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Initiating methanogenesis of vegetable waste at low inoculum-to-substrate ratio: Importance of spatial separation
Bioresource Technology 105 (2012) 169–173
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Initiating methanogenesis of vegetable waste at low inoculum-to-substrate ratio: Importance of spatial separation Fan Lü, Liping Hao, Min Zhu, Liming Shao, Pinjing He ⇑ Institute of Waste Treatment and Reclamation, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, PR China
a r t i c l e
i n f o
Article history: Received 25 August 2011 Received in revised form 24 November 2011 Accepted 26 November 2011 Available online 2 December 2011 Keywords: Acidification Anaerobic digestion Fermentation Gompertz modeling Start-up
a b s t r a c t With the goal of starting-up the methanogenesis of easily biodegradable waste with minimum inoculum, the present work evaluated different inoculum-to-substrate ratios (rI/S) in completely mixed systems and in the systems with spatial separation of inoculum and waste. It was found difficult to initiate methanogenesis in the completely mixed systems, even at high rI/S 1.105 on a volatile solid basis. Fermentation efficiencies were independent of rI/S. In the spatial-separation systems with a low total rI/S 0.053, the ultimate methane yield (35 °C, 1 atm) reached 445 mL/g-VS added for the inoculum-waste initially completely separated system. The yields decreased to 285, 181, and 34 mL/g-VS added, respectively, for partially separated systems with the ratios controlled at 1.105, 0.254, and 0.113 in the inoculum-containing reactors. This demonstrates the importance of setting spatial separation between inoculum and waste when inoculation is employed. An appropriate inoculation method would initiate methanogenesis rapidly even at low inoculum-to-substrate ratios. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Due to its high biodegradability, the anaerobic digestion of easily biodegradable waste, such as food waste and vegetable waste, is inclined to be blocked in an acid crisis, in which the reaction rates of hydrolysis and acidogenesis substantially exceed that of methanogenesis. As a result, methanogenesis is difficult to be initiated under acidic conditions. One of two common solutions is to reduce the organic loading of the feedstock, with the disadvantage of increasing the reactor volume requirement. The other solution is to add a larger quantity of microbial inoculum, with potential aims of reducing the burden on the unit microbial cell, alleviating inhibition by inhibitory substances, shortening the lag time for acclimation, or increasing the ultimate methane yield, the methane production rate, or the biodegradation rate of the waste. To assess the inoculation demand, the ratio of inoculum to substrate [rI/S, on a volatile solid (VS) basis], representing the ratio of the quantity of biomass added to the quantity of substrate, is usually used. In the literature, the effect of rI/S is still uncertain (summarized in Annex I). The tested rI/S differed from zero to 10.91 on a VS basis. Significant (Pandey et al., 2010) or insignificant changes (Raposo et al., 2006) were observed concerning the effects of inoculation on methane yield or methane production rate. Many factors regulate the effect of rI/S, including the substrate characteristics, the origin of the inoculum, the specific methanogenic activity of the inoculum, and the concentrations of inhibitory substances. As a result, it is difficult to determine an optimal value of ⇑ Corresponding author. Tel./fax: +86 21 65986104. E-mail address: [email protected] (P. He). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.11.104
rI/S suitable for all treatments, and the amount of inoculum needs to be estimated on a case-by-case basis (Angelidaki et al., 2009). Nevertheless, it is generally observed that a rI/S of 1 or less or even 2 is insufficient for rapidly-degradable substrates in a batch test (Xu et al., 2003; Neves et al., 2004; Wu et al., 2011). Even the wellknown continuously fed dry anaerobic digestion process DRANCO, usually mixes the feedstock waste with 6–7 times of digestate as inoculum. However, high rI/S will decrease the effective space in the reactor, resulting in a corresponding decrease in the organic loading rate of the reactor. Hence, it is important to find ways to rescue an acidified reactor or to accelerate the initiation of methane production from easily degradable waste at a minimal engineering cost, e.g. by selecting an appropriate inoculation method. The present work aimed to reconsider the role of rI/S on the anaerobic digestion of easily biodegradable waste by combining with spatial separation, so as to assess the feasibility of startingup the methanogenesis of easily biodegradable waste with minimum inoculum at an rI/S as low as 0.053. The rationale was to reduce the acid loading on the inoculum but retain the overall organic loading of the whole anaerobic system unimpaired, by means of regulating the transport rate of the organic acids. 2. Methods 2.1. Substrate and inoculum The vegetable waste was collected from a supermarket in Shanghai, China. It was ground to a particle size of less than 2 mm before feeding. The granular sludge for the inoculum was
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obtained from an upflow anaerobic sludge bed (UASB) reactor with liquid internal recirculation that was treating paper mill wastewater. The average size of the granular sludge was 3–5 mm. The characteristics of the vegetable waste were: total solids (TS) 7.5% and on a dry weight basis VS 88.1%, C 36.3%, N 3.1%, H 5.6%, starch 22.4%, lignocellulose 55.1% and protein 8.4%. The characteristics of the inoculum were: TS 9.3% and on a dry weight basis VS 89.4%, C 41.7%, N 8.1%, and H 6.4%.
2.2. Experimental set-up The digestion experiments were conducted separately in singlereactor and double-reactor mode in a thermostatic chamber at 35 °C. In the single-reactor mode, each 500 g sample of vegetable waste was completely mixed with different ratios of inoculum in a 1-L reactor, corresponding to rI/S = 0, 0.021, 0.053, 0.113, 0.338, and 1.105. The reactors were marked RS1, RS2, RS3, RS4, RS5, and RS6, respectively. Distilled water was added to make the total volume of the mixture 1 L. A reactor blank (RB) was filled only with inoculum and distilled water. Salts were added to meet the following concentrations: 0.4 g/L K2HPO43H2O, 0.2 g/L MgCl26H2O, 0.08 g/L CaCl22H2O, 0.2 g/L Na2S9H2O, 10 mL/L trace element solution, and 10 mL/L stock vitamin solution. The stock trace element and vitamin solutions were prepared according to Hao et al. (2011). In the double-reactor mode, for each treatment, a 500 g sample of vegetable waste was added and the inoculum was added to give a total rI/S of 0.053. Two 1-L reactors [a fermentation reactor (RF) and a methanogenesis reactor (RM)] were used for each treatment. The inoculum was wrapped using 0.5 mm pore string bags and placed only in RM, whereas the vegetable waste was divided between RF and RM. Four treatments were conducted with the rI/S in RM (rI/S,RM) controlled at 0.113, 0.254, 1.015, and 1 (i.e., no substrate), and were referred to as RD1, RD2, RD3, and RD4, respectively. Distilled water was added to make a mixture volume of 0.5 L in each reactor (i.e., total volume of 1 L for each treatment). The above-mentioned salts were added to the same concentrations. Each day, 100 mL of mixture were pumped from RF to RM, and another 100 mL of mixture were recycled back from RM to RF. Due to the relatively larger particle size of the granular sludge and the physical separation produced by the string bag, only vegetable waste could pass through the pipelines, whereas the granular sludge remained in RM. The contents of the reactors were mixed by turning them upside down every day. The scheme of the reactors is shown in Fig. 1. The mass of vegetable waste and inoculum used in each treatment is listed in Annex II.
2.3. Analytical methods and data analysis The biogas was collected using a 4-L foil gas sampling bag prior to sampling the liquid. The volume (corrected to 35 °C, 1 atm) and composition of the gaseous samples were then measured. The liquid from each reactor was sampled, and the pH, dissolved compound contents [as indicated by dissolved organic carbon (DOC), inorganic carbon, and dissolved nitrogen], volatile fatty acids (VFAs), and lactic acid concentration were measured. In addition, after 60 days of digestion, the mixture was removed from the reactor and tested for TS, VS, and the starch, protein, and lignocelluloses contents. The detailed analytical methods were documented in Annex III. A modified Gompertz three-parameter model (Eq. (1)) (Behera et al., 2010) was fitted to the experimentally observed cumulative methane production curves.
Rmax e ðk tÞ þ 1 MðtÞ ¼ P exp exp P
where M(t) is the cumulative methane production (mL/g-VS added) at time t (d); P is the ultimate methane yield (mL/g-VS added); Rmax is the maximum methane production rate (mL/g-VS addedd); k is the lag phase (d). P, Rmax and k were estimated by curve-fitting with minimum residual method using Sigmaplot v.12.0 (Systat Software Inc.). 3. Results and discussion 3.1. Anaerobic degradation in completely mixed single-reactor systems In the single-reactor systems, the ultimate biogas yield (35 °C, 1 atm) was 123, 39, 50, 4.1, 5.6, and 3.7 mL/g-VS added, and the ultimate methane yield was 6.2, 2.0, 2.2, 0.36, 0.25, and 3.2 mL/g-VS added, respectively for RS1, RS2, RS3, RS4, RS5, and RS6. Obviously, the CH4 production was negligible, whereas the production of nonmethane biogas, including CO2 and H2 dominated. Compared with the control RB (pH 8.09–8.76), the pH in the single-reactor systems declined quickly to as low as 4.2 during the first 8 days, and gradually increased afterwards but still remained in the acidic range [RS1: 4.46–5.56, RS2: 4.41–5.75, RS3: 4.2–6.32, RS4: 4.32–5.62, RS5: 4.2–6.61, RS6: 4.30–6.74]. DOC concentration (Fig. 2a) increased quickly during the first 4 days, afterwards, increased gradually and remained within the ranges 3.48–6.15 (RS1), 3.60–6.67 (RS2), 3.39–4.86 (RS3), 3.51–6.08 (RS4), 3.90–5.40 (RS5), and 2.87– 5.23 g-C/L (RS6). No clear relationship could be determined between DOC concentrations and rI/S values. Similarly, during the later
Vegetable waste
Gas bag
Gas bag
Granular sludge
Peristaltic pump
RS (RB)
Peristaltic pump Liquid sampling
Liquid sampling
(a) Single-reactor system (RS)
ð1Þ
RF
Liquid sampling
Peristaltic pump
RM
(b) Double-reactor system (RD)
Fig. 1. Scheme of experiment reactors.
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(a)
6
4
2
0
Acids-C concentration (g-C/L)
8
(b)
6
4
3.2. Anaerobic degradation in double-reactor systems with spatial separation
2
0 1.8
Dissolved nitrogen (g-N/L)
reached 11–34% in the inoculated treatments (34% in RS6). These acids were then deduced to be generated by the interaction of inoculum and substrate. Sanceda et al. (2003) found that certain branched VFAs were derived from the degradation of specific amino acids, such as iso-butyric acid from valine and iso-valeric acid from leucine. Therefore, these longer chain acids may have originated from the partial autolysis of the added sludge, which contained higher proportion of proteins than vegetable waste. Furthermore, the dissolved nitrogen concentrations reached 1.10–1.25 g-N/L in RS1– RS5, and 1.59 g-N/L in RS6 (Fig. 2c), higher than the fed waste nitrogen (1.163 g-N/L), suggesting that proteolysis was not inhibited and partial autolysis of the added sludge was significant in RS6. In sum, the waste fermentation efficiency was independent of rI/S. Due to high concentrated VFAs and lactate and low medium pH, it was difficult for methanogenesis to commence because of substrate inhibition, and the fermentation was also seriously suppressed because of product inhibition.
(c)
1.6 1.4 1.2 1.0 .8 .6 .4 .2 0.0 0
10
20
30
40
50
60
Time (day) Fig. 2. Changes of dissolved organic carbon and nitrogen in single-reactor systems. (a) Concentration of dissolved organic carbon, (b) concentration of carbon derived from organic acids, (c) concentration of dissolved nitrogen. (d) RB (rI/S = 1), (s) RS1 (rI/S = 0), (.) RS2 (rI/S = 0.021), (4) RS3 (rI/S = 0.053), (j) RS4 (rI/S = 0.113), (h) RS5 (rI/S = 0.338), () RS6 (rI/S = 1.105).
incubation stage, the acid concentrations (Fig. 2b) remained in the ranges 2.03–4.51 (RS1), 2.40–4.15 (RS2), 2.52–4.80 (RS3), 2.01– 3.81 (RS4), 2.47–4.70 (RS5), and 2.60–4.56 g-C/L (RS6). No clear relationship could be determined between acidogenesis efficiency and rI/S values. Assuming a stoichiometric coefficient of 0.66 for glucose fermentation to yield acetic or butyric acids, that means that approximately 8.98 g-C/L could be obtained for complete acidogenesis and 13.6 g-C/L for complete hydrolysis of the fed vegetable waste. Hence, acidogenesis and hydrolysis were nearly half inhibited. During the early stage, lactic acid production contributed to more than 70– 85% of the early acidogenesis, regardless of rI/S. Other species of organic acids were generated sequentially following the order: acetic, propionic and butyric, iso-butyric, valeric, and iso-valeric acid (shown in Annex IV). No apparent relationship could be observed between changes in their concentrations and rI/S. For RS1, only acetic, propionic, and butyric acids were generated, in constant proportions of 44.1 ± 2.5%, 4.4 ± 1.0%, and 51.4 ± 2.7%, respectively. Comparatively, the inoculated treatments had more diverse species of organic acids. The proportions of iso-butyric, valeric, and iso-valeric acids
Double-reactor systems showed active methanogenesis promoted by higher rI/S,RM values (Fig. 3). As estimated by the Gompertz model (Eq. (1)), the ultimate methane yield (P, mL/g-VS added) was highest in RD4 (445), followed by RD3 (285), RD2 (181), and RD1 (34). Maximum methane production rate (Rmax, mL/g-VS addedd) was also highest in RD4 (23.18), followed by RD3 (12.95), RD2 (12.57), and RD1 (4.29). Lag phase (k) was 9.2, 4.5, 21.8, and 31.5 days, respectively, for RD4, RD3, RD2, and RD1. The pH in methanogenesis reactors RM3 and RM4 (shown in Annex V) remained above 8. In the corresponding fermentation reactors RF3 and RF4, the pH increased gradually from 4.0 to above 7 over 20 days. Comparatively, there was more waste in RM1 and RM2, leading to the production rate being higher than the consumption rate of acids. Accordingly, the pH values dropped from 7–8 to 5, and increased again until day 20. The corresponding RF1 and RF2 retained low pH until day 30. The DOC in RM4 was lower than 0.54 g-C/L in the first 20 days, climbed to a maximum of 1.8 g-C/L in the following 20 days, and decreased below 0.17 g-C/L at the end (Fig. 4). The corresponding DOC in RF4 was maximal (10.1 gC/L) on day 4 and decreased continuously to 0.65 g-C/L. It suggested that the amount of the waste fed into RM4 was appropriate to guarantee the methanogens could consume most of organic acids for cell growth and avoid acid inhibition when the flow exchange rate was controlled at 100 mL/d (i.e. 0.2 m3/m3 d). Meanwhile, the recir-
500
CH4 production (mL/g-VS, at 35 oC, 1atm)
DOC concentration (g-C/L)
8
400 300 200 100 0 0
10
20
30
40
50
60
Time (day) Fig. 3. Cumulative methane production in double-reactor systems and Gompertz modeling. (d) RB (rI/S = 1, single-reactor system), (s) RS3 (rI/S = 0.053, singlereactor system), (.) RD1 (rI/S,RM = 0.113), (4) RD2 (rI/S,RM = 0.254), (j) RD3 (rI/S,RM = 1.015), (h) RD4 (rI/S,RM = 1). The lines closed to the data points are their corresponding Gompertz fitting curves.
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culation of RM4 effluent into RF4 led to the replacement of acidic liquids by neutral effluent; therefore, the pH in RF4 increased continuously and climbed above 7 after day 20, resultantly contributing to higher hydrolysis and acidogenesis efficiencies of the vegetable waste in RF4, as well as those of the vegetable waste transported to RM4. This could explain the observation that the decreasing rate of DOC in RF4 was lower and the DOC in RM4 increased after day 20. The ultimate methane yield of RD4 was 445 mL/g-VS added, a little higher than the theoretical methane production potential of glucose (421 mL/g-VS added) at 35 °C, suggesting the complete degradation of the carbohydrate-rich vegetable waste. The DOC changes of RD3 with rI/S,RM = 1.015 were similar to the above RD4 with rI/S,RM = 1. In contrast, the DOC increased to 3.7–4.6 g-C/L in RM2 (rI/S,RM = 0.254). After 20 days, the DOC decreased gradually, to 1.9 g-C/L at the end. The corresponding DOC in RF2 reached 8.4 g-C/L and then decreased continuously to 3.3 g-C/L. The DOC changes of RD1 with rI/S,RM = 0.113 were similar to the above RD2. The DOC increased to 6.0 g-C/L in RM1. After 27 days, the DOC decreased gradually, to 3.3 g-C/L at the end. The corresponding DOC in RF1 reached a maximum of 7.2 g-C/L and then decreased continuously to 3.9 g-C/L.
3.3. Elucidation and applications of the study The present work demonstrated that the complete homogenization of vegetable waste with inoculum was unfavorable for the methanogenesis initiation, even when the inoculum concentration was as high as the waste concentration of 33 g-VS/L in RS6. Since the fermentation of vegetable waste could occur spontaneously, owing to the indigenous microorganisms rather than the externally added inoculum, it was then independent of rI/S and resulted in conditions in which the methanogens were surrounded and strongly inhibited by organic acids and could not survive. The higher concentration of dissolved nitrogen and branched chain VFAs in RS6 implied the degradation of the microorganisms. Meanwhile,
DOC concentration (g-C/L)
12
(a)
10 8 6 4 2 0
DOC concentration (g-C/L)
12
(b)
10
the fermentation suffered from product inhibition by organic acids, leading to low fermentation efficiency around 33–56%. In comparison, the methanogens and the waste were effectively separated in the double-reactor system RD4, and connected by the controlled exchange of fermented waste, to ensure that the utilization rate of organic acids was higher than the feeding rate. In this way, the methanogens consumed the organic acids in time and survived without inhibition. Simultaneously, the recycling back of methanogenic effluent reduced the product inhibition of the fermentation. Noticeably, although the waste concentration was higher in RF4 (66 g-VS/L) than in the single-reactor systems (33 g-VS/L), the waste removal efficiency was higher, due to the neutralization of acidic conditions. The initial mixing of methanogens and vegetable waste was unfavorable, which impaired the methanogenic activity, as indicated by the reduced methane yield in RD3 where only 5% of waste was initially placed in RM3 and mixed with inoculum, and more seriously impaired in RD2 and RD1 where 20% and 55% of waste were initially placed in RM2 and RM1. By mathematical modeling, Vavilin and Angelidaki (2005) pointed out that the spatial separation of the initial methanogenic centers from active acidogenic areas was the key factor for efficient conversion of solids to methane at high organic loading rates, and the initial level of methanogenic biomass in the initiation centers was a critical factor for the survival of these centers. Their assumption could explain the present phenomena. The present study shows that the efficiency of the inoculation depended more on the inoculation method. It was important to initially separate inoculum and substrate or to reduce the concentration of acids around the inoculum, so as to allow development of methanogenic centers. When the inoculum was completely mixed with the waste, methanogenesis was not initiated, even at a high rI/S of 1.105. In contrast, when the inoculum was initially separated from the waste, the waste could be effectively transformed to methane even at a low rI/S of 0.053. The degree of separation positively affected the ultimate methane yield and the methane production rate. The rationale of the present double-reactor system resembled that of the routine two-phase anaerobic digestion system (Bouallagui et al., 2005) or the hybrid solid–liquid system with liquid recirculation (Wang et al., 2006). There were, however, several differences. First, only a small portion of granular sludge was needed, and a large volume methanogenic reactor like a UASB or anaerobic filter was then not required. Herein, the amount of granular sludge required was only 5.3% of the initial vegetable waste on a VS basis. Second, the pumped medium could be the vegetable waste directly but not only the acidified liquids. Third, the main purpose of the reactor set-up was to rescue an acidified reactor (e.g. anaerobic landfill bioreactor blocked in an ‘‘acid tomb’’) or to accelerate the initiation of methane production from easily degradable waste at a minimal engineering cost. Under such circumstances, a complete mixing of seed sludge with solid waste was highly not recommended, regardless of the amounts of seed sludge.
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4. Conclusions
6 4 2 0 0
10
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Time (day) Fig. 4. Changes of dissolved organic carbon in double-reactor systems. (a) Fermentation reactor; (b) methanogenesis reactor. (d) RD1 (rI/S,RM = 0.113), (s) RD2 (rI/S,RM = 0.254), (.) RD3 (rI/S,RM = 1.015), (4) RD4 (rI/S,RM = 1).
The work highlights the importance of spatial separation between inoculum and easily biodegradable waste when inoculation is employed. Inoculation will not be of much use for the fermentation of these types of waste. For initiating methanogenesis of these waste, it is strongly recommended not to homogenize inoculum and waste, which will dissipate and degrade the active methanogens. Multiple loading of the waste to the inoculum is superior to single shot loading if the total organic loading rate is the same. The present work suggests that an appropriate inoculation method could initiate methanogenesis rapidly even at a low inoculation rate.
F. Lü et al. / Bioresource Technology 105 (2012) 169–173
Acknowledgements This research was sponsored by NSFC (50878166; 51178327), Shanghai Subject Chief Scientist Program (10XD1404200), Shanghai Pujiang Program (11PJ1409200) and 863 Program of China (2008AA062401). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.11.104. References Angelidaki, I., Alves, M., Bolzonella, D., Borzacconi, L., Campos, J.L., Guwy, A.J., Kalyuzhnyi, S., Jenicek, P., van Lier, J.B., 2009. Defining the biomethane potential (BMP) of solid organic wastes and energy crops: a proposed protocol for batch assays. Water Sci. Technol. 59, 927–934. Behera, S.K., Park, J.M., Kim, K.H., Park, H.S., 2010. Methane production from food waste leachate in laboratory-scale simulated landfill. Waste Manage. 30, 1502– 1508. Bouallagui, H., Touhami, Y., Cheikh, R.B., Hamdi, M., 2005. Bioreactor performance in anaerobic digestion of fruit and vegetable wastes. Process Biochem. 40, 989– 995.
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Hao, L., Lü, F., He, P., Li, L., Shao, L., 2011. Predominant contribution of syntrophic acetate oxidation to thermophilic methane formation at high acetate concentrations. Environ. Sci. Technol. 45, 508–513. Neves, L., Oliveira, R., Alves, M.M., 2004. Influence of inoculum activity on the biomethanization of a kitchen waste under different waste/inoculum ratios. Process Biochem. 39, 2019–2024. Pandey, P., Ndegwa, P., Alldredge, J., Pitts, M., Soupir, M., 2010. Modeling effects of granules on the start-up of anaerobic digestion of dairy wastewater with Langmuir and extended Freundlich equations. Bioprocess Biosyst. Eng. 33, 833– 845. Raposo, F., Banks, C.J., Siegert, I., Heaven, S., Borja, R., 2006. Influence of inoculum to substrate ratio on the biochemical methane potential of maize in batch tests. Process Biochem. 41, 1444–1450. Sanceda, N.G., Suzuki, E., Kurata, T., 2003. Branched chain amino acids as source of specific branched chain volatile fatty acids during the fermentation process of fish sauce. Amino acids 24, 81–87. Vavilin, V.A., Angelidaki, I., 2005. Anaerobic degradation of solid material: importance of initiation centers for methanogenesis, mixing intensity, and 2D distributed model. Biotechnol. Bioeng. 89, 113–122. Wang, J.Y., Liu, X.Y., Kao, J.C., Stabnikova, O., 2006. Digestion of pre-treated food waste in a hybrid anaerobic solid–liquid (HASL) system. J. Chem. Technol. Biotechnol. 81, 345–351. Wu, D., Lü, F., Gao, H., Shao, L., He, P., 2011. Mesophilic bio-liquefaction of lincomycin manufacturing biowaste: the influence of total solid content and inoculum to substrate ratio. Bioresour. Technol. 102, 5855–5862. Xu, H.L., Wang, J.Y., Zhang, H., Tay, J.H., 2003. A comparative study of anaerobic digestion of food waste in a single pass, a leachate recycle and coupled solid/ liquid reactors. Water Sci. Technol. 47, 319–324.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Initiating methanogenesis of vegetable waste at low inoculum-to-substrate ratio: Importance of spatial separation Fan Lü, Liping Hao, Min Zhu, Liming Shao, Pinjing He ⇑ Institute of Waste Treatment and Reclamation, College of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, PR China
a r t i c l e
i n f o
Article history: Received 25 August 2011 Received in revised form 24 November 2011 Accepted 26 November 2011 Available online 2 December 2011 Keywords: Acidification Anaerobic digestion Fermentation Gompertz modeling Start-up
a b s t r a c t With the goal of starting-up the methanogenesis of easily biodegradable waste with minimum inoculum, the present work evaluated different inoculum-to-substrate ratios (rI/S) in completely mixed systems and in the systems with spatial separation of inoculum and waste. It was found difficult to initiate methanogenesis in the completely mixed systems, even at high rI/S 1.105 on a volatile solid basis. Fermentation efficiencies were independent of rI/S. In the spatial-separation systems with a low total rI/S 0.053, the ultimate methane yield (35 °C, 1 atm) reached 445 mL/g-VS added for the inoculum-waste initially completely separated system. The yields decreased to 285, 181, and 34 mL/g-VS added, respectively, for partially separated systems with the ratios controlled at 1.105, 0.254, and 0.113 in the inoculum-containing reactors. This demonstrates the importance of setting spatial separation between inoculum and waste when inoculation is employed. An appropriate inoculation method would initiate methanogenesis rapidly even at low inoculum-to-substrate ratios. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Due to its high biodegradability, the anaerobic digestion of easily biodegradable waste, such as food waste and vegetable waste, is inclined to be blocked in an acid crisis, in which the reaction rates of hydrolysis and acidogenesis substantially exceed that of methanogenesis. As a result, methanogenesis is difficult to be initiated under acidic conditions. One of two common solutions is to reduce the organic loading of the feedstock, with the disadvantage of increasing the reactor volume requirement. The other solution is to add a larger quantity of microbial inoculum, with potential aims of reducing the burden on the unit microbial cell, alleviating inhibition by inhibitory substances, shortening the lag time for acclimation, or increasing the ultimate methane yield, the methane production rate, or the biodegradation rate of the waste. To assess the inoculation demand, the ratio of inoculum to substrate [rI/S, on a volatile solid (VS) basis], representing the ratio of the quantity of biomass added to the quantity of substrate, is usually used. In the literature, the effect of rI/S is still uncertain (summarized in Annex I). The tested rI/S differed from zero to 10.91 on a VS basis. Significant (Pandey et al., 2010) or insignificant changes (Raposo et al., 2006) were observed concerning the effects of inoculation on methane yield or methane production rate. Many factors regulate the effect of rI/S, including the substrate characteristics, the origin of the inoculum, the specific methanogenic activity of the inoculum, and the concentrations of inhibitory substances. As a result, it is difficult to determine an optimal value of ⇑ Corresponding author. Tel./fax: +86 21 65986104. E-mail address: [email protected] (P. He). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.11.104
rI/S suitable for all treatments, and the amount of inoculum needs to be estimated on a case-by-case basis (Angelidaki et al., 2009). Nevertheless, it is generally observed that a rI/S of 1 or less or even 2 is insufficient for rapidly-degradable substrates in a batch test (Xu et al., 2003; Neves et al., 2004; Wu et al., 2011). Even the wellknown continuously fed dry anaerobic digestion process DRANCO, usually mixes the feedstock waste with 6–7 times of digestate as inoculum. However, high rI/S will decrease the effective space in the reactor, resulting in a corresponding decrease in the organic loading rate of the reactor. Hence, it is important to find ways to rescue an acidified reactor or to accelerate the initiation of methane production from easily degradable waste at a minimal engineering cost, e.g. by selecting an appropriate inoculation method. The present work aimed to reconsider the role of rI/S on the anaerobic digestion of easily biodegradable waste by combining with spatial separation, so as to assess the feasibility of startingup the methanogenesis of easily biodegradable waste with minimum inoculum at an rI/S as low as 0.053. The rationale was to reduce the acid loading on the inoculum but retain the overall organic loading of the whole anaerobic system unimpaired, by means of regulating the transport rate of the organic acids. 2. Methods 2.1. Substrate and inoculum The vegetable waste was collected from a supermarket in Shanghai, China. It was ground to a particle size of less than 2 mm before feeding. The granular sludge for the inoculum was
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obtained from an upflow anaerobic sludge bed (UASB) reactor with liquid internal recirculation that was treating paper mill wastewater. The average size of the granular sludge was 3–5 mm. The characteristics of the vegetable waste were: total solids (TS) 7.5% and on a dry weight basis VS 88.1%, C 36.3%, N 3.1%, H 5.6%, starch 22.4%, lignocellulose 55.1% and protein 8.4%. The characteristics of the inoculum were: TS 9.3% and on a dry weight basis VS 89.4%, C 41.7%, N 8.1%, and H 6.4%.
2.2. Experimental set-up The digestion experiments were conducted separately in singlereactor and double-reactor mode in a thermostatic chamber at 35 °C. In the single-reactor mode, each 500 g sample of vegetable waste was completely mixed with different ratios of inoculum in a 1-L reactor, corresponding to rI/S = 0, 0.021, 0.053, 0.113, 0.338, and 1.105. The reactors were marked RS1, RS2, RS3, RS4, RS5, and RS6, respectively. Distilled water was added to make the total volume of the mixture 1 L. A reactor blank (RB) was filled only with inoculum and distilled water. Salts were added to meet the following concentrations: 0.4 g/L K2HPO43H2O, 0.2 g/L MgCl26H2O, 0.08 g/L CaCl22H2O, 0.2 g/L Na2S9H2O, 10 mL/L trace element solution, and 10 mL/L stock vitamin solution. The stock trace element and vitamin solutions were prepared according to Hao et al. (2011). In the double-reactor mode, for each treatment, a 500 g sample of vegetable waste was added and the inoculum was added to give a total rI/S of 0.053. Two 1-L reactors [a fermentation reactor (RF) and a methanogenesis reactor (RM)] were used for each treatment. The inoculum was wrapped using 0.5 mm pore string bags and placed only in RM, whereas the vegetable waste was divided between RF and RM. Four treatments were conducted with the rI/S in RM (rI/S,RM) controlled at 0.113, 0.254, 1.015, and 1 (i.e., no substrate), and were referred to as RD1, RD2, RD3, and RD4, respectively. Distilled water was added to make a mixture volume of 0.5 L in each reactor (i.e., total volume of 1 L for each treatment). The above-mentioned salts were added to the same concentrations. Each day, 100 mL of mixture were pumped from RF to RM, and another 100 mL of mixture were recycled back from RM to RF. Due to the relatively larger particle size of the granular sludge and the physical separation produced by the string bag, only vegetable waste could pass through the pipelines, whereas the granular sludge remained in RM. The contents of the reactors were mixed by turning them upside down every day. The scheme of the reactors is shown in Fig. 1. The mass of vegetable waste and inoculum used in each treatment is listed in Annex II.
2.3. Analytical methods and data analysis The biogas was collected using a 4-L foil gas sampling bag prior to sampling the liquid. The volume (corrected to 35 °C, 1 atm) and composition of the gaseous samples were then measured. The liquid from each reactor was sampled, and the pH, dissolved compound contents [as indicated by dissolved organic carbon (DOC), inorganic carbon, and dissolved nitrogen], volatile fatty acids (VFAs), and lactic acid concentration were measured. In addition, after 60 days of digestion, the mixture was removed from the reactor and tested for TS, VS, and the starch, protein, and lignocelluloses contents. The detailed analytical methods were documented in Annex III. A modified Gompertz three-parameter model (Eq. (1)) (Behera et al., 2010) was fitted to the experimentally observed cumulative methane production curves.
Rmax e ðk tÞ þ 1 MðtÞ ¼ P exp exp P
where M(t) is the cumulative methane production (mL/g-VS added) at time t (d); P is the ultimate methane yield (mL/g-VS added); Rmax is the maximum methane production rate (mL/g-VS addedd); k is the lag phase (d). P, Rmax and k were estimated by curve-fitting with minimum residual method using Sigmaplot v.12.0 (Systat Software Inc.). 3. Results and discussion 3.1. Anaerobic degradation in completely mixed single-reactor systems In the single-reactor systems, the ultimate biogas yield (35 °C, 1 atm) was 123, 39, 50, 4.1, 5.6, and 3.7 mL/g-VS added, and the ultimate methane yield was 6.2, 2.0, 2.2, 0.36, 0.25, and 3.2 mL/g-VS added, respectively for RS1, RS2, RS3, RS4, RS5, and RS6. Obviously, the CH4 production was negligible, whereas the production of nonmethane biogas, including CO2 and H2 dominated. Compared with the control RB (pH 8.09–8.76), the pH in the single-reactor systems declined quickly to as low as 4.2 during the first 8 days, and gradually increased afterwards but still remained in the acidic range [RS1: 4.46–5.56, RS2: 4.41–5.75, RS3: 4.2–6.32, RS4: 4.32–5.62, RS5: 4.2–6.61, RS6: 4.30–6.74]. DOC concentration (Fig. 2a) increased quickly during the first 4 days, afterwards, increased gradually and remained within the ranges 3.48–6.15 (RS1), 3.60–6.67 (RS2), 3.39–4.86 (RS3), 3.51–6.08 (RS4), 3.90–5.40 (RS5), and 2.87– 5.23 g-C/L (RS6). No clear relationship could be determined between DOC concentrations and rI/S values. Similarly, during the later
Vegetable waste
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Fig. 1. Scheme of experiment reactors.
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(a)
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reached 11–34% in the inoculated treatments (34% in RS6). These acids were then deduced to be generated by the interaction of inoculum and substrate. Sanceda et al. (2003) found that certain branched VFAs were derived from the degradation of specific amino acids, such as iso-butyric acid from valine and iso-valeric acid from leucine. Therefore, these longer chain acids may have originated from the partial autolysis of the added sludge, which contained higher proportion of proteins than vegetable waste. Furthermore, the dissolved nitrogen concentrations reached 1.10–1.25 g-N/L in RS1– RS5, and 1.59 g-N/L in RS6 (Fig. 2c), higher than the fed waste nitrogen (1.163 g-N/L), suggesting that proteolysis was not inhibited and partial autolysis of the added sludge was significant in RS6. In sum, the waste fermentation efficiency was independent of rI/S. Due to high concentrated VFAs and lactate and low medium pH, it was difficult for methanogenesis to commence because of substrate inhibition, and the fermentation was also seriously suppressed because of product inhibition.
(c)
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Time (day) Fig. 2. Changes of dissolved organic carbon and nitrogen in single-reactor systems. (a) Concentration of dissolved organic carbon, (b) concentration of carbon derived from organic acids, (c) concentration of dissolved nitrogen. (d) RB (rI/S = 1), (s) RS1 (rI/S = 0), (.) RS2 (rI/S = 0.021), (4) RS3 (rI/S = 0.053), (j) RS4 (rI/S = 0.113), (h) RS5 (rI/S = 0.338), () RS6 (rI/S = 1.105).
incubation stage, the acid concentrations (Fig. 2b) remained in the ranges 2.03–4.51 (RS1), 2.40–4.15 (RS2), 2.52–4.80 (RS3), 2.01– 3.81 (RS4), 2.47–4.70 (RS5), and 2.60–4.56 g-C/L (RS6). No clear relationship could be determined between acidogenesis efficiency and rI/S values. Assuming a stoichiometric coefficient of 0.66 for glucose fermentation to yield acetic or butyric acids, that means that approximately 8.98 g-C/L could be obtained for complete acidogenesis and 13.6 g-C/L for complete hydrolysis of the fed vegetable waste. Hence, acidogenesis and hydrolysis were nearly half inhibited. During the early stage, lactic acid production contributed to more than 70– 85% of the early acidogenesis, regardless of rI/S. Other species of organic acids were generated sequentially following the order: acetic, propionic and butyric, iso-butyric, valeric, and iso-valeric acid (shown in Annex IV). No apparent relationship could be observed between changes in their concentrations and rI/S. For RS1, only acetic, propionic, and butyric acids were generated, in constant proportions of 44.1 ± 2.5%, 4.4 ± 1.0%, and 51.4 ± 2.7%, respectively. Comparatively, the inoculated treatments had more diverse species of organic acids. The proportions of iso-butyric, valeric, and iso-valeric acids
Double-reactor systems showed active methanogenesis promoted by higher rI/S,RM values (Fig. 3). As estimated by the Gompertz model (Eq. (1)), the ultimate methane yield (P, mL/g-VS added) was highest in RD4 (445), followed by RD3 (285), RD2 (181), and RD1 (34). Maximum methane production rate (Rmax, mL/g-VS addedd) was also highest in RD4 (23.18), followed by RD3 (12.95), RD2 (12.57), and RD1 (4.29). Lag phase (k) was 9.2, 4.5, 21.8, and 31.5 days, respectively, for RD4, RD3, RD2, and RD1. The pH in methanogenesis reactors RM3 and RM4 (shown in Annex V) remained above 8. In the corresponding fermentation reactors RF3 and RF4, the pH increased gradually from 4.0 to above 7 over 20 days. Comparatively, there was more waste in RM1 and RM2, leading to the production rate being higher than the consumption rate of acids. Accordingly, the pH values dropped from 7–8 to 5, and increased again until day 20. The corresponding RF1 and RF2 retained low pH until day 30. The DOC in RM4 was lower than 0.54 g-C/L in the first 20 days, climbed to a maximum of 1.8 g-C/L in the following 20 days, and decreased below 0.17 g-C/L at the end (Fig. 4). The corresponding DOC in RF4 was maximal (10.1 gC/L) on day 4 and decreased continuously to 0.65 g-C/L. It suggested that the amount of the waste fed into RM4 was appropriate to guarantee the methanogens could consume most of organic acids for cell growth and avoid acid inhibition when the flow exchange rate was controlled at 100 mL/d (i.e. 0.2 m3/m3 d). Meanwhile, the recir-
500
CH4 production (mL/g-VS, at 35 oC, 1atm)
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Time (day) Fig. 3. Cumulative methane production in double-reactor systems and Gompertz modeling. (d) RB (rI/S = 1, single-reactor system), (s) RS3 (rI/S = 0.053, singlereactor system), (.) RD1 (rI/S,RM = 0.113), (4) RD2 (rI/S,RM = 0.254), (j) RD3 (rI/S,RM = 1.015), (h) RD4 (rI/S,RM = 1). The lines closed to the data points are their corresponding Gompertz fitting curves.
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culation of RM4 effluent into RF4 led to the replacement of acidic liquids by neutral effluent; therefore, the pH in RF4 increased continuously and climbed above 7 after day 20, resultantly contributing to higher hydrolysis and acidogenesis efficiencies of the vegetable waste in RF4, as well as those of the vegetable waste transported to RM4. This could explain the observation that the decreasing rate of DOC in RF4 was lower and the DOC in RM4 increased after day 20. The ultimate methane yield of RD4 was 445 mL/g-VS added, a little higher than the theoretical methane production potential of glucose (421 mL/g-VS added) at 35 °C, suggesting the complete degradation of the carbohydrate-rich vegetable waste. The DOC changes of RD3 with rI/S,RM = 1.015 were similar to the above RD4 with rI/S,RM = 1. In contrast, the DOC increased to 3.7–4.6 g-C/L in RM2 (rI/S,RM = 0.254). After 20 days, the DOC decreased gradually, to 1.9 g-C/L at the end. The corresponding DOC in RF2 reached 8.4 g-C/L and then decreased continuously to 3.3 g-C/L. The DOC changes of RD1 with rI/S,RM = 0.113 were similar to the above RD2. The DOC increased to 6.0 g-C/L in RM1. After 27 days, the DOC decreased gradually, to 3.3 g-C/L at the end. The corresponding DOC in RF1 reached a maximum of 7.2 g-C/L and then decreased continuously to 3.9 g-C/L.
3.3. Elucidation and applications of the study The present work demonstrated that the complete homogenization of vegetable waste with inoculum was unfavorable for the methanogenesis initiation, even when the inoculum concentration was as high as the waste concentration of 33 g-VS/L in RS6. Since the fermentation of vegetable waste could occur spontaneously, owing to the indigenous microorganisms rather than the externally added inoculum, it was then independent of rI/S and resulted in conditions in which the methanogens were surrounded and strongly inhibited by organic acids and could not survive. The higher concentration of dissolved nitrogen and branched chain VFAs in RS6 implied the degradation of the microorganisms. Meanwhile,
DOC concentration (g-C/L)
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the fermentation suffered from product inhibition by organic acids, leading to low fermentation efficiency around 33–56%. In comparison, the methanogens and the waste were effectively separated in the double-reactor system RD4, and connected by the controlled exchange of fermented waste, to ensure that the utilization rate of organic acids was higher than the feeding rate. In this way, the methanogens consumed the organic acids in time and survived without inhibition. Simultaneously, the recycling back of methanogenic effluent reduced the product inhibition of the fermentation. Noticeably, although the waste concentration was higher in RF4 (66 g-VS/L) than in the single-reactor systems (33 g-VS/L), the waste removal efficiency was higher, due to the neutralization of acidic conditions. The initial mixing of methanogens and vegetable waste was unfavorable, which impaired the methanogenic activity, as indicated by the reduced methane yield in RD3 where only 5% of waste was initially placed in RM3 and mixed with inoculum, and more seriously impaired in RD2 and RD1 where 20% and 55% of waste were initially placed in RM2 and RM1. By mathematical modeling, Vavilin and Angelidaki (2005) pointed out that the spatial separation of the initial methanogenic centers from active acidogenic areas was the key factor for efficient conversion of solids to methane at high organic loading rates, and the initial level of methanogenic biomass in the initiation centers was a critical factor for the survival of these centers. Their assumption could explain the present phenomena. The present study shows that the efficiency of the inoculation depended more on the inoculation method. It was important to initially separate inoculum and substrate or to reduce the concentration of acids around the inoculum, so as to allow development of methanogenic centers. When the inoculum was completely mixed with the waste, methanogenesis was not initiated, even at a high rI/S of 1.105. In contrast, when the inoculum was initially separated from the waste, the waste could be effectively transformed to methane even at a low rI/S of 0.053. The degree of separation positively affected the ultimate methane yield and the methane production rate. The rationale of the present double-reactor system resembled that of the routine two-phase anaerobic digestion system (Bouallagui et al., 2005) or the hybrid solid–liquid system with liquid recirculation (Wang et al., 2006). There were, however, several differences. First, only a small portion of granular sludge was needed, and a large volume methanogenic reactor like a UASB or anaerobic filter was then not required. Herein, the amount of granular sludge required was only 5.3% of the initial vegetable waste on a VS basis. Second, the pumped medium could be the vegetable waste directly but not only the acidified liquids. Third, the main purpose of the reactor set-up was to rescue an acidified reactor (e.g. anaerobic landfill bioreactor blocked in an ‘‘acid tomb’’) or to accelerate the initiation of methane production from easily degradable waste at a minimal engineering cost. Under such circumstances, a complete mixing of seed sludge with solid waste was highly not recommended, regardless of the amounts of seed sludge.
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4. Conclusions
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Time (day) Fig. 4. Changes of dissolved organic carbon in double-reactor systems. (a) Fermentation reactor; (b) methanogenesis reactor. (d) RD1 (rI/S,RM = 0.113), (s) RD2 (rI/S,RM = 0.254), (.) RD3 (rI/S,RM = 1.015), (4) RD4 (rI/S,RM = 1).
The work highlights the importance of spatial separation between inoculum and easily biodegradable waste when inoculation is employed. Inoculation will not be of much use for the fermentation of these types of waste. For initiating methanogenesis of these waste, it is strongly recommended not to homogenize inoculum and waste, which will dissipate and degrade the active methanogens. Multiple loading of the waste to the inoculum is superior to single shot loading if the total organic loading rate is the same. The present work suggests that an appropriate inoculation method could initiate methanogenesis rapidly even at a low inoculation rate.
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Acknowledgements This research was sponsored by NSFC (50878166; 51178327), Shanghai Subject Chief Scientist Program (10XD1404200), Shanghai Pujiang Program (11PJ1409200) and 863 Program of China (2008AA062401). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2011.11.104. References Angelidaki, I., Alves, M., Bolzonella, D., Borzacconi, L., Campos, J.L., Guwy, A.J., Kalyuzhnyi, S., Jenicek, P., van Lier, J.B., 2009. Defining the biomethane potential (BMP) of solid organic wastes and energy crops: a proposed protocol for batch assays. Water Sci. Technol. 59, 927–934. Behera, S.K., Park, J.M., Kim, K.H., Park, H.S., 2010. Methane production from food waste leachate in laboratory-scale simulated landfill. Waste Manage. 30, 1502– 1508. Bouallagui, H., Touhami, Y., Cheikh, R.B., Hamdi, M., 2005. Bioreactor performance in anaerobic digestion of fruit and vegetable wastes. Process Biochem. 40, 989– 995.
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