Effect of propionate toxicity on methanogenesis of night soil at phychrophilic temperature Ram Kumar Dhaked, Chandra Kant Waghmare, Syed Imteyaz Alam, Dev Vrat Kamboj, Lokendra Singh * Defence Research and Development Establishment, Jhansi Road, Gwalior 474 002, India Received 26 January 2002; received in revised form 31 August 2002; accepted 18 September 2002 Abstract The effect of propionate concentrations on biodegradation of human waste (night soil) was studied at 10 �C. Propionate was toxic for the biomethanation at all the pH tested (6.0, 7.0 and 8.0). The maximum reduction in biogas production in presence of 200 mM propionate was observed at pH 7.0 followed by 8.0. The methane content in biogas also followed a similar trend and at pH 7.0 an 11.5% decrease was observed. Propionate caused the reduction of methanogenic count by an approximately 2 log value. Total volatile fatty acids increased with the increase in propionate concentration and particularly accumulation of propionate was ob- served. The results were also compared with the 30 �C fermentation. � 2002 Elsevier Science Ltd. All rights reserved. Keywords: Night soil; Biomethanation; Biodegradation; Propionate toxicity; Biogas; VFAs; Methane; Biodigester 1. Introduction Human waste disposal in highly populated and de- veloping countries such as India is an ever-growing problem. The improper disposal of waste causes a seri- ous threat of organic pollution to the environment and also several infectious diseases are bound to occur in epidemic proportion due to contamination of drinking water resources. The problem is more aggravated in high altitudes and snow bound areas of the Himalayan ranges and in Tundra where no proper treatment is practiced. In Antarctica, a similar problem is being en- countered by the expedition teams due to strict inter- national enforcement laws on the disposal of night soil and other wastes that pollute the pristine habitat of the continent. At present human waste is either incinerated or physically transported out of the continent. The technology of anaerobic biodegradation offers a rapid, effective and convenient mean of night soil disposal. However, the rate of biodegradation is reduced drasti- cally at low temperatures due to decreased growth of microorganisms. Acetic, propionic and butyric acid are the major volatile fatty acids (VFAs) formed during anaerobic biodegradation of night soil. These main substrates in the terminal step of methanogenesis are inhibitory to the process at higher concentrations (Boone and Xun, 1987; Zehnder, 1978). It has also been observed that some VFAs, for example propionate, are more toxic than others (Kugelman and Chin, 1971; McCarty and Mc- Kinney, 1961). During the biodegradation of organic compounds in anoxic methanogenic environments, propionate is a major intermediate. Studies showed that methanogenic propionate degradation takes place only by a collaboration of two different groups, propionate oxidizing bacteria and methanogenic bacteria. Propio- nate oxidizers such as Syntrophobacter wolinii oxidize propionate to acetate and carbon dioxide, producing hydrogen gas and formate as reduced products (Boone and Bryant, 1980). This reaction is endergonic under standard conditions, but methanogens or other mi- crobes, which use hydrogen or formate, can maintain concentrations of these molecules sufficiently low so that propionate degradation remains exergonic. Very limited work has been carried out on human waste disposal at low temperature (Sai Ram et al., 1993; Singh et al., 1995, 1999). Zeeman et al. (2000) recom- mended the use of an accumulation system for low Bioresource Technology 87 (2003) 299–303 *Corresponding author. Tel.: +91-751-233489; fax: +91-751- 341148. E-mail address:
[email protected] (R.K. Dhaked). 0960-8524/03/$ - see front matter � 2002 Elsevier Science Ltd. All rights reserved. PII: S0960-8524 (02 )00227-4 temperature treatment (20 and 15 �C) of a mixture of swill and night soil as against a completely stirred tank reactor system for mesophilic digestion (30 �C). How- ever, no investigation has been made on the effect of propionate at psychrophilic temperature. In our earlier studies, it was reported that human waste (night soil) degradation at low temperature resulted in decreased biogas production and digester failure due to accumu- lation of VFAs (Singh et al., 1993). Barredo and Evison (1991) observed the inhibition of methanogenesis at higher concentrations of propionate at mesophilic tem- perature. Several investigators have observed propio- nate inhibition under unbalanced anaerobic conditions, in increased and low hydrogen pressure (Archer et al., 1987; Boone and Xun, 1987; Fox et al., 1988; Mosey and Fernandes, 1988). Propionate utilising microbes appear to play an important role when fermentors are subjected to overload conditions (Boone, 1984; Boone and Bryant, 1980). The aim of the present study was to observe the toxic effect of propionate on biomethanation of night soil. The effect was studied at 10 �C and the results were compared with a 30 �C fermentation. 2. Methods 2.1. Inoculum The fermenting slurry from an anaerobic biodigester running on human night soil at 10 �C was used as in- oculum. It contained 4552 ppm total VFAs and 2.4% volatile solids. 2.2. Experimental set-up Batch type experiments were conducted in serum vials of 60 ml capacity containing 20 ml of night soil (1:1 diluted) with an equal volume of inoculum. Propionate (Sigma Chemicals) was added at 20–200 mM concen- trations. Bottles without propionate served as control. The pH of the mixture was set at 6.0, 7.0 or 8.0. All preparations were carried out under anaerobic condi- tions (N2, H2 and CO2 in the ratio of 8.5:1.0:0.5). Three sets of each treatment were incubated at 10 and 30 �C for 25 days. 2.3. Methanogens counts Methanogenic bacteria were enumerated by the most probable number (MPN) method by growing them in the media described by Balch et al. (1979). Serum vials containing 18 ml of liquid medium, prepared under an- aerobic conditions (N2, H2 and CO2 in the ratio of 8.5:1.0:0.5), were used for methanogenic counts. Ten- fold serial dilutions of the well mixed samples were pre- pared in the dilution medium containing NaH2PO4 � 2H2O, 2.964; Na2HPO4, 11.49; cystine HCl, 0.025; sodium sulphide, 0.025 and resazurin, 0.001 g l�1. Two milliliter of each dilution was inoculated in a set of five vials under aseptic conditions. The bottles, along with the control were incubated at 37 �C for 15 days. Gas from the headspace was analysed for methane on a gas chromatograph. Methanogenic MPN was computed on the basis of the bottles showing positive test and a standard table. 2.4. Analysis Total biogas was measured by plunger displacement of an airtight syringe. Methane contents and VFAs were determined by a Shimadzu gas chromatograph (GC 9A) equipped with a flame ionization detector and free fatty acid phase column. For methane estimation nitrogen flow was 50 ml/min and column and injector tempera- tures were 90 and 110 �C, respectively. VFAs concen- trations were determined after acidification and ether extraction of samples. The conditions used for VFAs estimations were nitrogen flow, 50 ml/min; column temperature, 120 �C and injector temperature, 140 �C. The results reported are the average values of triplicate determinations. 3. Results 3.1. Effect on biogas production The cumulative biogas production in the presence of different amount of propionate at 10 and 30 �C is shown in Figs. 1 and 2, respectively. The anaerobic fermenta- tion was faster at higher temperature (30 �C) with in- crease in biogas production by 2.0–3.3 times depending upon pH. The propionate was found to be toxic at both the temperatures and at all the pH tested. However, the Fig. 1. Effect of propionate on biodegradation of night soil at 10 �C. 300 R.K. Dhaked et al. / Bioresource Technology 87 (2003) 299–303 effect was more pronounced at 30 �C, with a drastic decrease in biogas production at pH 7.0 and 8.0. There was a gradual decrease in biogas production with the increase in propionate concentration at 10 �C. The trend was similar at 30 �C except at pH 7.0 and 8.0 where propionate at concentration of more than 80 mM se- verely inhibited biomethanation. The maximum reduc- tion in biogas production at 200 mM propionate was observed at pH 7.0 (60.4% at 10 �C and 77% at 30 �C) followed by pH 8.0 (54% at 10 �C and 70% at 30 �C). 3.2. Effect on methanogenic counts and methane content The total methanogenic counts were comparatively low at 10 �C on all the pH tested (Table 1). The highest counts were observed at 30 �C (pH 7.0). Addition of propionate to slurry resulted in lower numbers of methanogens and the counts decreased with rise in propionate concentration. The effect was more pro- nounced at 10 �C and appeared to be the pH indepen- dent. A two-log reduction in methanogenic counts was observed in the slurry fermenting at pH 6.0–8.0. How- ever, the reduction in the number of methanogens at 30 �C was pH dependent and the effect was most pro- nounced at pH 7.0 where counts decreased by 2 log, followed by a 1 log reduction at pH 6.0. Interestingly, the viable counts of methanogens remained unaffected at pH 8.0. Similar to the methanogenic counts, methane content of the biogas decreased with the increase in propionate concentrations (data not shown). The decrease in meth- ane content varied from 4.5% to 16.5% depending upon temperature and pH. The reductions in methane content were comparable at both the temperatures for the par- ticular pH values, except at 30 �C (pH 6.0) where maximum decrease at 200 mM propionate was recorded (16.5%). At 10 �C the methane content in the control was maximum at pH 7.0 (74%) followed by pH 8.0 (71%) and pH 6.0 (62.5%). It decreased with the in- creased propionate concentrations and in the presence of 200 mM propionate the methane contents were 53%, 62.5% and 65% at pH 6.0, 7.0 and 8.0, respectively. The same trend was recorded at 30 �C and methane contents decreased from 60.5% to 44% (pH 6.0), 72.5% to 62% (pH 7.0) and 69.5% to 65% (pH 8.0) in the presence of 200 mM propionate. 3.3. Accumulation of volatile fatty acids The VFA levels, after 25 days of batch fermentation, increased with propionate concentration ranging from 8860 to 42,468 ppm (Tables 2 and 3). The total VFA increased with propionate concentrations in a similar fashion at both temperatures, except at pH 6.0 where accumulation was more pronounced at 10 �C. Propio- nate was the major component in the accumulated VFA, ranging from 26% to 85% on a weight-by-weight bases. There was no accumulation of any other fatty acid up to caproate, and, rather, a decrease in acetate level was observed at both the temperatures, which was more appreciable at pH 7.0 and 8.0. 4. Discussion In general, studies have been confined to biogas production at mesophilic and thermophilic tempera- tures. Low temperature has a deleterious effect on methanogenesis leading to decreased biogas production and digester failure. Two to 3.3 times decrease in biogas production in the present investigation (Figs. 1 and 2) is in accordance with our earlier findings (Singh et al., 1993). Among different groups of bacteria involved in the process, methanogens are the most fastidious organisms. These bacteria are highly sensitive to the microenvi- ronment including H ions, O2, nutrients, metabolites, acetate, propionate and other short chain fatty acids. Fig. 2. Effect of propionate on biodegradation of night soil at 30 �C. Table 1 Effect of propionate at different pH on total methanogenic counts (ml�1) Propionate concentration 10 �C 30 �C pH 6.0 pH 7.0 pH 8.0 pH 6.0 pH 7.0 pH 8.0 0 mM (control) 1:2� 105 2:0� 105 3:0� 105 2:0� 106 2:5� 107 2:0� 107 100 mM 9:5� 103 2:5� 104 2:5� 104 4:5� 105 1:1� 107 1:6� 107 200 mM 4:5� 103 1:5� 103 1:5� 103 2:0� 105 4:5� 105 1:2� 107 R.K. Dhaked et al. / Bioresource Technology 87 (2003) 299–303 301 Propionate was the most dominant VFA accumu- lated at both mesophilic and psychrophilic temperatures (Tables 2 and 3). The effect of propionate toxicity has also been studied on pure cultures of methanogens in defined culture conditions by Barredo and Evison (1991). A decrease in methanogenic counts by two orders of magnitude was reported at propionate con- centrations above 80 mM. However, this study was re- stricted to mesophilic temperature. The effect of propionate on methanogenic counts in the present in- vestigation was also more or less similar at 10 �C where counts decreased by two orders of magnitude. However, the effect was pH dependent at 30 �C. Slurry fermenting at pH 8.0 showed very little ( would like to thank Mr. Arvind Tomar for his technical support. References Archer, D.B., Powell, G.E., Hilton, M.G., Tatton, M.J., 1987. Control of anaerobic digestion by H2 and acetate utilizing methanogenic bacteria. In: Wise, D. (Ed.), Bioenvironmental Systems, vol. 2. CRC Press, Inc., Boca Raton, FL, pp. 114–133. Balch, W.E., Fox, G.E., Margum, L.J., Woese, C.R., Wolfe, R.S., 1979. Methanogens: re-evaluation of a unique biological group. Microbiolol. Rev. 43, 260–276. Barredo, M.S., Evison, L.M., 1991. 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Table 3 Total and individual VFA at 30 �C P (mM) VFA (%) TVFA ppm A P IB B IV V C pH 6.0 0 32 26 7 16 9 5 1 12,855 20 21 23 5 12 7 5 0.8 14,762 40 23 41 1.7 14 8.7 8 2.6 15,440 60 15 48 1.1 15 9.2 9 1.0 14,610 80 13 53 0.7 14 16 6.2 2.1 15,303 100 12 56 0.7 14 9 6 0.7 19,168 150 11 60 0.5 13 7.8 6.1 0.9 23,549 200 8.4 63 nd 12 7.8 7.2 0.8 21,039 pH 7.0 0 8.4 41 9 20 11 7 1.1 8860 20 9.2 43 8 15 11 11 1.5 10,568 40 13 55 2 15 9 5.3 0.3 13,674 60 15 52 1.6 13 9.2 7 0.3 14,818 80 17 58 0.9 13 7 4 0.2 16,998 100 20 56 0.9 12 7 4 0.7 19,691 150 19 60 0.8 10 6 4 0.5 25,837 200 11 64 0.4 11 7 6.2 0.1 29,555 pH 8.0 0 22 34 10 17 11 6 0.2 13,224 20 20 47 1.8 15 11 9.2 3 14,134 40 18 52 1.5 15 8.8 4 1.3 15,901 60 9.3 57 0.9 16 10 6 0.5 15,910 80 9.1 57 0.7 15 10 7 1.1 17,752 100 8.1 57 0.5 14 10 8.4 2.0 18,712 150 7 64 15 8 5 0.1 0.7 21,943 200 1 70 15 8 nd 7 0.6 26,661 VFA ¼ volatile fatty acid, A ¼ acetate, P ¼ propionate, IB ¼ isobutyrate, B ¼ butyrate, IV ¼ isovalerate, V ¼ Valerate, C ¼ caproate, TVFA ¼ total volatile fatty acids and nd ¼ not detected. R.K. Dhaked et al. / Bioresource Technology 87 (2003) 299–303 303 Effect of propionate toxicity on methanogenesis of night soil at phychrophilic temperature Introduction Methods Inoculum Experimental set-up Methanogens counts Analysis Results Effect on biogas production Effect on methanogenic counts and methane content Accumulation of volatile fatty acids Discussion Acknowledgements References