Effects of Feed to Inoculum Ratio, Co-digestion, and Pretreatment on

(12) The changes of VFA and pH during digestion of cotton stalk were monitored, and the results are shown in Figure 1C,D. As ...... 2010, 101, 4042–...
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Effects of Feed to Inoculum Ratio, Co-digestion, and Pretreatment on Biogas Production from Anaerobic Digestion of Cotton Stalk Xi-Yu Cheng*,†,§ and Cheng Zhong‡,§ †

College of Life Science and Bioengineering, School of Science, Beijing Jiaotong University, Beijing 100044, People’s Republic of China ‡ Key Laboratory of Industrial Fermentation Microbiology (Ministry of Education), Tianjin University of Science & Technology, Tianjin 300457, People’s Republic of China ABSTRACT: Biogas yield from anaerobic digestion of cotton stalk (CS) at feed to inoculum (F/I) ratios of 2−4 reached 175− 180 mL/(g of VSadded) against 113 mL/(g of VSadded) observed at the F/I ratio of 6. CS was proven to be a promising cosubstrate in the digestion of swine manure (SM), and a CS/SM ratio of 50:50 with a C/N ratio of 25 was found to be the best in terms of the biogas production rate and yield with increases up to 1.8- and 1.9-fold, respectively, as compared to the control. The highest biogas yield (449 mL/(g of VSadded)) and production rate (0.65 L/(L·day)) with comparable technical digestion time were obtained in co-digestion of SM with CS pretreated by NaOH, which was 241−255% of those achieved in the control. This study indicates that co-digestion of SM with alkali-pretreated CS is a potential option for alleviating the insufficient substrate resource problems and improving the energy output.

1. INTRODUCTION China produced the largest numbers of cotton with a cotton stalk yield of 20 million tons/year.1 Traditionally, cotton stalk wastes have been combusted or discarded directly, causing a number of ecological and environmental problems. Anaerobic digestion, which has been successfully applied for biogas production at both home scale and large scale for more than 1 century and is considered as a promising method for renewable energy production from varied organic wastes,2−5 provided a viable alternative for the disposal of cotton stalk wastes.6−9 EIShinnawi et al. studied biogas production from anaerobic digestion of different kinds of crop residues (maize, rice, and cotton stalks) and observed that cotton stalks gave a lower value of methane yield than that obtained in the maize stalk.6 In a further study, constant amounts of maize, rice, and cotton stalks were mixed with cow dung and predigested at various time intervals before fermentation.7 The results showed that the biogas potential of the maize stalk mixture was also higher than that of cotton stalk. Methane yields of 65−86 mL of CH4/(g of cotton wastes) were observed from anaerobic digestion of cotton stalks and seed hulls in the presence of supplemented nutrients and trace elements.8 Improved methane yields (80− 242 mL of CH4/(g of VS)) were obtained in the digestion of cotton stalk pretreated with different agents.9 When considering its high carbon content, cotton stalk may also be used as a potential co-substrate of anaerobic digestion of swine manure. Substrates (e.g., swine manure) with too low of a carbon/nitrogen ratio (C/N) ratio would increase the risk of ammonia inhibition, while a too high ratio would not provide enough nitrogen for the maintenance of cell biomass.10−13 Some studies showed that the optimal C/N ratio for anaerobic digestion was between 20 and 35,12−14 and some other researchers revealed that it ranged from 16 to 25.15,16 Codigestion of agricultural wastes with manure wastes provided better performance than their monodigestion, and its main © 2014 American Chemical Society

benefits were that it could not only increase buffering capacity to help maintain an optimal pH for methangenic bacteria and provide a better C/N ratio in the feedstock but also dilute potentially toxic compounds, utilize the nutrients and bacterial diversities in various wastes, and so on.17−19 Although codigestion of manure wastes with many crop residues such as corn stalk, wheat straw, and rice straw was frequently studied,13,17−19 information on cotton stalk was still limited. Moreover, one of the major drawbacks when cotton stalks are being digested is its recalcitrant structure and higher lignin content than common lingocellulosic wastes such as corn stalk and wheat straw,4,13,20 which may hinder anaerobic digestion. Different pretreatments, which can reduce the crystallinity of cellulose and remove the lignin from lingocellulosic wastes, have been applied in attempts to improve the biodegrabability of lingocellulosic wastes.4,5 Several pretreatment studies of cotton stalk have also been reported such as saccharification by alkaline pretreatment and microwave assisted alkaline pretreatment, ethanol fermentation by pretreatment with various chemical agents, and biogas production by hydrothermal pretreatment with hot water, ammonia solution, and recycled liquid from anaerobic digestion.9,21,22 Among different pretreatment methods, the dilute acid pretreatment was frequently used because it is effective and inexpensive.23 And alkaline pretreatment, an efficient method for delignification of lignocellulose, has been proposed as a compatible chemical pretreatment for enhanced biogas production, since anaerobic digestion usually requires a pH adjustment by improving alkalinity.24 Given its large potential for biogas production, cotton stalk certainly deserves more research attention for being used as a feedstock in co-digestion with manures and the Received: December 29, 2013 Revised: April 17, 2014 Published: April 17, 2014 3157

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following process enhancement. However, there are currently limited publications reporting anaerobic co-digestion of swine manure with cotton stalk and the effect of acid and alkaline pretreatments. In addition, the feed to inoculum (F/I) ratio was considered as a key parameter affecting the efficiency of anaerobic digestion and the accuracy of the BMP assay.25−27 Significant decreases of methane yield were observed in digestion of various wastes such as straw, food, and green wastes when F/I ratios were over a certain level.25,26 At the same time, it is very important to find a suitable F/I ratio to minimize the requirement of active inoculum for the start-up of a biogas plant. In the present study, the biogas production performance in anaerobic mono- and co-digestion of cotton stalk was studied using batch digestion tests. The purpose of this study was to investigate the (i) effect of F/I ratio on the degradation rate and biogas production in anaerobic monodigestion of cotton stalk; (ii) effect of the mixing ratio of swine manure and cotton stalk and the C/N ratio on system performance of co-digestion of these wastes including biogas production, substrate degradation, and process stability, etc.; and (iii) effect of different pretreatments on the performance of co-digestion of swine manure and cotton stalk.

Table 2. Batch Experimental Conditions of Anaerobic Digestion proportion of the various mixtures (%TS)a exptl set first batch: effect of F/I ratio F1 F2 F3 F4 second batch: effect of codigestion C1 C2 C3 third batch: effect of pretreatment P1 P2

29.5 ± 0.2 78.9 ± 0.6 7.5 ± 0.3 2.42 ± 0.2 13

cotton stalk 93.1 89.0 − 1.15 50 38.3 19.5 21.6

6 4 3 2

100 100 100 100

0 0 0 0

no no no no

4 4 4

25 50 75

75 50 25

no no no

4 4

50 50

50 50

AHP AP

pretreatment

water was added, and pH was adjusted to 7 by using 1 M HCl or NaOH solution. No extra nutrient solutions were added in all batch experiments. After being flushed for 10 min with N2, all serum bottles were sealed with butyl rubber stoppers and aluminum crimps at once and then were incubated in a rotary shaker (110 rpm) at 35 °C. The blank trials containing the sludge inocula only were carried out to correct for the biogas produced from the inocula. Batch experiments were terminated on day 45 when a clear downward trend in daily biogas production was observed. Triplicate bottles were used in all experiments, and all values were the means of replicates of triplicate ± standard deviation (SD). For the first batch experiment (sets F1−F4) of studying the effect of the F/I ratio on biogas production, cotton stalk was used as the sole substrate and sludge inoculum was added at the F/I ratios of 2, 3, 4, and 6, respectively. The F/I ratios were chosen based on the previous studies.25,26 For the second batch experiment (sets C1−C3) of investigating the effect of co-digestion, all experimental conditions were the same as those of experimental set F2 except for the substrate. In brief, the F/I ratio was fixed at 4, and cotton stalk was co-digested with swine manure according to predetermined mixing ratios (Table 2) to reach a total TS concentration of substrate of 5% (w/v, based on the original substrate). The mixing ratios of substrates (Table 2) in codigestion were chosen to reach a different C/N ratio (18−35) based on the previous results.12−15 For the third batch experiment (P1 and P2) of investigating the effect of pretreatment, all experimental conditions were the same as those of experimental set C2 except for the substrate cotton stalk which was pretreated by acid or alkaline solution before it was codigested with swine manure at a mixing ratio of 50:50. All pretreatments were carried out in a 500 mL round-bottom glass flask with a water condenser. With ice water running through the condensation pipe of the system, 15 g of the grounded cotton stalk sample was mixed with 150 mL of acid or alkaline solutions in the flask. The flasks containing the mixtures were then gradually heated until 100 °C and maintained at the boiling point for 60 min. For acid hydrolysis pretreatment (AHP), the cotton stalk sample was placed in 0.9% (w/w) H2SO4 solution and then boiled at 100 °C for 60 min. For alkaline pretreatment (AP), the cotton stalk sample was placed in NaOH solution and boiled at 100 °C for 60 min. The alkali concentration in the AP was 6% (WNaOH/WStalk). The operational parameters in AHP and AP were chosen based on the previous studies.4,5,28 It should be mentioned that the pretreated samples were divided into two parts in each experimental set. One part of the samples (the mixtures of CS and water) was used for the following

Table 1. Characteristics of Cotton Stalk and Swine Manure swine manure

swine manure

Percentages are expressed in total solid (TS). AHP, acid hydrolysis pretreatment; AP, alkaline pretreatment.

2.1. Materials. Cotton stalk was collected from Hunan province, China. It was dried in the sun and then milled to 16-mesh powder by using a plant muller. The sample powder was dried in an oven at 60 °C for at least 48 h before use. The main components of cotton stalk were as follows (% (w/w), on a dry weight basis): cellulose, 38.3; hemicellulose, 19.5; lignin, 21.6. Swine manure used in the codigestion experiment was obtained from the Institute of Animal Sciences, Chinese Academy of Agricultural Sciences and kept refrigerated at 4 °C before use. The characteristics of cotton stalk and swine manure can be seen from Table 1.

param

cotton stalk

a

2. MATERIALS AND METHODS

TS (% fresh weight) VS (%TS) pH TKN (%TS) C/N cellulose (%TS) hemicellulose (%TS) lignin (%TS)

F/I

± 1.2 ± 0.9 ± 0.05 ± 1.0 ± 1.0 ± 0.9

2.2. Experimental Design and Method. Table 2 shows a summary of all of the experiments performed in this study. Experimental sets F (F1, F2, F3, and F4), C (C1, C2, and C3) and P (P1 and P2) were conducted to investigate the effect of F/I ratio, codigestion, and pretreatments on biogas production from batch anaerobic mono- and co-digestion of cotton stalk, respectively. All batch digestion experiments were carried out in a 500 mL serum bottle with a working volume of 300 mL. The mixtures of pond bed sludge and anaerobic sludge obtained from Gaobeidian Wastewater Treatment Plant of Beijing was used as seed, which contained total solid (TS) of 50 g/L and volatile solid (VS) 30 g/L. The TS concentrations of substrates (cotton stalk alone or cotton stalk together with swine manure) at the beginning of digestion of each experimental set were 5% (w/v, based on the original substrate). Sludge inoculum was added based on predetermined feed to inoculum ratios. The F/I ratio was calculated based on the initial VS concentrations of the substrate and the inoculum. Suitable distilled 3158

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average of the individual substrate’s methane yield. Weighted methane yield (weighted MY) was calculated as follows:

digestibility tests without solid separation, after their pH was adjusted to 7 by using 1 M HCl or NaOH solution. Another part of the samples was used for the analysis of the component change in the pretreatment process. 2.3. Analytical Methods. TS, VS, total Kjeldahl nitrogen, ammonium nitrogen, and soluble chemical oxygen demand (COD) were determined according to the standard methods described by the American Public Health Association (APHA) and the State Environmental Protection Administration of China (SEPAC).29,30 Total organic carbon was measured by following the Walkey−Black method.31 The C/N ratio was obtained by dividing the total organic carbon by the total Kjeldahl nitrogen.13 Weight loss after pretreatments and digestion was recorded at the end of batch digestion. The free ammonia concentrations at the end of the tests were calculated based on the previous method.11 Part of pretreated samples was centrifuged at 4500 rpm for 20 min, and the solid residues were then dried at 60 °C to constant weight for weight loss calculation and lignocellulosic component analysis. Cellulose, hemicellulose, and lignin were measured by the method described by Goering and Van Soest.32 In brief, neutral detergent fiber (NDF) and acid detergent fiber (ADF) are determined gravimetrically as the solid residue remaining after neutral and acid detergent extraction, respectively. Lignin is determined gravimetrically on a free ash basis after the ADF residue is extracted with 72% H2SO4 solution. Ash content was determined in an oven at 550 °C over 6 h. Cellulose is calculated by subtracting the preash lignin value from the ADF value. Hemicellulose is calculated by subtracting the ADF value from the NDF value. Weight loss and cellulose, hemicellulose, and lignin content are calculated on the basis of TS of cotton stalk samples. During anaerobic fermentation, the samples were collected at predetermined time points and centrifuged at 15000 rpm for 10 min. The supernatants were filtered by using a micropore membrane of 0.45 μm. The filtrates of 0.9 mL were treated by addition of 0.1 mL of 10% formic acid solution and then used for volatile fatty acids (VFAs) analysis. The concentrations of VFAs were determined using a gas chromatograph (Agilent GC 6890, Santa Clara, CA, USA) equipped with a flame ionization detector and a 30 m × 0.25 mm × 0.25 μm fused-silica capillary column (DB-FFAP). The temperatures of the injector and detector were 250 and 300 °C, respectively. The oven temperature was initially kept at 70 °C for 3 min, followed by a rampup of 20 °C/min for 5.5 min, and held at a final temperature of 180 °C for 3 min. Nitrogen was used as the carrier gas with a flow rate of 2.6 mL/min. The biogas composition was determined using gas chromatography (Agilent GC 6890) equipped with a thermal conductivity detector and two columns separated by a switching valve. The first column was a Plot Q polymer column, to separate CO2 and the compounds with a higher molecular weight, and the second was a molecular sieve column to separate the lower molecular weight gases such as H2, O2, N2, and CH4. Helium was used as the carrier gas at a flow rate of 23 mL/min. The oven, injector, and thermal conductivity detector temperatures were 50 °C, 150 °C, and 250 °C, respectively. Calibration curves of the above gas components were linear and reproducible. The volume of the biogas produced during anaerobic digestion of cotton stalk was measured using a glass syringe.33 The daily biogas production was expressed as biogas production per gram of VS of the substrate added in 1 day [mL/(day·(g of VSadded))]. The cumulative biogas production was expressed as the cumulative biogas production per gram of VS of the substrate added (mL/(g of VSadded)). Yields of biogas and methane were expressed as milliliters of biogas or methane produced per gram of VS of the substrate added (mL/(g of VSadded) and milliters of CH4 per gram of VSadded. Technical digestion time (T80) was defined as the time needed to produce 80% of the maximal digester gas production.2 In the present study, the final cumulative biogas production on day 45 was considered as the maximal digester gas production and used to calculate the technical digestion time. The synergistic effect of co-substrates was estimated based on the previous method.3,34 The synergistic effect could be seen as an additional methane yield for co-digestion substrates over the weighted

weighted MY = (MYCSR CS + MYSMR SM)/(R CS + R SM)

(1)

where weighted MY represents the weighted average of methane yield for co-substrates, MYCS and MYSM refer to the methane yields for CS and SM, and RCS and RSM are the VS fractions for CS and SM in the mixtures of co-digestion, respectively. Methane yield of swine manure (MYSM) is 277 mL/(g of VSadded), which was obtained in the preliminary experiment of this study. 2.4. Statistical Analysis. The experimental data were statistically analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test by using SPSS statistics 17 (IBM, Armonk, NY, USA). 2.5. Kinetic Modeling. The biogas production process in the anaerobic bottles can be modeled by modified Gompertz equation,35 which can be written as follows:

⎧ ⎡R e ⎤⎫ M(t ) = P exp⎨ − exp⎢ m (λ − t ) + 1⎥⎬ ⎣ P ⎦⎭ ⎩

(2)

The kinetics of biogas production from cotton stalk can be well described using the above Gompertz equation, where M(t) represents cumulative biogas volume per gram of VS substrate added (mL/(g of VSadded)), P is the biogas production potential (mL/(g of VSadded)), Rm is the maximum biogas production rate [mL/(day·(g of VSadded))], λ is the lag time (days), and e equals about 2.718. The cumulative biogas production curve was nonlinearly fitted by the equation with Origin 8.0 pro.

3. RESULTS AND DISCUSSION 3.1. Effect of F/I Ratios on Biogas Production in Anaerobic Monodigestion of Cotton Stalk. Biogas production in anaerobic digestion of cotton stalk at four F/I ratios was investigated, and the results are shown in Figure 1. Rapid biogas production started from day 4−5 at the F/I ratios of 2−4 (Figure 1A). Biogas production decreased to some extent with the increase of F/Is. The highest daily biogas production reached 17.1, 14.8, and 14.2 mL/(day·(g of VSadded)) on days 5, 7, and 10 of digestion at the F/I ratios of 2, 3, and 4, respectively. When compared with those obtained at these three F/I ratios, a significant decrease of biogas production was noticed at a higher F/I ratio of 6. The daily biogas production fluctuated during the first 20 days of digestion with a highest level of 8.5 mL/(day·(g of VSadded)) and then dropped to a low level after day 21. It is to be noted that cumulative biogas production in the digesters operated at F/I ratios of 2, 3, and 4 increased from day 3 until about day 21 and then gradually leveled off thereafter (Figure 1B). In the case of the F/I ratio of 6, the cumulative biogas production began to increase slowly until day 5 (Figure 1B). After 45 days of digestion, the biogas yields of cotton stalk at the F/I ratios of 2−4 reached 175−180 mL/(g of VSadded) against 113 mL/(g of VSadded) achieved at the F/I ratio of 6 (Table 3). Biogas yield of raw cotton stalk was 65 mL/(g cotton stalk).8 Another studies reported that biogas yields of cotton stalk reached 157 mL/(g of TS) (approximately 177 mL/(g of VSadded)) and 89.6 mL/(g of VSadded), respectively.9,20 The biogas yield obtained in this study is comparable with those results. The average methane contents of the biogas at the four F/I ratios of 2, 3, 4, and 6 were 58.5%, 58.2%, 57.8%, and 56.5%, respectively (Table 3). And the corresponding methane yields at these F/I ratios were calculated to be 106, 102, 102, and 64 mL of CH4/(g of VS added), respectively. The lower methane content and yield at the F/I ratio of 6 indicated that there may 3159

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digestion was also lower than those obtained at the F/I ratios of 2−4 (30.8% vs 37.1−39.3%). Technical digestion time, which is defined as the time needed to produce 80% of the maximal digester gas production,2 is another indicator of biogas production performance. In the present study, the final cumulative biogas production on day 45 was considered as maximal digester gas production and used to calculate technical digestion time based on formula 1. As shown in Table 3, technical digestion times at the F/I ratios of 2−4 were obviously shorter than that obtained in the case of the F/I ratio of 6 (16−20 days vs 23 days). The biogas and methane yields of cotton stalk achieved in this study are lower than those of some other crop residues such as corn stalk and wheat straw.8 This might be due to the relatively higher lignin content of cotton stalk (21.6%), which hinders attack of enzymes to degradable hemicellulose and cellulose components. As shown in Figure 1 and Table 3, both daily biogas production and biogas yield obtained a significant increase when the F/I ratios were decreased from 6 to 2−4. Low efficiency of biogas fermentation at a high F/I ratio might be due to the insufficient methanogens and/or low methanogenic activity of sludge, which could cause the accumulation of the volatile fatty acids in the bioreactor and the subsequent drop of pH.15 The optimal pH values for anaerobic digestion range from 6.5 to 7.5, and methanogenic bacteria will be inhibited at a lower level of pH.12 The changes of VFA and pH during digestion of cotton stalk were monitored, and the results are shown in Figure 1C,D. As observed in the digestion of cotton stalk at the F/I ratio of 6, the pH in the digester decreased to 6.3 on day 8 and remained at a low level until day 12 (Figure 1D), corresponding well with higher VFA concentrations (Figure 1C) of fermentation broth in this period than those obtained at the F/I ratios of 2−4. Acetic acid was the main VFA present in all four batch tests (Figure 1C). The VFAs accumulation leads to the decrease of pH, therefore affecting methanogenic activity during the anaerobic digestion process.12,36 As a result, an inhibited steady state with a low methane yield will be observed.12,36 Previous studies indicated that the methane yield of wheat straw showed an obvious decrease at F/I ratio over 4.25 Another study verified that markedly lower methane yields were observed in anaerobic digestion of food wastes and/or green wastes (grass clippings) at a F/I ratio of 5 than those obtained at F/I ratios of 2−4.26 It is true that the peak of daily biogas production at F/I = 2 was slightly higher and technical digestion time was relatively shorter (16 days vs 19 days, Table 3) than those obtained at F/I = 4, but differences in biogas and methane yield between these two F/I ratios were not statistically significant (p < 0.01; Table 3). Swine manure may also contain some active microorganisms for biogas production, and sufficient inoculum was therefore anticipated in the co-digestion at these two F/I ratios. On this condition, a relatively higher F/I ratio will allow one digester to dispose of more wastes when an ideal total TS concentration of substrate and inoculum is fixed. It will really bring economical benefit for improving efficiency or treatment capacity of one existing digester and reducing disposal cost of wastes. Also, given that it will be very difficult to get a huge amount of active inoculum for start-up of a biogas plant in the future, the F/I of 4 with low requirement for the amounts of inoculum was chosen for the following studies of co-digestion and the effect of pretreatment. 3.2. Effect of Mixing Ratios on Biogas Production in Co-digestion of Swine Manure with Cotton Stalk. In an

Figure 1. Effect of the F/I ratios on daily biogas production (A), cumulative biogas production (B), VFA accumulation (C), and pH (D) from anaerobic digestion of cotton stalk.

be an inhibition of methanogenic bacteria in this case. The corresponding weight loss of substrate at the end of the 3160

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Table 3. Effect of F/I Ratios on Biogas Production from Anaerobic Monodigestion of Cotton Stalka F/I ratios (exptl set)

a

param

6 (F1)

4 (F2)

3 (F3)

2 (F4)

biogas yield (mL/(g of VSadded)) methane content (%) methane yield (mL of CH4/(g of VSadded)) weight loss after digestion (%TS) technical digestion time (days)

113 ± 4 56.5 ± 0.4 64 ± 2a 30.8 ± 0.4 23 ± 1.0

176 ± 9 57.8 ± 0.5 102 ± 5b 37.9 ± 0.6 19 ± 0.6

175 ± 9 58.2 ± 0.3 102 ± 5b 37.1 ± 0.5 20 ± 1.0

180 ± 9b 58.5 ± 0.4 106 ± 5b 39.3 ± 0.9 16 ± 0.6

a

b

b

Values followed by different superscript lowercase letters are significantly different at p < 0.01 according to Duncan’s test.

CH4/(g of VSadded), respectively, corresponding to increases up to 1.7−2.2-fold. When compared with the control, the advantages of codigesting cotton stalk and swine manure are obvious with increases up to 1.8, 1.9, and 2.2 times in biogas production rate, biogas yield, and methane yield, respectively. It is worth noting that the extended lag phase of more than 10 days in the digestion of swine manure alone observed in previous studies was shortened.13 The earlier peak of daily biogas production observed from the co-digestion at higher CS/SM ratio of 75:25 than those from other treatments is probably because parts of the VS in stalks may be more easily degradable than the VS in manure.37 However, the highest biogas yield of 341 mL/(g of VSadded) was observed from the co-digestion at the CS/SM ratio of 50:50. When the CS/SM ratios were 25:75, 50:50, and 75:25, the corresponding C/N ratios were 18, 25, and 35, respectively. These results suggested that the ideal C/N ratio is 25 in the co-digestion of swine manure and cotton stalk, which was consistent with the optimum C/N range of 20−30 reported before.13,14 Possible mechanisms behind the improved performance of the co-digestion process included not only providing a better carbon/nitrogen ratio in the feedstock but also diluting potentially toxic compounds, utilizing the nutrients and bacterial diversities in various wastes, and increasing buffering capacity to help maintain an optimal pH for methangenic bacteria, etc.17−19 Changes of the VFA concentration and pH were therefore determined, and the results are given in Figure 2C,D. As can be seen from Figure 2C, acetic acid was the main VFA present in all tested conditions. In the case of monodigestion of cotton stalk (set F2), VFA concentration on day 4 was about 320 mg/L and then decreased to an extremely low level (