Biochemical Engineering Journal 21 (2004) 59–62

Short communication

Anaerobic degradation of cellulose by rumen microorganismsat various pH values

Zhen-Hu Hu, Gang Wang, Han-Qing Yu∗Laboratory of Environmental Biotechnology, School of Chemistry, The University of Science and Technology of China, Hefei, Anhui 230026, China

Received 19 February 2004; received in revised form 3 May 2004; accepted 18 May 2004

Abstract

Batch experiments were performed to investigate the anaerobic degradation of crystalline cellulose of 10 g l−1 by rumen microorganismsat pH from 4.8 to 7.3. The degradation efficiency increased with pH and the highest value of about 78% was achieved at pH 6.8 and 7.3.Acetate and propionate were the major aqueous products at all pH values. With increasing pH, the molar percentage of acetate decreased,whereas that of propionate increased. A modified Gompertz equation was able to adequately model the fermentation of cellulose by rumenmicroorganisms. The results from this study might provide useful information for the application of rumen cultures for conversion ofcellulosic wastes into value-added products.© 2004 Elsevier B.V. All rights reserved.

Keywords:Cellulose; Degradation; Modelling; Rumen microorganisms; pH

1. Introduction

Cellulose is mainly produced by conventional agricultureand forestry practices in much large quantities than carbo-hydrates. Chemical and biological methods have been pro-posed and explored for the conversion of cellulosic materi-als [1]. Compared with the chemical means, the biologicalconversion is considered environmentally friendly and lessenergy intensive[2]. Cellulose hydrolysis is the key processfor biological conversion of cellulosic materials[3]. How-ever, due to the low cellulolytic activities in swamps, thebio-degradation rate of cellulose is slow. Recently, culture re-sources, such as anaerobic sludge, rumen and other microor-ganisms, were applied to degrade cellulose and its wastesinto volatile fatty acids (VFA) and/or gases[4–6]. The re-sults showed that rumen microorganisms are superior overother microbes for the degradation of cellulosic materials,which was attributed to higher cellulolytic activities[4]. Themixed rumen microorganisms, composed of bacteria andfungi, have complete enzyme components and high enzymeactivities for the degradation of cellulosic materials. In addi-tion, fermentation of waste materials with mixed microbes isnon-sterile and therefore is expected to be cost-effective[7].

∗ Corresponding author. Tel.:+86 551 360 7592;fax: +86 551 360 1592.E-mail address:hqyu@ustc.edu.cn (H.-Q. Yu).

Anaerobic digestion of cellulose is affected by many pa-rameters, such as cellulose structural feature, culture source,medium pH, temperature, and nutrients[8]. Since pH af-fects growth rate of microorganisms, pH changes may causedrastic shifts in the relative numbers of different species in aheterogeneous population such as is present in the fermen-tative reactor[9]. Many aspects of microbial metabolism aregreatly influenced by pH variations over the range withinwhich the microorganisms can grow. These aspects includeutilisation of carbon and energy sources, efficiency of sub-strate degradation, synthesis of proteins and various typesof storage material, and release of metabolic products fromcells [8]. Despite the significance of pH in these importantfermentations, there is little information about the pH ef-fect on the fermentation of cellulose by rumen microorgan-isms and its kinetics. In this paper, the fermentation of cel-lulose by mixed rumen microorganisms at various pH val-ues was evaluated, and the fermentation kinetics was alsoanalysed.

2. Materials and methods

2.1. Culture and media

Rumen solutions obtained from a fistulated goat weretransferred to the laboratory as seed microorganisms. It was

1369-703X/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.bej.2004.05.004

60 Z.-H. Hu et al. / Biochemical Engineering Journal 21 (2004) 59–62

squeezed through four-layer gauze and the vials with rumensolutions were purged with N2 gas.

Rumen microorganisms were grown in batch culture withcellulose (Avicel PH 102, Ajiao Co., China) as the sole car-bon and energy source. Microorganisms were cultured ina 5.0 l bioreactor with a working volume of 3 l inoculatedwith strained rumen fluid of 300 ml. The reactor tempera-ture was maintained at 40± 1◦C, and pH was controlled at4.8, 5.3, 5.8, 6.3, 6.8 and 7.3, respectively, by the automaticaddition of 4 N NaOH. Agitation was kept at 120 rpm. Sub-strate of 10 g l−1 was used in all experiments. The mediumused in these experiments contained the same constituentssuggested by Pavlostathis et al.[3].

2.2. Analytical methods

Cellulose, reducing sugars, and cell dry weight were de-termined as described by Pavlostathis et al.[3]. Protein wasmeasured by a modification of Bradford method[10]. Liq-uid samples of 4 ml taken from the reactor were centrifugedat 10,000 rpm for 15 min, and the supernatant was passedthrough a 0.45�m membrane filter for the analysis of solu-ble total organic carbon (S-TOC) and VFA. S-TOC of sam-ples was measured using a TOC analyser (TOC-VCPN, Shi-madzu Co., Japan), whereas VFA was determined by a gaschromatography (GC-6890N, Agilent Inc., USA) equippedwith a flame ionisation detector and a 30 m× 0.25 mm× 0.25�m fused-silica capillary column. Biogas produc-tion was measured with water displacement method, whilethe content of the biogas was analysed by a gas chromato-graph (SP-6800A, Lunan Instrument Co., China) equippedwith a thermal conductivity detector and a 1.5 m× 2 mmstainless-steel column.

3. Results and discussion

3.1. Cellulose degradation

The fermentation patterns of cellulose at various pH val-ues are illustrated inFig. 1a. At pH 6.8, the cellulose hydrol-ysis began to occur after 12 h of inoculation. The hydrolysisrate increased up to about 60 h, and then slowed down. A de-crease in pH resulted in the delay of lag-time and the reduc-tion of hydrolysis rate. However, as pH increased from 6.8to 7.3, a longer lag-time was observed, but the overall cel-lulose hydrolysis was not affected by the delayed lag-time.As shown inFig. 1b, the cellulose degradation efficiencyincreased with pH. No cellulose degradation was observedat pH ≤ 4.8. The reduced cellulose degradation at low pHvalues could be partially attributed to the limited number ofcellulose-degrading microorganisms.

As shown inFig. 1c, low-concentration reducing sug-ars were detected in the supernatant. Their concentrationslightly increased in the mid phase of fermentation, and then

5.0 5.5 6.0 6.5 7.0 7.540

60

80

100

120

5.0 5.5 6.0 6.5 7.0 7.50

25

50

75

100

0 24 48 72 96 1200

2500

5000

7500

10000

Red

ucin

g s

uga

rs(m

gl−1

)

pH

Deg

rada

tion

effic

ienc

y (%

)pH

Con

cent

ratio

n (m

gl−1

)

Time (hour)

pH 7.3 pH 6.8 pH 6.3 pH 5.8 pH 5.3

(a)

(b)

(c)

Fig. 1. The cellulose degradation at various pH values (a) celluloseconcentration (b) degradation efficiency, and (c) concentration of reducingsugars.

began to decrease near the end of fermentation. The con-tribution of reducing sugars to S-TOC was less than 2%.Low levels of reducing sugars suggest that there was no ac-cumulation of the hydrolytic end products of cellulose inthe course of fermentation with rumen microorganisms[5].This result was in agreement with the previous observationsthat limiting-rate step was the hydrolysis of insoluble sub-strate to soluble carbohydrates in anaerobic fermentation ofcellulose[3,11].

3.2. VFA production

In all batches, acetate and propionate were found to bethe two major aqueous products of cellulose fermentation.In addition, butyrate and iso-butyrate were also detected, butin low levels. Valerate was detected only at pH 5.8 and 5.3.

Z.-H. Hu et al. / Biochemical Engineering Journal 21 (2004) 59–62 61

Fig. 2 shows the effect of pH on the production of (a)acetate and propionate (b) butyrate and iso-butyrate. At pH≤ 6.3, the concentration of acetate increased with pH. At pH> 6.3, the concentration of acetate slightly decreased as pHincreased. For propionate, its concentration increased withan increase of pH. However, at pH > 6.3, the increasing rateslowed down. The change pattern of butyrate concentrationwas similar to that of acetate (Fig. 2b).

Fig. 2c illustrates the distribution of individual VFA inmolar as a function of pH. The concentrations of individualVFA were determined from the final concentration at theend of fermentation. Although the acetate concentration in-creased with pH (Fig. 2a), its molar percentage decreasedfrom 73.5% at pH 5.3 to 48.4% at pH 7.3. In contrast toacetate, the molar percentage of propionate increased from17.9 to 46.0% with increasing pH. This suggests that thevariation of pH led to the change of distribution of fermen-tative products.

Fig. 2. Aqueous fermentation products (a) acetate and propionate (b)butyrate and iso-butyrate, and (c) percentage of acetate, propionate andbutyrate.

The yields of total VFA, acetate, propionate and butyratewere determined by the acid concentration and the corre-sponding substrate concentration during the fermentation.The results are summarised inTable 1. Acetate yield gener-ally decreased with increasing pH, whereas propionate yieldappeared an opposite trend to that of acetate. At pH≥ 6.8,propionate yield was higher than acetate yield, whereas atpH < 6.8, acetate yield exceeded propionate yield. However,limited change in the total VFA yield was observed with thevariation of pH. This illustrates that the distribution of in-dividual VFA was sensitive to pH variation, but total VFAyield was independent of pH.

3.3. Cell yield and specific growth rate

The cell yields at various pH values were determined fromgrowth data at the exponential phase by plotting the amountof cell produced versus cellulose degraded. The slope of theresulting line equals the cell yield. As shown inTable 1, thecell yield increased generally with pH at pH≤ 6.8. Reducedcell yields at lower pH values might be due to the increasingmaintenance energy requirements[12]. As pH changed from6.8 to 7.3, the cell yield remained relatively constant around0.23 g g−1 cellulose degraded. Comparing with the cell yieldof a single bacterium[4], mixed rumen microorganisms havehigher yield values.

The effect of pH on specific growth rate (µ) is alsoillustrated in Table 1. The maximum µ (µmax) was0.069 h−1, which was achieved at pH 6.8, correspondingto a re-generation time of 11.7 h. The shift of pH led toa decrease of specific growth rate. In degradation systemof complex organics containing cellulose, cellobiose andglucose, the hydrolysis of cellulose is the limiting-step[11]. This is partially due to the low specific growth rate ofbacteria on cellulose. Therefore the degradation efficiencywould be improved by increasing specific growth rate ofcellulolytic microorganisms. This might be realised par-tially by adjusting the pH of bioreactor to optimum rangefor cell growth.

3.4. Modelling

A modified Gompertz equation (Eq. (1)) has been used todescribe the cumulative hydrogen production process[13].In this study, the cumulative VFA production data were usedto fit the modified Gompertz equation. The correlation co-efficient values varied between 0.982 and 0.998, indicatingthat this equation was able to adequately estimate the VFAproduction from cellulose by rumen microorganisms:

V(t) = P exp

[−exp

(Rm · e

P(λ − 1) + 1

)](1)

whereV(t) is the cumulative total VFA production (mg g−1

cellulose),P the total VFA production potential (mg g−1 cel-lulose),Rm is the maximum VFA production rate (mg h−1),

62 Z.-H. Hu et al. / Biochemical Engineering Journal 21 (2004) 59–62

Table 1Product yields at various pH values

pH µ (h−1) Yx (g g−1) Yv (g g−1) Ya (g g−1) Yp (g g−1) Yb (g g−1)

7.3 0.054 0.236 0.631 0.254 0.326 0.0256.8 0.069 0.232 0.627 0.242 0.309 0.0246.3 0.051 0.171 0.678 0.307 0.296 0.0565.8 0.034 0.137 0.634 0.325 0.279 0.0515.3 0.004 0.142 0.637 0.332 0.270 0.048

µ, specific growth rate;Yx, cell yield; Yv, total VFA yield; Ya, acetate yield;Yp, propionate yield;Yb, butyrate yield. All units in g g−1 cellulose degraded.

Table 2Gompertz parameters calculated usingEq. (1)

pH P (mg g−1 cellulose) Rm (mg h−1) λ (h) R2

7.30 489.1 10.67 32.5 0.99826.80 495.4 11.37 9.2 0.99666.30 472.1 9.93 15.3 0.99815.80 336.6 8.87 13.4 0.99415.30 84.5 3.25 27.5 0.9818

P, the total VFA production potential;Rm, the maximum VFA productionrate;λ, the lag-phase time;R2, correlation coefficient.

e = 2.71828,λ the lag-phase time (h), andt is the incuba-tion time (h).

As shown inTable 2, the highest VFA production po-tential was predicted as 495.4 mg g−1 cellulose at pH 6.8,whereas the maximum VFA production rate was estimatedas 11.37 mg h−1. As pH increased from 6.3 to 7.3, bothmaximum total VFA production potential and maximumVFA production rate did not change significantly, suggest-ing that high cellulose degradation would be achieved in thispH range. However, at pH< 5.8, both of which decreasedmarkedly, 17.1 and 28.6% lower than those at pH 6.8, re-spectively. On the other hand, the shortest lag-phase time,9.2 h, was also predicted at pH 6.8. The change of pH wouldlead to the delay of lag-time.

4. Conclusions

These experimental results showed that efficient degra-dation of cellulose could be achieved in batch cultures in-oculated with rumen microorganisms. The degradation ef-ficiency increased with pH and the highest value of about78% was achieved at pH 6.8 and 7.3. Acetate and propionatewere the major aqueous products at all pH values. Butyrateand iso-butyrate was also formed, but in low levels. Withincreasing pH, the molar percentage of acetate decreased,whereas that of propionate increased. A modified Gompertzequation was able to adequately model the anaerobic degra-dation of cellulose by rumen microorganisms.

Acknowledgements

The authors wish to thank the Natural Science Founda-tion of China (Grant Nos. 20122203 and 20377037) for thefinancial support of this study.

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