 2
.56
a
Swin
e0.
3-0
.57
± 0
.45
de
-0.6
4 ±
0.5
6 e
-0.0
8 ±
0.6
2 c-
e-0
.82
± 0
.37
f-0
.75
± 0
.52
ef-0
.25
± 0
.51
c-f
1-0
.26
± 0
.48
c-e
-0.2
7 ±
0.5
5 c-
e-0
.01
± 0
.46
c-e
-0.4
4 ±
0.5
3 d
-f-0
.43
± 1
.02
d-f
-0.1
1 ±
0.7
5 b
-f
20.
07 ±
0.4
1 c-
e0.
14 ±
0.5
6 b
-d0.
44 ±
0.1
1 b
c-0
.36
± 0
.69
c-f
0.02
± 0
.53
a-f
0.16
± 0
.36
a-e
30.
10 ±
0.2
5 b
-e0.
22 ±
0.2
4 b
c0.
86 ±
0.4
5 ab
0.13
± 0
.46
a-f
0.36
± 0
.62
a-d
0.74
± 0
.48
ab
40.
39 ±
0.1
7 b
c0.
39 ±
0.3
6 b
c1.
38 ±
0.6
6 a
0.42
± 0
.41
a-d
0.56
± 0
.68
a-c
0.87
± 0
.38
a
1 D
ata
wer
e co
llect
ed fr
om
four
th e
xper
imen
tal s
tud
y.
2 R
educ
tions
wer
e d
eter
min
ed re
lativ
e to
initi
al v
alue
s in
co
mp
ost
mix
ture
s.3
Valu
es (m
ean
± S
.D.)
with
in e
ach
man
ure
sour
ce fo
r th
is p
atho
gen
follo
wed
by
a d
iffer
ent
lett
er a
re s
igni
fican
tly d
iffer
ent
(P <
0.0
5).
4 C
om
po
st m
ixtu
res
wer
e ex
po
sed
dai
ly t
o li
ght
co
nditi
ons
of 2
89 t
o 3
59 μ
mo
l/m
2 /se
c fo
r 12
h a
nd t
o d
ark
cond
itio
ns fo
r 12
h.
5 C
om
po
st m
ixtu
res
wer
e ex
po
sed
dai
ly t
o li
ght
co
nditi
ons
of 5
24 t
o 5
73 μ
mo
l/m
2 /se
c fo
r 12
h a
nd t
o d
ark
cond
itio
ns fo
r 12
h.
234 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
compost mixtures initially but the low-moisture con-
ditions present in these mixtures inhibit the indig-
enous microflora from generating additional ammo-
nia. The low moisture conditions, however, do not
directly contribute to inactivation of desiccation-re-
sistant Salmonella (Pedersen et al., 2008; Tamura et
al., 2009), whereas a substantial proportion of the L.
monocytogenes population is susceptible to either
ammonia or desiccation stress.
The last study addressed the influence of light in
the visible and infrared spectrum on inactivation of
pathogens in manure-based compost mixtures held
at sublethal temperatures. Compost mixtures pre-
pared with chicken litter, dairy cow manure or swine
manure and held at 20°C were exposed to one of
three lighting conditions simulating dark, sunny, or
cloudy conditions. Preliminary studies were con-
ducted with each of these compost mixtures to de-
termine the approximate time interval over which
samples should be taken to obtain measurable
pathogen reductions and determine whether light
conditions could significantly affect their inactiva-
tion. Unfortunately, the storage time intervals select-
ed for swine compost mixture were underestimated
and the greatest pathogen reduction was only
slightly greater than 1 log CFU/g (Table 4). Despite
this limitation, significant trends were determined
for the swine compost mixture data. In particular,
both pathogen populations increased during the
first week of storage under all lighting conditions in
swine compost mixtures. Following 2 weeks of stor-
age, Salmonella remained at elevated populations
for the swine compost mixtures that were held in
the dark, whereas under cloudy or sunny conditions,
Salmonella populations decreased slightly. Further
reductions in Salmonella populations occurred dur-
ing the next two weeks of storage, but there were no
statistical differences in response to light exposure
(P > 0.05). For L. monocytogenes in swine compost
mixtures, only sunny conditions at week 4 had signifi-
cantly greater reductions of this pathogen compared
to mixtures held under cloudy or dark conditions (P
< 0.05). The inability to detect significant differences
in the reduction of either pathogen under dark and
cloudy conditions is likely due to the relatively mini-
mal reductions that occurred during the short time
period that was examined for swine compost mix-
tures. In contrast, over all time periods, reductions in
L. monocytogenes populations were statistically sig-
nificant for both cloudy and sunny conditions com-
pared to dark conditions for chicken compost mix-
tures stored for 6 weeks or in cow compost mixtures
stored for 14 weeks (Table 4, P < 0.05).
A completely different set of responses to light
was observed for Salmonella in chicken- or cow
compost mixtures. In chicken compost mixtures,
only sunny conditions led to statistically greater re-
ductions in populations than dark conditions, and
these occurred midway through the storage trial. In
contrast but similar to the response in swine com-
post mixtures, light conditions did not affect the re-
ductions in Salmonella populations in cow compost
mixtures at any sampling time (Table 4).
A number of factors could contribute to light-
mediated inactivation of L. monocytogenes in the
compost mixtures. As a component of sunlight,
both long wave (UVA, 315 to 400 nm) and medium
wave (UVB, 280 to 315 nm) ultraviolet light has been
shown to damage the genetic material of microor-
ganisms (Davies and Evison, 1991; Jagger, 1985;
Jiang et al., 2009); however, in our environmental
chambers, ultraviolet light was filtered out by the
diffusive ceiling light panels and hence had no role.
Alternatively, exogenous sensitizers in the compost
materials such as humic substances (Chien et al.,
2007) may be activated by visible light energy. Such
a mechanism has been demonstrated for inactiva-
tion of the Gram-positive, Enterococcus faecalis in
waste stabilization pond water whereas the Gram-
negative E. coli was inherently less susceptible to
this pathway (Kadir and Nelson, 2014). Although L.
monocytogenes has previously displayed some des-
iccation resistance, surviving for three months in a
simulated food processing environment (Vogel et
al., 2010), it is not as resistant as Salmonella based
on the lower recoveries of L. monocytogenes com-
pared to Salmonella in aerosols of meat processing
plants (Okraszewska-Lasica et al., 2014). Hence, a
third mechanism by which increased intensities of
light may have led to increased inactivation of the L.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 235
monocytogenes isolates used in this study may be
through localized heating, additional dehydration at
surface locations, and in turn increased desiccation
stress. In support of this explanation, the moisture
content in the compost mixtures decreased as the
mixtures were exposed to higher levels of light and
the highest levels of dehydration occurred in the
dairy compost mixtures followed by the swine com-
post mixtures (Table 5). In addition to affecting the
moisture content of the compost mixtures, light ex-
posure led to decreased pH in the chicken and cow
compost mixtures (Table 5). L. monocytogenes sur-
vival could have been improved under less alkaline
conditions; however, that response was likely to be
minimal in these compost mixtures due to the con-
current stress imposed by low moisture contents and
light exposure. In the case of Salmonella, however,
it is known that it is extremely resistant to desiccation
(Pedersen et al., 2008; Tamura et al., 2009). Given
that dehydration has induced cross-tolerance to a
number of other stressors (Gruzdev et al., 2011), such
a state could also have been responsible for our in-
ability to discern an effect of light on inactivation of
Salmonella in the swine or cow compost mixtures.
In summary, both Salmonella and L. monocyto-
genes may survive in compost mixtures that are ex-
posed to sublethal temperatures for extended pe-
riods of time. As ambient temperatures increased,
the persistence of pathogens decreased which
may be attributed to increased competitive activity
by the more dominant indigenous microflora. At-
tempts to maintain the moisture content of compost
mixtures on a weekly basis was challenging because
rapid evaporation resulted in very dry mixtures in
most cases. Under these conditions, L. monocyto-
genes appeared to be more susceptible to desicca-
tion stress than Salmonella based on their relative re-
duction in populations in chicken and cow compost
mixtures over time. L. monocytogenes populations
also decreased more rapidly when compost mix-
tures were exposed to light conditions, described as
sunny or cloudy, compared to dark conditions, but
Table 5. % Moisture content and pH in compost mixtures held at 20°C and exposed to different light conditions across all five storage time periods examined with each type of manure1, 2.
% Moisture3 pH3
Manure source Dark Cloudy4 Sunny5 Dark Cloudy Sunny
Chicken 45.6 ± 24.4 a 33.8 ± 17.1 b 25.0 ± 16.8 b 9.5 ± 0.3 a 9.3 ± 0.3 ab 9.2 ± 0.6 b
Dairy cow 32.1 ± 19.9 a 21.4 ± 13.4 b 12.3 ± 5.7 c 8.7 ± 0.6 a 8.1 ± 0.8 b 8.3 ± 0.5 b
Swine 35.1 ± 17.2 a 29.9 ± 12.5 a 19.9 ± 13.5 b 9.0 ± 0.5 a 9.0 ± 0.5 a 9.0 ± 0.4 a
1 Data were collected from fourth experimental study.2 Swine, chicken, and dairy cow compost mixtures were stored for 4, 12, and 18 weeks, respectively.3 Values in each row of an attribute followed by a different letter are significantly different (P < 0.05).4 Compost mixtures were exposed daily to light conditions of 289 to 359 μmol/m2/sec for 12 h and to dark
conditions for 12 h.5 Compost mixtures were exposed daily to light conditions of 524 to 573 μmol/m2/sec for 12 h and to dark
conditions for 12 h.
236 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
this response did not occur with Salmonella in cow
or swine compost mixtures. It is suggested that the
drier conditions encountered in light-exposed cow
and swine compost mixtures may have induced a
cross-tolerance response by Salmonella to the light
stress. If cross-tolerance responses by Salmonella
are generated in low moisture compost mixtures
held at sublethal temperatures, it will therefore be
important to apply an intervention treatment to
those compost mixtures prior to the activation of
that response.
ACKNOWLEDGEMENTS
The project was supported by the National Re-
search Initiative of the USDA Cooperative State
Research, Education, and Extension Service, grant
# 2008-35201-18658. We gratefully thank Derrick
Huckaby, Lindsey Davey, and Jessica Colvin for tech-
nical assistance.
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regulations on pathogen inactivation during com-
posting. J. Environ. Engr. Sci. 6:573-586.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 239
240 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.
VOLUME 4 ISSUE 1
Preventing Post-Processing Contamination in a Food Nugget Processing Line When Lan-guage Barriers ExistJ. A. Neal, C. A. O’Bryan and P. G. Crandall
20
A Personal Hygiene Behavioral Change Study at a Midwestern Cheese Production PlantJ. A. Neal, C. A. O’Bryan and P. G. Crandall
13
Behavioral Change Study at a Western Soup Production Plant
C. A. O’Bryan, J. A. Neal, and P. G. Crandall
27
Salmonella in Cantaloupes: You Make Me Sick!B. A. Almanza
35
The Hurricane Sandy DilemmaB. A. Almanza
43
Introduction Special IssueP. G. Crandall
8
Case Studies
Instructions for Authors61
Introduction to Authors
Intellect-u-ale: A Smart Approach to Quality Assurance in a Micro-BreweryA. J. Corsi, M. Goodman, and J. A. Neal
50
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 241
Antibiotic Use in Livestock ProductionBroadway, P. R., J. A. Carroll, and T. R. Callaway
76
Effects of Co-nutrients in Foods and Bioremediation in the Environment on Methylmercury
P. G. Crandall, C. A. O’Bryan
86
Alternative antimicrobial supplements that positively impact animal health and food safety Broadway, P. R., J. A. Carroll, and T. R. Callaway
109
Human Health Benefits of Isoflavones from Soybeansk. Kushwaha, C. A. O’Bryan, D. Babu, P. G. Crandall, P. Chen, and S.-O. Lee
122
REVIEW
Contribution of Chemical and Physical Factors to Zoonotic Pathogen Inactivation during Chicken Manure CompostingM.C. Erickson, J. Liao, X. Jiang, and M.P. Doyle
96
ARTICLES
Instructions for Authors147
Introduction to Authors
The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.
VOLUME 4 ISSUE 2
242 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 243
MANUSCRIPT SUBMISSION
Authors must submit their papers electronically
([email protected]). According to instruc-
tions provided online at our site: www.afabjournal.
com. Authors who are unable to submit electroni-
cally should contact the editorial office for assistance
by email at [email protected].
INSTRUCTIONS TO AUTHORS
• Aerobic microbiology
• Aerobiology
• Anaerobic microbiology
• Analytical microbiology
• Animal microbiology
• Antibiotics
• Antimicrobials
• Bacteriophage
• Bioremediation
• Biotechnology
• Detection
• Environmental microbiology
• Feed microbiology
• Fermentation
• Food bacteriology
• Food control
• Food microbiology
• Food quality
• Food Safety
• Foodborne pathogens
• Gastrointestinal microbiology
• Microbial education
• Microbial genetics
• Microbial physiology
• Modeling and microbial kinetics
• Natural products
• Phytoceuticals
• Quantitative microbiology
• Plant microbiology
• Plant pathogens
• Prebiotics
• Probiotics
• Rumen microbiology
• Rapid methods
• Toxins
• Veterinary microbiology
• Waste microbiology
• Water microbiology
CONTENT OF MANUSCRIPT
We invite you to consider submitting your re-
search and review manuscripts to AFAB. The jour-
nal serves as a peer reviewed scientific forum for to
the latest advancements in bacteriology research
on Agricultural and Food Systems which includes
the following fields:
244 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
With an open access publication model of this
journal, all interested readers around the world can
freely access articles online. AFAB publishes origi-
nal papers including, but not limited to the types
of manuscripts described in the following sections.
Papers that have been, or are scheduled to be, pub-
lished elsewhere should not be submitted and will
not be reviewed. Opinions or views expressed in pa-
pers published by AFAB are those of the author(s)
and do not necessarily represent the opinion of the
AFAB or the editorial board.
MANUSCRIPT TYPES
Full-Length Research Manuscripts
AFAB accepts full-length research articles con-
taining four (4) figures and/or tables or more. AFAB
emphasizes the importance of sound scientific ex-
perimentation on any of the topics listed in the focus
areas followed by clear concise writing that describes
the research in its entirety. The results of experi-
ments published in AFAB must be replicated, with
appropriate statistical assessment of experimental
variation and assignment of significant difference.
Major headings to include are: Abstract, Introduc-tion, Materials and Methods, Results, Discussion (or Results and Discussion), Conclusion, Acknowl-edgements (optional), Appendix for abbreviations (optional), and References.
Manuscripts clearly lacking in language will be re-
turned to author without review, with a suggestion
that English editing be sought before the paper is
reconsidered. AFAB offers a fee based language
service upon request. Please contact [email protected] for more information about our fees
and services.
Rapid Communications
Under normal circumstances, AFAB aims for re-
ceipt-to-decision times of approximately one month or less. Accepted papers will have priority for publi-
cation in the next available issue of AFAB. However,
if an author chooses or requires a much more rapid
peer review, the journal editorial office has the capa-
bility to shorten the review timing to one week or less.
Any type of manuscript whether it be a full length
manuscript, brief communication or review paper can
be submitted as a rapid communication. There will be
additional costs for processing and page charges will
be double the normal rate. Authors who choose this
option must select Rapid Communications as the pa-
per type when submitting the paper and the editors
will judge whether a rapid review is possible and let
the author know immediately.
Brief Communications
Brief communications are short research notes giv-
ing the results of complete experiments but are con-
sidered less comprehensive than full-length articles
with three (3) figures and/or tables or less. Manuscripts
should be prepared with the same subheadings as full
length research papers. The running head above the
title of the paper is “Brief Communications.”
Unsolicited Review Papers
Review papers are welcome on any topic listed in
the focus section and have no page limits. Reviews
are assessed the same pages charges as all other
manuscripts. All AFAB guidelines for style and form
apply. Major headings to include are: Abstract, In-troduction, Main discussion topics and appropri-ate subheadings, Conclusions, Acknowledgements (optional) and References. Review papers shorter
than 20 pages of double spaced text and references
will be considered mini-reviews with the subhead-
ing above the title on the first page. The running
head above the title of the paper is either “Review”
or “Mini-review”.
Solicited Review Papers
Solicited reviews will have no page limits. The
editor-in-chief will send invitations to the authors
and then review these contributions when they are
submitted. Nominations or suggestions for potential
timely reviews are welcomed by the editors or edito-
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 245
rial board members and should be sent to submit@
afabjournal.com. There will be no page charges for
solicited review papers but the solicitation must origi-
nate from the editor-in-chief or one of the editors. Re-
quests from authors will automatically be classified as
unsolicited review papers. The running head above
the title of the paper will be “Invited Review.”
Conference and Special Issues Reviews
AFAB welcomes opportunities to publish papers
from symposia, scientific conference, and/or meet-
ings in their entirety. Conference organizers need
simply to contact AFAB at [email protected]
and a rapid decision is guaranteed. If in agreement,
the conference organizers must guarantee delivery
of a set number of peer reviewed manuscripts within
a specified time and submitted in the same format
as that described for unsolicited review papers. Con-
ference papers must be prepared in accordance with
the guidelines for review articles and are subject to
peer review. The conference chair must decide
whether or not they wish to serve as Special Issue
Editor and conduct the editorial review process. If
the conference chair/organizer chooses to serve as
special issue editor, this will involve review of the pa-
pers and, if necessary, returning them to the authors
for revision. The conference organizer then submits
the revised manuscripts to the journal editorial of-
fice for further editorial examination. Final revisions
by the author and recommendations for acceptance
or rejection by the chair must be completed by a
mutually agreed upon date between the editor and
the conference organizer. Manuscripts not meeting
this deadline will not be included in the published
symposium proceedings but if submitted later can
still be considered as unsolicited review papers. Al-
though offprints and costs of pages are the same
as for all other papers, the symposium chair may be
asked to guarantee an agreed upon number of hard
copies to be purchased by conference attendees. If
the decision is not to publish the symposium as a
special issue, the individual authors retain the right
to submit their papers for consideration for the jour-
nal as ordinary unsolicited review manuscripts.
Book Reviews
AFAB publishes reviews of books considered to
be of interest to the readers. The editor-in-chief ordi-
narily solicits reviews. Book reviews shall be prepared
in accordance to the style and form requirements of
the journal, and they are subject to editorial revision.
No page charges will be assessed solicited reviews
while unsolicited book reviews will be assigned the
regular page charge rate.
Opinions and Current Viewpoints
The purpose of this section will be to discuss, cri-
tique, or expand on scientific points made in articles
recently published in AFAB. Drafts must be received
within 6 months of an article’s publication. Opinions
and current perspectives do not have page limits.
They shall have a title followed by the body of the
text and references. Author name(s) and affiliation(s)
shall be placed between the end of the text and list
of references. If this document pertains to a par-
ticular manuscript then the author(s) of the original
paper(s) will be provided a copy of the letter and of-
fered the opportunity to submit for consideration a
reply within 30 days. Responses will have the same
page restrictions and format as the original opinion
and current viewpoint, and the titles shall end with
“Opinions.” They will be published together. Letters
and replies shall follow appropriate AFAB format
and may be edited by the editor-in-chief and a tech-
nical editor. If multiple letters on the same topic are
received, a representative set of opinions concern-
ing a specific article will be published. A disclaimer
will be added by the editorial staff that the opinion
expressed in this viewpoint is the authors alone and
does not necessarily represent the opinion of AFAB
or the editorial board.
COPYRIGHT AGREEMENT
The copyright form is published in AFAB as space
permits and is available online (www.afabjournal.com).
246 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
AFAB grants to the author the right of re-publication
in any book of which he or she is the author or edi-
tor, subject only to giving proper credit to the original
journal publication of the article by AFAB. AFAB re-
tains the copyright to all materials accepted for pub-
lication in the journal. If an author desires to reprint
a table or figure published from a non-AFAB source,
written evidence of copyright permission from an au-
thority representing that source must be obtained by
the author and forwarded to the AFAB editorial office.
PEER REVIEW PROCESS
Authors will be requested to provide the names
and complete addresses including emails of five (5) potential reviewers who have expertise in the research
area and no conflict of interest with any of the authors.
Except for manuscripts designated as Rapid Commu-
nication each reviewer has two (2) weeks to review
the manuscript, and submit comments electronically
to the editorial office. Authors have three (3) weeks
to complete the revision, which shall be returned to
the editorial office within six (6) weeks after which the
authors risk having their manuscript removed from
AFAB files if they fail to ask the editorial office for
an extension by email. Deleted manuscripts will be
reconsidered, but they will have to be processed as
new manuscripts with an additional processing fee as-
sessed upon submission. Once reviewed, the author
will be notified of the outcome and advised accord-
ingly. Editors handle all initial correspondence with
authors during the review process. The editor-in chief
will notify the author of the final decision to accept or
reject. Rejected manuscripts can be resubmitted only
with an invitation from the editor or editor-in chief. Re-
vised versions of previously rejected manuscripts are
treated as new submissions.
PRODUCTION OF PROOFS
Accepted manuscripts are forwarded to the edito-
rial office for technical editing and layout. The manu-
script is then formatted, figures are reproduced, and
author proofs are prepared as PDFs. Author proofs
of all manuscripts will be provided to the correspond-
ing author. Author proofs should be read carefully and
checked against the typed manuscript, because the
responsibility for proofreading is with the author(s).
Corrections must be returned by e-mail. Changes
sent by e-mail to the technical editor must indicate
page, column, and line numbers for each correction
to be made on the proof. Corrections can also be
marked using “track changes” in Microsoft Word or
using e-annotation tools for electronic proof correc-
tion in Adobe Acrobat to indicate necessary chang-
es. Author alterations to proofs exceeding 5% of the
original proof content will be charged to the author. All
correspondence of proofs must be agreed to by the
editorial office and the author within 48 hours or proof
will be published as is and AFAB will assume no re-
sponsibility for errors that result in the final publication.
PUBLICATION CHARGES
AFAB has two publication charge options: conven-
tional page charges and rapid communication. The
current charge for conventional publication is $25 per printed page in the journal. There is no additional
charge for the publication of pages containing color
images, micrographs or pictures. For authors who
wish to have their papers processed as a rapid com-
munication, authors will pay the rapid communication
fee when proofs are returned to the editorial office
in addition to twice the conventional page charges.
Charges for rapid communications are $1000 per manuscript for guaranteed peer review within one
week and $100 per journal page.
HARD COPY OFFPRINTS
If you are wishing to obtain a physical hard copy of
the AFAB journal, offprints are available in any quan-
tity at an additional charge: $100/page for black-white
and $150/page for color prints. You may order your
offprints at any time after publication on our website.
Scientific conference organizers may be expected to
agree to a set number of offprints as a part of their
agreement with AFAB.
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 247
MANUSCRIPT CONTENT REQUIREMENTS
Preparing the Manuscript File
Manuscripts must be written in grammatically
correct English. AFAB offers a fee based language
service upon request ([email protected]).
Manuscripts should be typed double-spaced, with
lines and pages numbered consecutively. All docu-
ments must be submitted in Microsoft Word (.doc or
.docx, PC or Mac). All special characters (e.g., Greek,
math, symbols) should be inserted using the sym-
bols palette available in this font. Tables and figures
should be placed in separate sections at the end of
the manuscript (not placed in the text). Failure to fol-
low these instructions will cause delays of the pro-
cessing and review of the manuscript.
Title Page
At the very top of the title page, include a title of
not more than 100 characters. Format the title with
the first letter of each word capitalized. No abbre-
viations should be used. Under the title, the authors
names are listed. Use the author’s initials for both first
and middle names with a period (full-stop) between
initials (e.g., W. A. Afab). Underneath the authors, a
list affiliations must be listed. Please use numerical
superscripts after the author’s names to designate
affiliation. If an authors address has changed since
the research was completed, this new information
must be designated as “Current address:”. The cor-
responding author should be indicated with an aster-
isk e.g., * Corresponding author. The title page shall
include the name and full address of the correspond-
ing author. Telephone and e-mail address must also
be provided for the corresponding author, and email-addresses must be provided for all authors.
Editing
Author-derived abbreviations should be defined
at first use in the abstract and again in the body of
the manuscript. If abbreviations are extensive au-
thors may need to provide a list of abbreviations
at the beginning of the manuscript. In vivo, in vitro
and bacterial names must be italicized (obligatory).
Authors must avoid single sentence paragraphs and
merge such paragraphs appropriately. Authors must
not begin sentences with “Figure or Table shows…”
as these are inanimate objects and cannot “show”
anything. When number are reported in text or in ta-
bles, always put a zero in front of decimal numbers:
“0.10” instead of “.10”.
MANUSCRIPT SECTIONS
Abstract
The abstract provides an abridged version of the
manuscript. Please submit your abstract on a sepa-
rate page after the title page. The abstract should
provide a justification of your work, objectives, meth-
ods, results, discussion and implications of study or
review findings . Your abstract must consist of com-
plete sentences without references to other work or
footnotes and must not exceed 250 words. On the
same page as your abstract, please provide at least ten (10) keywords to be used for linking and index-
ing. Ideally, these keywords should include signifi-
cant words from the title.
Introduction
The introduction should clearly present the foun-
dation of the manuscript topic and what makes the
research or the review unique. The introduction
should validate why this topic is important based on
previously published literature, and the relevance of
the current research. Overall goals and project ob-
jectives must be clearly stated in the final sentence
of the last paragraphs of the introduction.
Materials and Methods
Information on equipment and chemicals used
must include the full company name, city, and state
(country if outside the United States or Province if
in Canada) [i.e., (Model 123, ACME Inc., Afab, AR)].
248 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Variability, Replication, and Statistical Analysis
To properly assess biological systems indepen-
dent replication of experiments and quantification
of variation among replicates is required by AFAB.
Reviewers and/or editors may request additional
statistical analysis depending on the nature of the
data and it will be the responsibility of the authors
to respond appropriately. Statistical methods com-
monly used in the bacteriology do not need to be
described in detail, but an adequate description
and/or appropriate references should be provided.
The statistical model and experimental unit must
be designated when appropriate. The experimen-
tal unit is the smallest unit to which an individual
treatment is imposed. For bacterial growth stud-
ies, the average of replicate tubes per single study
per treatment is the experimental unit; therefore,
individual studies must be replicated. Repeated
time analyses of the same sample usually do not
constitute independent experimental units. Mea-
surements on the same experimental unit over time
are also not independent and must not be consid-
ered as independent experimental units. For analy-
sis of time effects, assess as a rate of change over
time. Standard deviation refers to the variability
in the biological response being measured and is
presented as standard deviation or standard error
according to the definitions described in statistical
references or textbooks.
Results
Results represent the presentation of data in
words and all data should be described in same
fashion. No discussion of literature is included in
the results section.
Discussion
The discussion section involves comparing the
current data outcomes with previously published
work in this area without repeating the text in the
results section. Critical and in-depth dialogue is
encouraged.
Results and Discussion
Results and discussion can be under combined or
separate headings.
Conclusions
State conclusions (not a summary) briefly in one
paragraph.
Acknowledgments
Acknowledgments of individuals should include
institution, city, and state; city and country if not U.S.;
and City or Province if in Canada. Copies being re-
viewed shall have authors’ institutions omitted to re-
tain anonymity.
References
a) Citing References In Text
Authors of cited papers in the text are to be pre-
sented as follows: Adams and Harry (1992) or Smith
and Jones (1990, 1992). If more than two authors of
one article, the first author’s name is followed by the
abbreviation et al. in italics. If the sentence structure
requires that the authors’ names be included in pa-
rentheses, the proper format is (Adams and Harry,
1982; Harry, 1988a,b; Harry et al., 1993). Citations to a
group of references should be listed first alphabeti-
cally then chronologically. Work that has not been
submitted or accepted for publication shall be listed
in the text as: “G.C. Jay (institution, city, and state,
personal communication).” The author’s own un-
published work should be listed in the text as “(J.
Adams, unpublished data).” Personal communica-
tions and unsubmitted unpublished data must not
be included in the References section. Two or more
publications by the same authors in the same year
must be made distinct with lowercase letters after
the year (2010a,b). Likewise when multiple author ci-
tations designated by et al. in the text have the same
first author, then even if the other authors are differ-
ent these references in the text and the references
section must be identified by a letter. For example
Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014 249
“(James et al., 2010a,b)” in text, refers to “James,
Smith, and Elliot. 2010a” and “James, West, and Ad-
ams. 2010b” in the reference section.
b) Citing References In Reference Section
In the References section, references are listed in
alphabetical order by authors’ last names, and then
chronologically. List only those references cited in the
text. Manuscripts submitted for publication, accepted
for publication or in press can be given in the refer-
ence section followed by the designation: “(submit-
ted)”, “(accepted)’, or “(In Press), respectively. If the
DOI number of unpublished references is available,
you must give the number. The year of publication fol-
lows the authors’ names. All authors’ names must be
included in the citation in the Reference section. Jour-
nals must be abbreviated. First and last page num-
bers must be provided. Sample references are given
below. Consult recent issues of AFAB for examples
not included in the following section.
Journal manuscript:
Examples:
Chase, G., and L. Erlandsen. 1976. Evidence for a
complex life cycle and endospore formation in the
attached, filamentous, segmented bacterium from
murine ileum. J. Bacteriol. 127:572-583.
Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van
Doesburg, and A. J. M. Stams. 2009. A typical
one-carbon metabolism of an acetogenic and
hydrogenogenic Moorella thermioacetica strain.
Arch. Microbiol. 191:123-131.
Book:
Examples:
Hungate, R. E. 1966. The rumen and its microbes
Academic Press, Inc., New York, NY. 533 p.
Book Chapter:
Examples:
O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010.
Assessing consumer concerns and perceptions
of food safety risks and practices: Methodologies
and outcomes. In: S. C. Ricke and F. T. Jones. Eds.
Perspectives on Food Safety Issues of Food Animal
Derived Foods. Univ. Arkansas Press, Fayetteville,
AR. p 273-288.
Dissertation and thesis:
Maciorowski, K. G. 2000. Rapid detection of Salmo-
nella spp. and indicators of fecal contamination
in animal feed. Ph.D. Diss. Texas A&M University,
College Station, TX.
Donalson, L. M. 2005. The in vivo and in vitro effect
of a fructooligosacharide prebiotic combined with
alfalfa molt diets on egg production and Salmo-
nella in laying hens. M.S. thesis. Texas A&M Uni-
versity, College Station, TX.
Van Loo, E. 2009. Consumer perception of ready-to-
eat deli foods and organic meat. M.S. thesis. Uni-
versity of Arkansas, Fayetteville, AR. 202 p.
Web sites, patents:
Examples:
Davis, C. 2010. Salmonella. Medicinenet.com.
http://www.medicinenet.com/salmonella /article.
htm. Accessed July, 2010.
Afab, F. 2010, Development of a novel process. U.S.
Patent #_____
Author(s). Year. Article title. Journal title [abbreviated].
Volume number:inclusive pages.
Author(s) [or editor(s)]. Year. Title. Edition or volume (if
relevant). Publisher name, Place of publication. Number
of pages.
Author(s) of the chapter. Year. Title of the chapter. In:
author(s) or editor(s). Title of the book. Edition or vol-
ume, if relevant. Publisher name, Place of publication.
Inclusive pages of chapter.
Author. Date of degree. Title. Type of publication, such
as Ph.D. Diss or M.S. thesis. Institution, Place of institu-
tion. Total number of pages.
250 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 4, Issue 3 - 2014
Abstracts and Symposia Proceedings:
Fischer, J. R. 2007. Building a prosperous future in
which agriculture uses and produces energy effi-
ciently and effectively. NABC report 19, Agricultural
Biofuels: Tech., Sustainability, and Profitability. p.27
Musgrove, M. T., and M. E. Berrang. 2008. Presence
of aerobic microorganisms, Enterobacteriaceae and
Salmonella in the shell egg processing environment.
IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10)
Vianna, M. E., H. P. Horz, and G. Conrads. 2006. Op-
tions and risks by using diagnostic gene chips. Pro-
gram and abstracts book , The 8th Biennieal Con-
gress of the Anaerobe Society of the Americas. p.
86 (Abstr.)
Data Presentation in Tables and Figures
Figures and tables to be published in AFAB must
be constructed in such a fashion that they are able
to “stand alone” in the published manuscript. This
means that the reader should be able to look at
the figure or table independently of the rest of the
manuscript and be able to comprehend the experi-
mental approach sufficiently to interpret the data.
Consequently, all statistical analyses should be very
carefully presented along with variation estimates
and what constitutes an independent replication
and the number of replicates used to calculate the
averages presented in the table or figure.
Each table and figure must be on a separate
page in the submitted paper. In addition, you will
need to submit all data for charts, tables and
figures in native format when possible (e.g., Mi-
crosoft Excel, Powerpoint). Photographs should
be submitted as high-resolution (600 dpi) .jpg or
tif. files. All figures should be clearly presented with
well defined axis and units of measurement. Sym-
bols, lines, and bars must be made distinct as “stand
alone” black and white presentations. Stippling,
dashed lines etc. are encouraged for multiple com-
parison but shades of gray are discouraged. Color
images, micrographs, pictures are recommended
and there is no additional fee for their submission.
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