cid

d g

Ch

rsity

form

egrad

when dosed with a concentration range from 1 to 20 g l of a synthetic mixture of volatile fatty acids (VFA). Biogas production from the VFA

mix and from reactors without VFA additions was used as a baseline control against which the results were compared and interpreted. The 1-l

Process Biochemistry 40 (200mesophilic (35 8C) reactors were seeded with an actively digesting sludge of sewage origin and monitored for biogas production, gascomposition, volatile fatty acid concentration, glucose content and the cellulolytic enzymes carboxymethylcellulase and avicelase. Cellulose

reduction was measured from initial and final samples of the reaction mix in each case. VFA caused inhibition of the cellulolytic activity at

concentrations 2 g l1, and therefore of the rate of cellulose hydrolysis. The fermentation of glucose was slightly inhibited at VFAconcentrations above 4 g l1. The inhibitory effect on the production of biogas and also on the methane to carbon dioxide ratio was evidentabove 6 g l1 VFA in the initial mixture when used as the sole substrate. In combination with paper as primary substrate, biogas productiondue to the paper was more than halved above 1 g l1 initial VFA, indicating inhibition of the hydrolysis process. Where glucose was theprimary substrate biogas production was more than halved above 8 g l1 which indicated that the fermentation was less sensitive to inhibitioncaused by VFA.

# 2005 Elsevier Ltd. All rights reserved.

Keywords: Anaerobic digestion; Cellulose; Glucose; Enzyme; VFA; Inhibition

1. Introduction

Anaerobic digestion involves a series of metabolic

reactions in which complex components in the feed are

sequentially reduced to a mixture of methane and carbon

dioxide as the principal end products. These reactions are

often simply referred to as: hydrolysis, fermentation and

methanogenesis [1,2]. The methanogenic phase is normally

considered the limiting step of the process due to the slow

growth rate of the methanogenic bacteria. Where the

substrate is particulate and comprises predominantly

cellulose then hydrolysis can be the controlling step in

the conversion process [3,4].

Various physicalchemical conditions affect the production

of methane, and inhibition of bacterial activity by either

substrate or productmaybe expectedwhen their concentrations

are increased to extremes. For example, high volatile fatty acid

(VFA) concentrations in the system cause the inhibition of

methanogenesis [1,58]. Under conditions of overloading and

in the presence of inhibitors, methanogenic activity cannot

remove hydrogen and volatile organic acids as quickly as they

are produced. The result is the accumulation of acids, the

depletion of buffering capacity and the depression of pH to

levels that also inhibit the hydrolysis/acidogenesis phase [9]. It

has also been shown that even when process pH is optimal, the

accumulation of VFAs may contribute to a reduced rate of

hydrolysis of the solid organic substrate [10], or even to

inhibitionat extremelyhigh levels (>10 g l1) [9]. Inhibition ofthe fermentative bacterial population by its main product VFAs

when using glucose as the main substrate [11] has also been

observed.

One of the most important ways of maintaining the

carbon and electron flow during anaerobic digestion is to* Corresponding author. Tel.: +44 23 805 94650; fax: +44 23 806 77519.

E-mail address: cjb@soton.ac.uk (C. Banks).

1359-5113/$ see front matter # 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.procbio.2005.01.025The effect of volatile fatty a

digestion of cellulose an

Irene Siegert,

School of Civil Engineering and the Environment, Unive

Received 15 April 2004; received in revised

Abstract

Batch anaerobic reactor experiments were set up in which the d1additions on the anaerobic

lucose in batch reactors

arles Banks *

of Southampton, Highfield, Southampton SO17 1BJ, UK

27 October 2004; accepted 19 January 2005

ation of the primary substrates cellulose and glucose was assayed

www.elsevier.com/locate/procbio

5) 34123418

reactions a soluble substrate in the form of D-glucose powder

recorded daily and corrected to standard temperature and

pressure.

2.5. Experimental design

Three different experiments were set up to study the

effect of a synthetic mixture of volatile fatty acids between

the concentrations of 1 and 20 g l1 in the different phases ofthe anaerobic digestion process. Experiment 1 used

14.85 g l1 of the paper as the primary substrate; experiment2 used 15.7 g l1 of glucose as the primary substrate;experiment 3 used VFA as the only substrate. In each case,

the reactors were set up with a 20% (v/v) inoculum with the

volume being made up to 710 ml with the dilution medium.

All experiments were set up in duplicate, with controls

Bioche(VWR International, UK) was used as the main carbon

source. The synthetic mixture of volatile fatty acids

consisted of: 18% acetic acid, 50% propionic acid, 5% n-

butyric acid, 12% iso-butyric acid, 5% n-valeric acid, 5%

iso-valeric acid, 2% caproic acid and 3% heptanoic acid. All

VFA components were obtained from Sigma Ltd., UK.

2.2. Inoculum

Anaerobic digester sludge was obtained from Millbrook

wastewater treatment plant, Southampton, UK. The sludge

had a pH of 7.207.28 and contained on average 0.47% total

solids and 0.18% total volatile solids.

2.3. Dilution medium

The dilution medium contained the following (per litre of

deionised water): 2.7 g KH2PO4, 3.5 g K2HPO4, 0.53 gcontrol the organic loading to the system, creating a

favorable environment for the mixed culture of micro-

organisms, to ensure VFA production and utilisation rates

are balanced [1,12]. To some extent the problem associated

with the accumulation of process intermediates encountered

in conventional single phase digesters has been overcome by

the use of two phase digestion systems that alleviate the

build up of VFAs within the hydrolysis/acidogenesis phase

[13].

The current work investigates the effect of VFAs in batch

systems on each phase of the anaerobic digestion process,

i.e. hydrolysis, acidogenesis and biogas production (metha-

nogenesis), by using a synthetic mixture of VFA as an

intermediate substrate. Both low lignin papercellulose and

glucose have been used as primary substrates thus allowing

the effect of VFA on hydrolysis and acidogenesis to be

determined independently.

2. Materials and methods

2.1. Substrates

The cellulosic substrate used as a main carbon source to

assay hydrolysis reactions was paper with a weight of

23 g m2. This was manufactured using two parts ofhardwood pulp from Wisa forest (containing 91.76%

cellulose and 1.1% lignin), and one part softwood from

Lagonia pine (with 91.70% cellulose and 1.36% lignin). The

paper contained no fillers, binders or surface coatings and

was said to be typical in its pulp composition to paper

manufactured for magazines and stationary, but not typical

of that used for newsprint, which has a higher lignin content.

The finished sheet paper, which was 0.5 mm thick, was

shredded into 4 mm strips of 150 mm length and dried

overnight at 100 8C prior to use. To assay the fermentative

I. Siegert, C. Banks / ProcessNH4Cl, 0.08 g CaCl22H2O, 0.1 gMgCl26H2O, 1.0 ml traceelements solution and 1.0 mg resazurin. The trace elements

solution contained (per litre of distilled water): 5.1 ml HCl

36%; 1.5 g FeCl24H2O; 60 mg H3BO3; 100 mgMnCl24H2O; 120 mg CoCl26H2O; 70 mg ZnCl2; 25 mgNiCl26H2O; 15 mg CuCl22H2O; 25 mg NaMoO42H2O[14]. Before use, the dilution medium was either autoclaved

for 5 min at 121 8C or boiled and then cooled whilst spargingwith nitrogen to remove dissolved oxygen. When cool

1.2 g l1 of NaHCO3 was added.

2.4. Reactors

The reactors were of a 1-l batch suspended growth design

without mechanical mixing (Fig. 1). Each comprised of a

sealed vessel maintained in a water bath at constant

temperature of 35 8C and agitated manually at least twice aday to avoid stratification. Each digester had a gas sampling

valve which allowed samples to be taken without

interference with head space composition and was

connected to a gas collection system of two acidified water

displacement bottles connected in series for gas volume

measurement. After filling with test substrate and inoculum

the reactors were sparged with nitrogen and kept isolated

from the atmosphere throughout the experiment. Samples

for analysis were taken every 2 days, and gas volumes were

mistry 40 (2005) 34123418 3413

Fig. 1. Schematic representation of the 1-l digesters and water displacement

gasometers.without substrate (cellulose or glucose) and without initial

VFA additions. In all experiments, a reactor pH of 7 was

maintained throughout by adding the appropriate amount of

5 M KOH. Mass balances were obtained for the three

experimental studies.

2.6. Analytical methods

Total solids (TS) and total volatile solids (TVS) were

determined according to Standard Methods [15]. Cellulose

content was determined by homogenizing a 20 ml sample,

which was then centrifuged at 3500 rpm for 15 min. The

supernatant was discarded and the pellet resuspended in

15 ml of an aceticnitric acid reagent (prepared by mixing

150 ml of 80% acetic acid and 15 ml of concentrated nitric

using a Fisons cathometer head space analyser with columns

for separation of methane (CH4), carbon dioxide (CO2) and

nitrogen (N2). Enzyme assays were carried out as follows:

carboxymethylcellulase (CMC) activity was determined as

previously described [19,20] using 1% CMC solution in

0.1 M citrate buffer at pH 6.0; avicelase activity was

determined as previously described [17,20] using a solution

of 5 mg ml1 avicel plus 0.1 mg ml1 glucose in 0.1 Mcitrate buffer at pH 6.0. One unit of activity was defined as

the amount of enzyme, which releases 1 mmol of reducingsugars min1, measured using the DNSmethod with glucoseas the standard. Protein content was determined by the

method of Bradford [21] with bovine serum albumin as a

standard.

primary substrate was present but no initial VFAs were

I. Siegert, C. Banks / Process Biochemistry 40 (2005) 341234183414

te con

l1, (acid). The suspension was then boiled in a water bath for

30 min. The sample was cooled, centrifuged and the

supernatant discarded. The pellet was then resuspended in

distilled water. This washing procedure was repeated a

further two times to ensure that all non-cellulosic material

was removed. The final sample was resuspended in distilled

water and filtered through a pre-dried pre-weighed 0.45 mmglass fibre filter (Whatman GFC). The filter was then dried at

100 8C to a constant weight and the weight difference wastaken to be the cellulose/hemicellulose content of the

original 20 ml sample. The control reactor without cellulose

was also analysed for cellulose content and the result

subtracted from the cellulose content of the samples [16,17].

Glucose content was determined by the dinitrosalicylic acid

(DNS) method [18]. Volatile fatty acids were quantified and

differentiated using a Varian star 3400 CX gas chromato-

graph (GC) with a free fatty acid phase (FFAP) 25 m fused

silica capillary column of 0.5 mm film thickness and an i.d.of 0.53 mm. The GC oven temperature was programmed to

increment from 60 to 100 8C in 10 min, and then to maintaina final temperature for 5 min, with a final hold time of 2 min

at a temperature of 210 8C to clean the column of anyresidues. Carrier gas flow to the GC was at a rate of

6 ml min1 and peak detection was by means of a flameionisation detector. Samples were pre-treated by acidifying

to 10% with formic acid. Gas composition was determined

Fig. 2. Carboxymethylcellulase activity induced at an initial primary substra

of: (&) 1 g l1, (&) 2 g l1, (~) 4 g l1, (~) 6 g l1, (^) 8 g l1, (^) 10 g

and contained primary cellulosic substrate (*) and no primary substrate (*).centration of 14.85 g l1 of the paper with VFA additions at concentrations+) 12 g l1, () 16 g l1, (*) 20 g l1. Controls were without VFA additions3. Results and discussion

The enzymatic assay results from experiment 1 in which

14.85 g l1 of the paper was used as the primary substratetogether with VFA additions in the range 120 g l1 areshown in Figs. 2 and 3. The cellulolytic activity, as assayed

by the carboxymethylcellulase (a) and avicelase (b)

methods, decreased with the increase in synthetic VFA

concentrations initially added to the system. No carbox-

ymethylcellulase (or endoglucanase) activity was observed

at above 16 g VFA l1 and no avicelase (or exoglucanase/b-glucosidase) activity was observed above 10 g VFA l1.This is borne out by the fact that the enzymic activities

measured were lower than those measured in the control

(control 2) in which no primary substrate was present. An

inhibitory effect on the hydrolysis of cellulose was also

observed at concentrations above 2 g l1 VFA as indicatedby the reduction in cellulose content measured (Table 1)

with no apparent reduction in the initial cellulose load at

VFA concentrations of 16 and 20 g l1. The maximumcarboxymethylcellulase and avicelase activities were

observed after 6 days. The maximum concentration of

soluble proteins (1.09 mg ml1) was produced when

I. Siegert, C. Banks / Process Biochemistry 40 (2005) 34123418 3415

Fig. 3. Avicelase activity induced at an initial primary substrate concentration of 14.85 g l1 of the paper with VFA additions at concentrations of: (&) 1 g l1,(&) 2 g l1, (~) 4 g l1, (~) 6 g l1, (^) 8 g l1, (^) 10 g l1, (+) 12 g l1, () 16 g l1, (*) 20 g l1. Controls were without VFA additions and containedprimary cellulosic substrate (*) and no primary substrate (*).

Table 1

on fro

2 ratio

luloseAverage reductizzon in cellulose content, and average gas and VFA producti

Initial

VFA (g l1)Cellulose

reduction (%)

Cellulose

digested

(ml biogas g1)

CH4:CO

from cel

digestion

1 61.83 251 1:1.30added (control 1) and at 1 g l1 VFA (0.93 mg ml1). AtVFA concentrations of 2 g l1 and above protein productiondecreased from 0.86 to 0.57 mg ml1 with increasing VFAconcentration.

2 46.67 95 1:1.36

4 40.84 77 1:1.50

6 32.32 34 1:1.64

8 24.75 11 1:1.90

10 8.77 0

12 5.49 0

16 0 0

20 0 0

Control 1 66.45 287

Gas composition ratios from cellulose digestion are shown where biogas was pr

Fig. 4. Glucose reduction from an initial substrate concentration 15.7 g l1 at VFA8 g l1, (^) 10 g l1, (+) 12 g l1, () 16 g l1, (*) 20 g l1 with controls contam digestion of cellulose and glucose at different initial VFA concentrations

VFA production

from cellulose

digestion (g l1)

Glucose

digested

(ml biogas g1)

VFA production

from glucose

digestion (g l1)

4.80 122 11.33The results support the general theory that catabolic

enzymes are repressed by immediate or distant products of

their activity. One possibility is by feedback inhibition,

where a product of an enzyme or a series of enzymes in a

5.11 107 11.45

4.04 102 12.04

3.82 99 11.75

2.34 78 12.07

1.09 38 13.79

0.60 33 10.00

0 16 13.25

0 16 13.83

4.45 126 10.75

oduced.

concentrations of: (&) 1 g l1, (&) 2 g l1, (~) 4 g l1, (~) 6 g l1, (^)ining glucose (*) and no glucose (*) with no initial VFA additions.

pathway acts on an enzyme earlier in the pathway and stops

its activity. This type of inhibitor is generally an organic

molecule and its effect on the enzyme in most cases

corresponding increase in VFA concentration. The compo-

sition of the biogas was also found to change in the cellulose

fed digester with the CH :CO ratio changing from 1:1.30 to

I. Siegert, C. Banks / Process Biochemistry 40 (2005) 341234183416

Table 2

Biogas production from the digestion of cellulose, glucose and synthetic volatile fatty acids at different initial VFA concentrations added

Initial addition

of VFA (g l1)Volume of biogas

produced from

cellulose digestion (ml)

Volume of biogas

produced from

glucose digestion (ml)

Volume of biogas

produced from the VFA

mixture initially added (ml)

1 2548 1948 195

2 900 1706 199

4 710 1631 139

6 512 1591 129

8 407 1256 89

10 286 641 79

12 185 560 79

16 60 307 76

20 0 307 77

Control 1 2829 2016

Control 2 244 67 100increases with the concentration of the inhibitor; the effects

are usually reversible [2224].

The results of experiment 2, where glucose was used as

the primary substrate, are shown in Fig. 4. There was an

immediate response shown by a reduction in glucose

concentration in reactors exposed to initial VFA concentra-

tions 4 g l1. The glucose for all the treatments wascompletely digested after 68 h, however, with reactors at

higher initial VFA concentrations showing a delay in the

onset of fermentation by as much as 16 h. These results

agree with previous research [25], in which it was found that

a mixture of acetic, propionic and butyric acids at 6 g l1

was slightly toxic to acid forming bacteria but did not show

any inhibitory effect on methanogenesis.

In experiment 1 with the increased VFA load, there was a

decrease in the amount of biogas associated with the

degradation of the primary substrate produced and aFig. 5. Cumulative gas production without addition of primary substrate and addit

4 g l1, (~) 6 g l1, (^) 8 g l1, (^) 10 g l1, (+) 12 g l1, () 16 g l1, (*) 204 2

1:1.90 with an increase in VFA concentration from 1 to

8 g l1 (Table 1). In experiment 2, the digestion of glucoseat different VFA concentrations led mainly to the

production of carbon dioxide. A slight increase in the

amount of VFAs produced was also observed associated

with the lowest concentration of VFA initially added

(1 g l1).Table 2 shows the effect of the synthetic mixture of VFAs

on the production of gas from the different substrates used.

The volatile intermediates produced from the digestion of

cellulose and glucose and the overall balance of VFA

suggests that the primary substrates are more easily

transformed to biogas by the methanogenic microorganisms

than the synthetic VFAs initially added, although the

possibility of an interchange within the overall VFA pool

cannot be discounted. None of the experiments allowed for

an acclimation of the inoculum prior to the tests and theion of VFA at concentrations of: (*) 0 g l1, (&) 1 g l1, (&) 2 g l1, (~)g l1.

6 g l VFA. The CH4:CO2 ratio for the VFA concentrations

system pH, VFA caused the inhibition of the cellulolytic1

I. Siegert, C. Banks / Process Biochemistry 40 (2005) 34123418 3417activity at concentrations 2 g l , and therefore of the rateof cellulose hydrolysis. The fermentation of glucose was

slightly inhibited at VFA concentrations above 4 g l1. Theinhibitory effect on the production of biogas and also on the

methane to carbon dioxide ratio was evident above 6 g l1

VFA in the initial mixture when used as the sole substrate. In

combination with paper as primary substrate, biogas

production due to the paper was more than halved above

1 g l1 initial VFA, indicating inhibition of the hydrolysisprocess. Where glucose was the primary substrate biogas

production was more than halved above 8 g l1 whichindicated that the fermentation was less sensitive to

inhibition caused by VFA.

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I. Siegert, C. Banks / Process Biochemistry 40 (2005) 341234183418

The effect of volatile fatty acid additions on the anaerobic digestion of cellulose and glucose in batch reactorsIntroductionMaterials and methodsSubstratesInoculumDilution mediumReactorsExperimental designAnalytical methods

Results and discussionConclusionsReferences