bacterial survivors of agrowastes anaerobiosis: …

Ndubuisi-Nnaji et al: Bacterial Survivors of Agrowastes Anaerobiosis: Their Hemolytic Activity and Antibiotic Susceptibility Patterns

World Journal of Applied Science and Technology, Vol. 10 No. 1 (2018). 86 - 90 86

ISSN: 2141 – 3290 www.wojast.com

BACTERIAL SURVIVORS OF AGROWASTES ANAEROBIOSIS: THEIR HEMOLYTIC ACTIVITY AND ANTIBIOTIC

SUSCEPTIBILITY PATTERNS NDUBUISI-NNAJI, U. U., *OFON, U. A.

AND GEORGE, Q. S. Department of Microbiology, University of Uyo, AkwaIbom State

[email protected]

ABSTRACT This study evaluated the effect of anaerobic digestion on the bacteriological quality, hemolytic activity and antibiotics susceptibility patterns of heterotrophic bacteria and anaerobic bacteria, indicator organisms (total coliforms, faecal coliforms) and putative pathogens (Salmonella, Shigella,Staphylococcusaureus and Vibrio species) in agro-industrial wastes.Experiments were conducted in four batch reactors treating palm oil mill effluent and brewery wastes (spent grains) with different inocula:cow dung,pig slurry and poultry droppings at thermophilic temperature (400C) for 30 days. Anaerobic digestion showed a significant reduction (p<0.05) in indigenous populations of total heterotrophic bacteria (69%), total anaerobic bacteria (24%), total coliforms (45.31%) and faecal coliform (47.15%).Similarly, there were notable reductions(p<0.05)in population of Salmonella-Shigella(41.86%),S. aureus (45.58%) and undetectable levels of Vibrio species(100%). Hemolytic assay of isolated bacteria indicated that 28.8% were α-hemolytic, 56.2% γ-hemolytic (non-pathogenic) and 15.1%β-hemolytic. Generally, antibiotic resistance of isolates was very low, except for a few species of Bacillus, Clostridium and Salmonella. Although anaerobic digestion remains a veritable tool for the reduction and/or elimination of pathogens, the presence of indicator/pathogenic bacteria above recommended limits (≤ 3.0 log CFUg-1) suggests that further treatment be considered before land application of digestate as biological soil amendment in order to reduce or eliminate health risk associated with these pathogens.

INTRODUCTION

Anaerobic digestate is the resultant material following anaerobic digestion of biodegradable materials. It is a nutrient rich materialconsisting of left over indigestible materials like animal faeces, dead or living organisms and plant remains. It has been acknowledged as sustainable potential alternatives to conventional inorganic and other undigested organic fertilizers (Coelho et al., 2018).However, digestates are not innocuous products (Liu et al., 2018), their safety measured by the concentration of pathogens present is of great concern to its users as pathogens like species of Clostridium,Salmonella, Shigella,Klebsiella, Escherichia colietc., may contaminate the biogas slurry. Some of these pathogens are hardly destroyed during anaerobic digestion as they become hardy resisting the prevailing milieu in the digesters. The safe application of digestates to soils and crops may be limited by the potential risk of pathogen contamination. This study therefore seeks to add to the existing knowledge pool on the effect of anaerobic digestion on the persistence and antibiotic susceptibility patterns of bacteria associated with anaerobic digestate from codigestion of palm oil mill effluent (POME) and brewery spent grain (BSG) amended with livestock manure. It also appraises the haemolytic patterns of these pathogens which based on published data are hitherto unavailable.

MATERIALS AND METHODS Experimental design and sample collection Four laboratory scale batch bioreactors operating at mesophilic temperature (40oC) with a working volume of 100 ml over a hydraulic retention time of 30 days were employed in the study. The reactors were labelled A – D as follows: Batch A = 10g BSG + 55ml POME + 10g cow dung; Batch B = 10g BSG + 55ml POME + 5g pig dung; Batch C = 10g BSG +



55mlPOME + 10mg poultry dropping; Batch D (control) = 10g BSG + 55ml POME. The description of the bioreactors is as detailed in Eduoket al. (2018). While Brewery Spent Grain (BSG) was collected from Champion Brewery PLC, Palm Oil Mill Effluent (POME) was obtained from a local milland Fresh livestock manure (cow dung, pig slurry and poultry dropping) were collected from an integrated farm all located within Uyo in Akwa Ibom State, Nigeria. Accordingly the digesters were charged with the homogenous mixture of BSG, POME and livestock manure in the ratio of 1:5:1. Bacteriological Analysis Sampling was conducted at the initial time (t0) and final time (tf) for all digesters. A total of 8 samples, (4 influent and 4 effluent)were collected, serially diluted and cultivated on nutrient agar (Oxoid, UK) and Schaedler agar media (Oxoid, UK) using pour plate method ([UP1]de Diaz et al., 2016) for the estimation of total heterotrophic and total anaerobic bacteria loads respectively. Potential pathogens of interest: Staphylococcus, Vibrio, Salmonella species, Escherichiacoli (faecal coliform)and other coliforms were isolated on various selective media including Mannitol Salt Agar, Thiosulphate Citrate Bile Salt Sucrose Agar, Salmonella Shigella Agar, Eosin Methylene Blue Agar and McConkey Agar respectively. Haemolysin Production Assay of Bacterial Isolates This test was carried out using the blood agar to assay for the production of haemolysin. The plates were streaked with a loopful of the bacterial cultures and incubated at 37oC for 24 hours. Clear zones around bacterial colonies indicated haemolysin production. Antibiotic Susceptibility Assay of Bacterial Isolates This was carried out and interpreted using Modified Kirby-Bauer disc diffusion technique as described by Cheesbrough (2006). Commercially available discs (Rapid Labs CM-12-8NR100)were selected on the basis of their clinical relevance as follows: Amoxicillin, Streptomycin, Norfloxacin, Chloramphenicol, Ciprofloxacin, Levofloxacin, Erythromycin, Gentamycin, Ampiclox and Rifampicin for gram positive bacteria;Tarivid, Peflacin, Ciprofloxacin, Augmentin, Gentamycin, Streptomycin, Ceporex, Nalidixic acid, Septrin and Ampicillin for gram negative bacteria. Theywere placed on the surface of the Mueller Hinton agar plates and incubated at 37°C for 24 hours. The zones of inhibition were measured and recorded as S = Sensitive/Susceptible (≥ 21 mm); R = Resistant (≤15 mm); I = Intermediate (16 to 20 mm) (Cheesbrough, 2006).

RESULTS AND DISCUSSION The result of the bacterial load profile of the feedstock and digestate as presented in Figure 1a and 1b revealed a general decrease in the loadsof all the bacterial groups after digestion. Notably, anaerobic treatment resulted in undetectable levels (100 % reduction) of Vibrio species. Simultaneously, a decline in total coliforms (45.31 %), faecal coliform (47.15 %), SalmonellaShigella count (41.86%) and Staphylococcus count (45.58 %) were also observed across all digesters. This marked reduction in bacterial loadsranging from 0.41 to 1.0 log CFU/ml was statistically significant (P< 0.05) and substantiates the earlier report of Alfa et al. (2014). However it varied with the report of Cote et al. (2006) who attributed to the differencesto variation in waste composition, environmental factors, digester design and prevailing operating digester condition. Haemolytic activity and antibiotic susceptibility patterns of the putative pathogens isolated from feedstock and digestate samples as shown in Tables 1, 2a and 2b revealed that 52.6% of the isolates did not produce hemolysin (γ-hemolytic) while only 15.1% produced beta (β) haemolysin and 28.8% produced alpha (α) haemolysin. Based on their haemolytic activity Aeromonas sp., Shigella sp., Pseudomonas aeruginosa, Streptococcus sp., Enterococcus faecium and Enterobactersp were the principal pathogens isolated from the waste as they produced beta-haemolysin with zones of complete clearance on blood agar.



The antibiotic susceptibility patterns of gram positive and gram negative bacteria are shown in Tables 2a and 2b respectively. Precisely four strains (5.48 %) of bacteria were resistant to at least one of the tested antibiotics.

Table 1: Haemolytic Patterns of Bacteria Isolated from Feedstock and Digestate Samples

Bacterial Isolates

Total Number Tested

Haemolytic Patterns of Isolates No (%) Alpha (α) Beta (β) Gamma (γ)

Clostridiumsp 6 1(16.7) 0 5(83.3) Bacillussp 4 1(25.0) 0 3(75.0) S. aureus 4 4(100) 0 0 S. epidermidis 4 0 0 4(100) Bacillus megaterium 8 2(25.0) 0 6(75.0) Lactobacillus sp 4 1(25.0) 0 3(75.0) Micrococcus sp 4 3(75.0) 0 1(25.0) Aeromonassp 3 0 2(66.7) 1(33.3) Salmonella sp 8 3(37.5) 0 5(62.5) Shigella sp 1 0 1(100) 0 P. aeruginosa 5 1(20.0) 1(20.0) 3(60.0) P. fluorescens 3 1(33.3) 0 2(66.7) Streptococcus sp 2 0 2(100) 0 Citrobacter sp 2 1(50.0) 0 1(50.0) Klebsiellasp 2 0 0 2(100) Enterococcus faecium 2 0 2 0 Enterobacter sp 7 1(14.3) 1(14.3) 5(71.4) Vibrio cholerae 4 2(50.0) 2(50.0) 0 Total 73 21(28.8) 11(15.1) 41(56.2)

The four resistant strains were represented by Clostridium, Streptococcus and Salmonella species and their resistance was observed for Chloramphenicol (2.5 %), Rifampicin (4.88 %) and Tarivid (3.13 %). On the contrary, 94.5 % of the isolates were highly sensitive (100 %) to all the commercial antibiotics under evaluation. However, the persistence of P. aeruginosa and Enterobacter species after (anaerobic digestion (AD) process was not of concern as the encountered pathogens were not resistant but highly (100%) sensitive to all the common antibiotics (Ciprofloxacin , Gentamycin and Ampicillin ) tested thus presenting the digestate as an innocuous substance that would pose no environmental risk. This finding did not corroborate Resende et al. (2014) who detected the prevalence of multiple drug resistant (MDR) bacteria in influent and effluent samples from biodigesterstreating cattle manure.

Figure 1a: Figure 1b:



Table 2a: Antibiotic Susceptibility Patterns of Gram Positive Bacterial Isolates Bacterial Isolates Total Number

Tested Antibiotic Susceptibility Patterns No (%) AMX S NB CH CPX LEV E CN APX RD

Clostridiumsp 6 6(100) 6(100) 6(100) 5(83.3) 6(100) 6(100) 6(100) 6(100) 6(100) 5(83.3) Bacillus sp 4 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 3(75.0) 4(100) 4(100) 4(100) S. aureus 4 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) S. epidermidis 4 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) B. megaterium 8 8(100) 8(100) 8(100) 8(100) 8(100) 8(100) 8(100) 8(100) 8(100) 8(100) Lactobacillus sp 4 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) Micrococcus sp 4 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) 4(100) Aeromonassp 3 3(100) 3(100) 3(100) 3(100) 3(100) 3(100) 3(100) 3(100) 3(100) 3(100) Streptococcus sp 2 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 1(50.0) Enterococcus faecium 2 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) Total 41 41(100) 41(100) 41(100) 40(97.6) 41(100) 41(100) 41(100) 41(100) 41(100) 39(95.1) Key: AMX= Amoxicillin, S= Streptomycin, NB= Norfloxacin, CH= Chloramphenicol, CPX= Ciproflox, LEV= Levofloxacin E= Erythromycin CN= Gentamycin, APX= Ampiclox, RD= Rifampicin Table 2b: Antibiotic Susceptibility Patterns of Gram Negative Bacterial Isolates Bacterial Isolates Total Number

Tested Antibiotic Susceptibility Patterns No (%) OFX PEF CPX AU CN S CEP NA SXT PN

Salmonella sp 8 7(87.5) 8(100) 8(100) 8(100) 8(100) 8(100) 8(100) 8(100) 8(100) 8(100) Shigella sp 1 1(100) 1(100) 1(100) 1(100) 1(100) 1(100) 1(100) 1(100) 1(100) 1(100) P. aeruginosa 5 5(100) 5(100) 5(100) 5(100) 5(100) 5(100) 5(100) 5(100) 5(100) 5(100) P. fluorescens 3 3(100) 3(100) 3(100) 3(100) 3(100) 3(100) 3(100) 3(100) 3(100) 3(100) Citrobacter sp 2 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) Klebsiellasp 2 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) 2(100) Enterobacter sp 7 7(100) 7(100) 7(100) 7(100) 7(100) 7(100) 7(100) 7(100) 7(100) 7(100) Vibrio cholerae 4 4(100) 4(100) 3(100) 3(100) 3(100) 4(100) 4(100) 4(100) 4(100) 4(100) Total 32 31(96.9) 32(100) 32(100) 32(100) 32(100) 32(100) 32(100) 32(100) 32(100) 32(100) KEY: OFX= Tarivid, PEF= Peflacin, CPX= Ciprofloxacin, AU= Augmentin, CN= Gentamycin, S= Streptomycin, CEP= Ceporex, NA= Nalidixic Acid, SXT= Septrin PN= Ampicilin



A few studies (Beneragama et al., 2011; Beneragama et al., 2012; Beneragama et al., 2013) have reported the prevalence of antimicrobial resistance among bacteria isolated from effluents of anaerobic digesters and their results suggest that temperature was critical in the reduction of drug resistant pathogens thussupporting the findings of this study.

CONCLUSION

The presence of pathogens above the European Union permissible limit for land application of digestate in agriculture calls for concern, hence further treatment should be encouraged. Though the study revealed that anaerobic digestate can be a source of virulent (beta-hemolytic) pathogens, antimicrobial resistance was not common among these pathogens. The inhibition of bacterial pathogens by antibiotics can help mitigate potential health risk posed by anaerobic digestate as soil amendment.

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Beneragama, N., Iwasaki, M., Lateef, S. A., Yamashiro, T., Ihara, I., Umetsu, K. (2012). The survival of multidrug-resistant bacteria in thermophilic and mesophilic anaerobic co-digestion of dairy manure and waste milk. Animal Science Journal, 84(5): 426 – 433.

Beneragama, N., Moriya, Y., Yamashiro, T., Iwasaki, M., Lateef, S. A., Ying, C. and Umetsu, K. (2013). The survival of cefazolin-resistant bacteria in mesophilic co-digestion of dairy manure and waste milk. Waste Management and Research, 31(8): 843 – 848.

Coelho, J. J., Prieto, M. L., Dowling, S., Hennessy, A., Casey, Woodcock, T. and Kennedy, N. (2018).Physical-chemical traits, phytotoxicity and pathogen detection in liquidanaerobic digestates.Waste Management, 78: 8 – 15.

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Cote, C., Masse, D. I. andQuessy, S. (2006). Reduction of indicator and pathogenic microorganismsby psychrophilic anaerobic digestion in swine slurries. Bioresource Technology, 97: 686 – 681.

de Diego-Díaz, B., Fernández-Rodríguez, J., Vitas, A. I. and Peñas, F. J. (2016). Biomethanization of solid wastes from the alcoholic beverage industry: malt and sloe. Kinetic and microbiological analysis. Chemical Engineering Journal, 33(2): 274 – 315.

Eduok, S., John, O., Ita, B., Inyang, E. and Coulon, F. (2018). Enhanced biogas production from anaerobic codigestion of lignocellulosic biomass and poultry feces using source separated human urine as buffering agent. Frontiers in Environmental Science, 6(67): 1 – 9.

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Resende, J. A., Silva, V. L., Oliveira, T. L. R., Fortunato, S. O.,Carneiro, J. C., Otenio, M. H., Diniz, C. G. (2014). Prevalence and persistence of potentially pathogenic and antibioticresistant bacteria during anaerobic digestion treatment of cattle manure. Bioresource Technology, 153: 284 – 291.

bacterial survivors of agrowastes anaerobiosis: …

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