Ind. Eng. Chem. Res. 2003, 42, 1845-1849
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Methanogenic Digestion of Lignocellulose Residues under Conditions of High-Rate Acidogenic Fermentation B. C. Qi,† G. M. Wolfaardt,‡ C. Aldrich,*,† and L. Lorenzen† Departments of Chemical Engineering and of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa
A semicontinuous module reactor was operated under methanophilic conditions to effect rapid solid anaerobic digestion of lignocellulose substances for waste utilization and stabilization. This was achieved by recovering the methanogenic phase from the high-rate acidogenic fermentation phase. The influence of a total carbon to nitrate ratio (C/N(NO3)) of the lignocellulose feed on the development of methanogens from a high-rate acidification fermentation phase was first investigated. The effects of pH adjustment and dilution of the high-rate acidification fermentation phase using the two-phase anaerobic digestion configuration were subsequently evaluated. The results showed that an increase in the C/N(NO3) ratio of the feed substrate could promote the development of methanogens as a result of the restraint of denitrification during or after the acidification fermentation phase. In-process neutralization of the digestion pH and dilution of the digestion intermediate products could reduce the inhibition from the high-rate acidification fermentation phase. The integrated digestion process promoted the biotransformation of lignocellulose residues from the high-rate acidification phase to the methanogenic phase and ultimately stabilized digestion. 1. Introduction Lignocellulose is the principal form of biomass in the biosphere and therefore the predominant renewable resource in the environment. However, owing to the chemical and structural complexity of lignocellulose substrates,1 the sustainable utilization of lignocellulose wastes is limited. A potential strategy to overcome this problem is the application of anaerobic digestion, a technology that is gaining environmental and economical importance in organic solid waste management and utilization. Owing to efficient resource recovery and lessened environmental impact, anaerobic digestion compares favorably with alternative treatments, such as incineration and landfill.2 Previous work in our laboratory demonstrated that the solubility of lignocellulose substrates could be increased by a combined pretreatment method, thus improving the efficiency of the acidification fermentation process.3 However, the anaerobic conversion of lignocellulose to methane is a complex process involving many species of bacteria, which predominantly include acidogens and methanogens (including acetogens). The acidogens and methanogens differ widely in physiological and nutritional requirements, with the methanogens the most sensitive to environmental changes such as pH and high content of un-ionized acids, or other concentrated toxins that are often encountered in a high-rate acidification digester.4,5 After successful hydrolysis, the fermentative acidogens normally have higher growth rates than acetogens and methanogens. This may lead to a subsequent accumulation of intermediary products with a resultant * To whom correspondence should be addressed. Tel.: +27(21)8084487. Fax: +27(21)8082059. E-mail:
[email protected]. † Department of Chemical Engineering. ‡ Department of Microbiology.
fall in pH, thus giving rise to unbalanced fermentation and diminishing the stability of the process. To ensure an efficient and reliable biological treatment, it is critical to establish an appropriate microbial community structure and to maintain the continued stability of that community under varying loads and environmental conditions. In conventional one-stage thermophilic digesters, long hydraulic retention times are to be used to depress the volatile fatty acid production rates of fast-growing acidogens and balance the volatile fatty acid conversion rates of slow-growing acetogens and methanogens.6 However, a long hydraulic retention time will suppress hydrolysis and the production of volatile fatty acids that are substrates of acetogens and methanogens, thereby affecting the efficiency of the high-rate digester.7 Furthermore, compared with the hydrolysisacidification fermentative microorganisms, acetogenicmethanogenic fermentation is more sensitive to the changes in environmental conditions. It was reported that separation of the hydrolysis-acidification and coupled acetogenic-methanogenic fermentation (under optimal environments for each group) substantially enhanced the associated biological reactions and provided higher stabilization of organics and gasification rates8-11 and greater pathogen mortality than the onephase high-rate digestion configuration.12 Furthermore, the denitrification process is thermodynamically favorable above the methanogenic process. In the presence of nitrates, denitrifiers compete with methanogens for the carbon substrates (acetates) and hydrogen from the acidification process, especially at the acidification stage featuring low pH and high H2 concentration (H+ and H2 accumulation). Denitrification is therefore unavoidable during the anaerobic stabilization of nitrate-containing biosludge. Fang and Zhou13 investigated the interactions of methanogens and denitrifiers in the treatment of phenolic wastewaters. They found that methanogenesis occurred only at chemical
10.1021/ie0206094 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/03/2003
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oxygen demand/NO3-N ratios greater than approximately 3.3. At ratios less than approximately 3.3, methanogenesis ceased and denitrification became incomplete because of an insufficient supply of the substrate. Oh and Silverstein14 studied the acetate limitation and nitrate accumulation during denitrification; they found that if acetate is added to the denitrifying activated sludge mixed liquor to obtain a C/N ratio in the range of 2:1 to 3:1, nitrate was completely consumed at the same rate with no nitrite accumulation. In the literature, most of the investigations were carried out on wastewaters and generally a simple carbon source (such as acetate, ethanol, sucrose, phenol, etc.) was used. When lignocellulose residues, which contain high proportions of nitrate, were anaerobically digested, there also existed the competition between denitrifiers and methanogens for a carbon source and electrons. Qi has previously observed that methanogenesis of lignocellulose could be severely hindered by the accumulated volatile fatty acids and other concentrated toxins (such as NO3-, high molecular weight substances, such as humic substances, one of the most probable intermediate digestion components of the lignocellulose biomass, etc.).3 To succeed in the development of methanogenesis from the high-rate acidification fermentation process, efforts should be made to neutralize these inhibiting factors. This can be done by provision of a proper carbon to nitrogen (C/N) ratio, neutralization of digestion pH, and dilution of the digestion medium. Compared with the simple carbon source used in most previous works, it is difficult to calculate the available carbon source (such as acetate, ethanol, butyrate, phenol, etc.) of the lignocellulose for a proper C/N ratio of the feed at the beginning of the digestion, so it should be more practical to use the total carbon content instead of the available carbon content when the solid anaerobic digestion of lignocellulose residues was concerned. In addition, all methanogens seem to be universally capable of using hydrogen as an electron donor and carbon dioxide as an electron acceptor;15 therefore, profoundly anaerobic techniques are required, particularly for growth of methanogens. In this paper the following are discussed: (1) the influence of the C/N(NO3) ratio of the lignocellulose feed on the development of methanogens from a high-rate acidification fermentation phase; (2) the effects of pH adjustment and dilution of the high-rate acidification fermentation phase on the promotion of the methanogenic phase using a two-phase anaerobic digestion configuration. 2. Experimental Work 2.1. Materials. Tobacco dust from the British American Tobacco Co. in South Africa was used as the source of lignocellulose. Activated sludge from the municipal sewage treatment plant in Stellenbosch, South Africa, was used as the microorganisms seed and the nutrient source for establishment of a degradative microbial community. The feedstock was prepared by mixing activated sludge with tobacco dust (2:1, w/w) in a porcelain roller for 2 h before it was transferred to the anaerobic reactor. The characteristics of the tobacco dust and activated sludge are shown in Table 1. The different C/N ratios expressed as the C/N(NO3) were obtained by mixing the high-nitrate-content lignocellulose residues (tobacco dust; 308 mg/kg of dry base) with the low-nitrate-content lignocellulosic residues (dry
Figure 1. Schematic diagram of the laboratory setup: (A1) batch digestion reactor; (A2) two-phase digestion reactor; (B) leachate collecting container; (C) water bath; (D) leachate recycle pump; (E) water pump; (F) oxygen-free gas cylinder; (G) biogas sampling vessel; (H) wet-gas volume meter; (I) water seal; (J) water jacket; (K) temperature controller; (L) temperature-controlled water flow; (M) batch reactor feed inlet. Table 1. Characteristics of the Tested Samples characteristics total solids (TS, %) VS (% of TS) ash (% of TS)
activated sludge 24.6 50.71 49.29
grass
tobacco dust
91.2 80.11 19.89
94.2 71.97 28.03
Element Composition of Dry Mass C (%) 29.3 54.9 N (%) 3.22 2.44 NO3-N (mg/kg) 41 10 NH4-N (mg/kg) 1245 1221 K (%) 1.13 1.51 Ca (%) 1.78 1.19 Mg (%) 0.22 0.26 B (mg/kg) 28 23 Cu (mg/kg) 754 13 Fe (mg/kg) 1.42 0.29 Zn (mg/kg) 1068 72 Na (mg/kg) 1216 915
43.0 2.37 308 761 1.70 4.29 0.70 26 57 0.57 761 288
grass; 10 mg/kg of dry base). The net weight of the activated sludge in the feed is about 34%. The contents of the total carbon and nitrate of the activated sludge were both considered to calculate the C/N(NO3) ratio of the feed. 2.2. Experimental Setup. The optimal C/N(NO3) ratio was investigated in an acidification batch reactor (1.0 L) by monitoring the N2 (one of the end products) proportion in the biogas of the reactor (A1), which was flushed with a 92% helium and 8% nitrogen gas mixture at the start-up of digestion. The experimental setup of the batch reactor is shown in Figure 1a. The digestion temperature was thermostatically controlled at 50-55 °C by a water bath (C). The feedstock was first mixed
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with tap water (pH 9.46) at a solid to solution ratio of 1:1. Then the gastight reactor (A1) was flushed with oxygen-free gas (92% He, 8% N2) from a gas cylinder (F) before it was placed in the water bath. The fermentation gas was sampled through a vessel (G) and measured with a wet-gas volume meter (H). The optimal C/N(NO3) ratio obtained from the batch reactors was then applied to the lignocellulose feeding substrates of the acidification stage for the two-phase digestion system given in Figure 1b. It consists of two gastight reactors (A2) in series (each with a volume of approximately 1.5 L), which contained the biomass mixture to be digested. Preparation and loading of the feed in each of the vessels were identical with those of the batch reactor. The temperatures in these two vessels were maintained at constant levels by temperature-buffered water that was circulated from a water bath (C) to water jackets (J) around the vessels (A2). The experimental procedures included development of the acidogenic phase and initiation of the methanogenic process as well as leachate preconditioning and dilution. The procedures consisted of the following: (1) development of the acidogenic phase under thermophilic conditions (5055 °C) for 2-3 weeks; (2) development of mesophilic conditions (35-38 °C) for various periods of digestion; (3) dilution of the leachate collected from the acidification digester with tap water; (4) conditioning of the pH and oxidation-reduction potential (ORP) of the leachate with concentrated NaOH (1%) or HCl (1%) (Merck, AR) and cysteine-HCl (0.25 g/L; Sigma, AR) solutions; (5) purging of the leachate container with an oxygen-free gas mixture (approximately 80% CO2 and 20% H2); (6) recycling the leachate through the digestion system every other hour for various periods of digestion time. To maintain strict anaerobic conditions in the system, flushing with pressurized oxygen-free gas (92% helium and 8% nitrogen gas mixture) from cylinder (F) was performed at the start of each experiment before the system was completely sealed. The biogas (0.4 mL) developed in the system during digestion was sampled through a gastight vessel (G). A water seal (I) was used to balance any pressure above the atmosphere raised in the system and to prevent air contamination to the system. The leachate (B) collected from the bottom reactor was recycled to the top reactor through a pump (D) to ensure an even distribution of the soluble compounds of the biomaterial and to unify the pH-OPR conditions of the system as much as possible. The solid digestion biomaterial was maintained in the reactors to cultivate an ideal microbial community. The yield of the biogas developed in the reactors was measured using a wet-gas meter (H) (Alexander Weight & Co. (Westminster) Ltd.; maximum working pressure 65 mbar) under ambient conditions. The compositions of CO2, CH4, and N2 (during denitrification) in the biogas were analyzed using gas chromatography (GOWMAC GC 580; Bridgewater, MA, TCD detector, 60/80 carboxen-1000 packing, 5 m × 3 mm stainless steel columns; 45/60 carboxen-1000 packing, 670 mm × 3 mm stainless steel column), where helium gas was used as the carrier gas and the flow rate was set at 30 mL/min at 300 kPa. The column and detector temperatures were set at 100 and 200 °C, respectively. 3. Results and Discussion The effects of both the variable C/N(NO3) ratio and the digestion time on the denitrification of lignocellulose
Figure 2. Effects of C/N(NO3) ratios on the accumulative N2 percent of the biogas in the acidification batch reactors.
Figure 3. Effects of C/N(NO3) ratios on the accumulative CO2 percent of the biogas in the acidification batch reactors. Table 2. pH, ORP, and TVFA in the Digesters with Different C/N(NO3) Ratios on the 70th day of Acidogenic Digestion reactor C/N(NO3) pH ORP (mV) TVFA (mg/L) acetic propionic isobutyric butyric isovaleric
1
2
3
4
16 × 102 6.1 -186 8986.33 4680 0 1209.67 2846 251.33
31 × 102 5.8 -209 10364 7226.67 610.67 137.33 2088 301.33
63 × 102 5.1 -291 17106.01 9930.67 2090 150.67 4608 326.67
91 × 102 4.9 -297 24132 11230.33 3265.67 166 9102.33 368.33
residues in a series of acidification batch reactors were considered. The accumulative N2 and CO2 contents in the biogas were given in Figures 2 and 3, respectively. The methane contents were low throughout the experiment process. Less than 2% of methane was detected in the biogas after 5 weeks of digestion. The pH, ORP, and total volatile fatty acids (TVFA) at the end of the experiment (on the 70th day of digestion) are listed in Table 2. The results in Figure 2 show that both the C/N(NO3) ratio of the feeding substrate and the period of digestion influenced denitrification. Denitrification normally appeared after 2 weeks of digestion, increased with the period of digestion, and decreased with an increase in the C/N(NO3) ratio. Considering the acidification efficiency, the changes of the CO2 content in Figure 3 and the TVFA content in Table 2 show that the C/N(NO3) ratio also influenced acidification. For a given period of digestion, higher
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Figure 4. Changes in pH with a period of methanogenic digestion at different dilution rates.
Figure 5. Changes in ORP with a period of methanogenic digestion at different dilution rates.
Table 3. Biogas Yield (m3/kg VS Added) and CH4 Content on the 25th day of Methanophilic Digestion pH controlled dilution rate gas yield (m3/kg of VS added) CH4 content (%)
7.2 1:3 0.084
7.2 1:5 0.113
7.2 1:7 0.102
8.2 1:3 0.072
8.2 1:5 0.107
8.2 1:7 0.098
37.2
51.9
44.2
35.1
48.5
39.4
TVFA and CO2 contents were observed in the reactor with higher C/N(NO3) ratios. The relatively high ORP (-186 mV) at a lower C/N(NO3) ratio of 16 × 102, compared with a lower ORP (-297 mV) at a higher C/N(NO3) ratio of 91 × 102, implied incomplete denitrification. However, a greater accumulation of propionic and butyric acids at higher C/N(NO3) ratios of 63 × 102 and 91 × 102 was probably due to intermediate acid product inhibition to acetogenic fermentation. It could be postulated that a rapid, complete denitrification together with mild acidification was desirable for the transformation of the acidification phase into the methanogenic phase. At the lowest C/N(NO3) ratio, incomplete denitrification was probably the dominant inhibitor. At the highest C/N(NO3) ratio under high-rate acidogenic fermentation, the accumulation of an intermediate acid product was probably the dominant inhibitor. On the other hand, mild denitrification was also beneficial to acidification in that denitrification could consume some products of acidification, thereby promoting the process. Therefore, to develop efficient integrated two-phase digestion, the C/N(NO3) ratio of the feeding substrate should be taken into account. Under the given experimental conditions of this study, a C/N(NO3) ratio in the range of 31 × 102-63 × 102 gave the best results. A C/N(NO3) ratio of 63 × 102 was used for the following experiments in this study. At this C/N(NO3) ratio, a digestion period of 18 days was used for the acidification stages. The changes in pH, ORP of the digestion leachate, and biogas composition were examined under different pH conditions and dilution rates as the digestion progressed. Some typical results are given in Figures 4-8. The biogas yields [m3/kg of volatile solid (VS) added] on the 25th day of methanophilic digestion are given in Table 3. As indicated in Figure 6 and Table 3, the recovery of the methanogenic phase from the acidification phase was achieved by adjusting the pH and diluting the inhibitory components. The CH4 content was increased from 5% to approximately 40-45% with pH adjustment and dilution of the acidification leachate. The results
Figure 6. Changes in CH4 (%) with a period of methanogenic digestion at different dilution rates (pH controlled at 7.2).
Figure 7. Changes in CO2 (%) with a period of methanogenic digestion at different dilution rates (pH controlled at 7.2).
in Table 3 show that the dilution rate had a greater influence on the biogas yield than the pH for the same digestion time; e.g., at the same dilution rate of 1:5, the biogas yield was changed from 0.113 to 0.107 and the CH4 content from 51.9% to 48.5% when the pH was changed from 7.2 to 8.2. At pH 7.2, the biogas yield was changed from 0.084 to 0.113 and the CH4 content from 37.2% to 51.9% when the dilution rate was changed from 1:3 to 1:5. This indicates that while the pH was controlled in the range of 7-8, the dilution rate had a stronger influence on the development and growth of methanogens. In addition, from Figures 4 and 5, it can be seen that there were no significant changes in pH and ORP values during digestion with different dilution
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Figure 8. Changes in N2 (%) with a period of methanogenic digestion at different dilution rates (pH controlled at 7.2).
rates. This suggests that the digestion system had a high pH buffering capacity, which provided the system with an ability to resist rapid fluctuations in pH resulting from dilution or microbial activities. Therefore, it appears as if the inhibitory effect from concentrated nonionized volatile acids or other more complicated organic toxins were more detrimental to the growth of methanogens. From Figure 7, it can be seen that the CO2 content of the biogas was markedly higher during the initial stage of methanogenic digestion with dilution than without dilution, while the CH4 content (Figure 6) generally increased with an increase in the dilution rates and retention time. This shows that diluting the intermediary acidification products through recycling had positive effects on both the acidogenic fermentation and the methanogenic phase. However, when the dilution rate increased from 1:5 to 1:7, only a small increase in CH4 production was observed. This suggests that dilution could promote the conversion of intermediary acidification products to simple digestible acids, such as volatile fatty acids, and reduce the concentrations of the intermediary acidification products, such as lignin-relative dissolved phenolic and humic acids. Simultaneously, dilution decreased the availability of the nutrients for the growth of the microorganisms during digestion. The inhibition of acid accumulation and the toxification of intermediary acidification products normally occurred shortly after the high-rate thermophilic acidogenic digestion stage. After the methanogens had developed, a sufficient quantity of the substrate (such as acetic acid, CO2) was required. Thus, there is an optimal dilution ratio, and the high dilution ratio does not always lead to improvement of the methanogenic phase by prolonging retention time. Denitrification is thermodynamically more stable than the methanogenic process. As a consequence, denitrification is difficult to avoid during the anaerobic digestion of nitrate-bearing organic substances. From the changes of the N2 content in the biogas (see Figure 8), it can be seen that denitrification was not completely suppressed in the high-rate acidification. Nonetheless, it was still encountered in the methanophilic stage. However, with the retention time prolonged, the denitrification quickly leveled off after approximately 15 days of methanogenic acclimation. 4. Conclusions Successful development of methanogenesis could be achieved by provision of a proper carbon to nitrogen
ratio. It is more practical to use a C/N(NO3) ratio to investigate the influence of the carbon to nitrogen ratio on the methanogenic digestion of lignocellulose. The methanogenic phase could be recovered from the high-rate acidogenic phase by diluting the acidification system and adjusting the pH of the recycled leachate. The variance in the pH of the recycling leachate between 7 and 8 did not present an evident influence on the methanogens. This implied a pH buffering capacity in the acidification digestion sludge system. Simultaneously, dilution decreased the availability of nutrients to the microorganisms during digestion. Thus, a higher dilution rate did not necessarily promote the growth of the methanogenic phase at longer retention times. The integrated digestive process composed of a highrate thermophilic acidogenic stage, followed by a dilution-pH-ORP-controlled mesophilic stage promoted the biotransformation of the lignocellulose residues to the methanogenic phase and thus ultimately stabilized the digestion system. Literature Cited (1) Eriksson, K. E. L.; Blanchete, R. A.; Ander, P. Microbial and Enzymatic Degradation of Wood and Wood Components; Springer: Berlin, Germany, 1990. (2) Pavlostathis, S. G.; Misra, G.; Prytula, M.; Yeh, D. Anaerobic Process. Water Environ. Res. 1995, 67 (4), 459-470. (3) Qi, B. C. Bio-disposal of Lignocellulose Substances with Activated Sludge. Ph.D. Dissertation, University of Stellenbosch, Stellenbosch, South Africa, 2001. (4) Mawson, A. J.; Earle, R. L.; Larsen, V. F. Degradation of Acetic and Propionic Acids in the Methane Fermentation. Water Res. 1991, 25 (12), 1549-1554. (5) Parkin, G. F.; Owen, W. F. Fundamentals of Anaerobic Digestion of Wastewater Sludge. J. Environ. Eng. 1986, 112 (1), 867-920. (6) Gosh, S.; Bouy, K.; Dressell, L.; Miller, T.; Wilcox, G.; Loos, D. Pilot-and Full-scale Two-phase Anaerobic Digestion of Municipal Sludge. Water Environ. Res. 1995, 67 (2), 206-214. (7) Gosh, S.; Pohland, F. G. Kinetics of Substrate Assimilation and Product Formation in Anaerobic Digestion. J. Water Pollut. Control Fed. 1974, 46 (4), 748-759. (8) Yeoh, B. G. Two-phase Anaerobic Treatment of Canemolasses Alcohol Stillage. Water Sci. Technol. 1997, 36 (6-7), 441-448. (9) Cohen, A. Two-phase Digestion of Liquid and Solid Wastes. Proceedings of the 3rd International Symposium on Anaerobic Digest, Cambridge, MA, 1983; p 123. (10) Gosh, S.; Ombregt, J. P.; Pipyn, P. Methane Production from Industrial Wastes by Two-phase Anaerobic Digestion. Water Res. 1985, 29, 1083-1088. (11) Gosh, S. Improved Sludge Gasification by Two-phase Digestion. J. Environ. Eng. 1987, 113 (6), 1265-1284. (12) Lee, K. M.; Bruunner, C. A.; Farrel, J. B.; Eralp, A. E. Destruction of Enteric Bacteria and Viruses during Two-phase Digestion. J. Water Pollut. Control Fed. 1989, 61, 1421-1429. (13) Fang, H. H. P.; Zhou, G. M. Interaction of Methanogens and Denitrifiers in Degradation of Phenols. J. Environ. Eng. 1999, 125 (1), 57-63. (14) Oh, J.; Silverstein, J. Acetate Limitation and Nitrite Accumulation during Denitrification. J. Environ. Eng. 1999, 125 (3), 234-242. (15) Klass, D. L. Biomass for Renewable Energy, Fuels and Chemicals; Academic Press: London, 1998.
Received for review August 7, 2002 Revised manuscript received January 13, 2003 Accepted February 20, 2003 IE0206094
