ELSEVIER 0 9 6 0 - 8 5 2 4 ( 9 5 ) 0 0 0 7 5 - 5

Bioresource Technology 53 (1995) 147-149 © 1995 Elsevier Science Limited

Printed in Great Britain. All fights reserved 0960-8524/95/$9.50

PRODUCTION OF BIOGAS FROM NIGHT SOIL AT PSYCHROPHILIC TEMPERATURE

Lokendra Singh,* M. S. Maurya, K. V. Ramana & S. I. Alam

Biotechnology Division, Defence R&D Establishment, Gwalior-474 002, India

(Received 21 November 1994; revised version received 2 May 1995; accepted 11 May 1995)

Abstract Anaerobic digestion of night soil was carried out in a 25 l digester at 10°C using an adapted inoculum. Bio- gas production was studied at 20, 25, 30, 35 and 40 days hydraulic retention time. Digesters operated at 25 day hydraulic retention time produced 69"72 l biogas/kg VS/day with a methane content of 73"0%. Microbial counts and enzyme activities are also reported.

Key words: Psychrophilic, night soil, biogas, adapta- tion, hydrolytic bacteria, enzymes.

INTRODUCTION

Biogas is one of the best renewable energy sources and it also has a special significance in the context of depleting energy reserves in populous countries like India. Most of the studies on farm or village biogas production have concentrated on cowdung as a raw material. Only a few studies have been carried out with night soil (Hashimoto et al., 1982a;b; Sai Ram et al., 1993). Approximately 60 million tonnes of night soil is produced annually in India. The bio- conversion of this material into methane could mitigate the energy problem of the country to some extent.

In general, studies have been confined to biogas production at mesophilic and thermophilic tempera- tures. Low temperature has a deleterious effect on methanogenesis and can cause decreased gas yields and digester failure. In our previous studies (Singh et al., 1993) we used as the inoculum slurry from digesters operating at mesophilic temperatures, which was adapted gradually to 20°C. The fermenta- tion of night soil was carried out at the adapted temperature since tests at lower temperatures did not prove successful.

The present study, therefore, deals with the fer- mentation of night soil at 10°C using an adapted inoculum from cowdung digesters established in hilly areas of India. Attempts were also made to optimise the HR T in 25 1 digesters.

*Author to whom correspondence should be addressed.

METHODS

Adaptation of slurry The slurry was collected from four different digest- ers operating with cowdung as the substrate in a hilly area of north India. These digesters were com- prised of a floating-dome with cowdung, fixed-dome with cowdung, fixed-dome with cowdung plus Agar- atum and fixed-dome with cowdung plus Lantana. The slurries were initially adapted to night soil in 2 1 flasks (semi-continuous) at 20°C for 3 months. Sub- sequently, the slurry exhibiting maximal biogas production was further adapted in 251 digesters (semi-continuous) to a minimal temperature. The slurry was subjected to decreasing temperature by 2°C at each 4 month interval until it reached 10°C. Thus five steps (at 4 month intervals) for adaptation to decreasing temperature were undertaken. The best slurry was used to inoculate the following digesters.

Digestion of night soil Semi-continuous fermentation was carried out in 25 1 metal digesters of the floating-dome type with a working volume of 20 I. Night soil was diluted 1:1 (wet wt/vol) with tap water and feeding was done every 24 h at 20, 25, 30, 35 and 40 days HRT using two digesters for each HRT. Digestion was carried out at 10°C in an environmental chamber.

Analysis Gas production was monitored daily. Volatile fatty acids, pH of the slurry and methane content in the biogas were measured at weekly intervals. Enzymes and microbial counts in the slurry were monitored at the end of the experiment.

Volatile fatty acids (VFA) and gas analysis Methane and the VFA were determined by using a gas chromatograph (Shimadzu) fitted with a flame ionisation detector and FFAP column.

147

Microbial counts and enzyme assays Proteolytic, lipolytic, cellulolytic and amylolytic bac- teria were enumerated by inoculating the diluted

148 L. Singh, M. S. Maurya, K. V. Ramana, S. I. A lam

slurry into casein agar, tributyrin agar, carboxyme- thyl cellulose agar and starch agar. The plates were incubated in anaerobic jars for 3 days at 10°C under Nz+H2 in the ratio of 8:2. Sulphate-reducing and methanogenic bacteria were determined by an MPN technique using media as described by Pfennig et al. (1981) and Balch et al. (1979), respectively. Cellulase or amylase activities were determined by incubating 1 ml of slurry supernatant with 1 ml of 1% CMC or soluble starch in 2 ml of acetate buffer (0.1 M, pH 6"0) for 1 h at 30°C. The reducing sugars released were determined using DNS reagent (Miller et al., 1959). Protease activity was determined by the method described by Malathi and Chakraborty (1991) and lipase activity as per Sztajer and Malis- zewska (1988).

RESULTS AND DISCUSSION

During batch fermentation of night soil in 21 flasks, slurry from Agaratum and Lantana (residues) digest- ers produced 2.2-2.3 1 biogas in a period of 17 days at 20°C, but slurries from cowdung digesters yielded only 1.46-1.81 biogas. Hence, the mixture of Agar- atum and Lantana slurries was adopted for further studies. The results indicated in the study are aver- ages of two replicates. The biogas production from night soil at 10°C is shown in Fig. 1. There was a rise in biogas production in the first week in all the digesters, but the 20 day HRT digester showed a continuous decrease in biogas production after the first week. The digesters reached a stable biogas

8

6

t

2

I r I I I I I I [ I I I I 0 I 2 3 4 5 6 7 8 9 iO I I 12 13

Weeks

Fig. 1. Biogas production from night soil at 10°C at dif- ferent hydraulic retention times: x 20 days, • 25 days, 30

days, * 35 days, [] 40 days.

production by about 9-10 weeks. Maximum biogas was produced at 25 day HRT and was followed by 30, 35 and 40 days HRT digesters. The pH of the digesting slurry varied from 7-2 to 7.4 during the course of digestion at 25-40 day HRT. The pH of the 20 day HRT-digester slurry dropped to 6-8 by the end of the second week. VFA in the digesting slurry in the last week of the experiment are shown in Table 1. At 20 day HRT, VFA accumulation became much higher than in digesters working at any other HRT. At 20 day HRT, propionate concen- tration was about three times higher than that of acetate, whereas at higher HRT both VFA were maintained at almost equal concentration. Propio- nate has been reported to be toxic for methanogens (Ranade et al.., 1987) and this fact coupled with the fall in pH might have caused the lower gas produc- tion in the 20 day HRT digester. Acetate accounted for only 17% of the total fatty acid content. It appears that conversion of higher fatty acids to acet- ate, a substrate for methanogens, was inhibited. Data on biogas production showed that digesters operating at 25 day HRT produced the maximum amount of biogas at 10°C. Hence, hydrolytic bacte- rial counts and enzyme quantification were carried out with 25 days HRT digester slurry. Among the hydrolytic microbial population, proteolytic bacteria were the most dominant (1 x 107/ml) followed by lipolytic (1.2 x 106/ml), amylolytic (1.1 x 105/ml) and cellulolytic (9 x 103/ml) bacteria. Dominance of pro- teolytic bacteria was as expected due to the high protein content of night soil. The methanogens were 100 times higher than sulphate reducers. Analysis of hydrolytic enzymes at 10-30°C showed maximum activity at 30°C. Amylase, CMCase and lipase activ- ities at 10°C were about one-third of the activities of the same samples incubated at 30°C. Protease activ- ity at 10°C, however, was one quarter that at 30°C. This showed that the adapted microbial consortium had developed the ability of anaerobic digestion at 10°C but the ability to produce hydrolytic enzymes active at low temperatures had not increased. Table 2 shows that biogas yield increased with the increas- ing HRT and thus decreasing loading rates. Accordingly, the highest yield was obtained at 40 day HRT (14.0 l/kg wet NS/day; 79.36 l/kg VS/day), which was because of the optimum utilisation of substrate at low loading rate. One of the objectives

Table 1. Volatile fatty acids (ppm) during anaerobic digestion of night soil at different HRT

VFA Hydraulic retention time

20 days 25 days 30 days 35 days 40 days

Acetate 1515 347 161 100 80 Propionate 4081 363 159 92 60 Isobutyrate 387 26 ND ND ND Butyrate 1933 54 40 ND ND Isovalerate 230 ND ND ND ND Valerate 602 40 ND ND ND

Psychrophilic digestion of night soil 149

of anaerobic digestion is to reduce the HRT; thus by lowering the volume of digester for a constant amount of substrate it reduces the cost of installa- tion and operation. Though digesters at all the five HRT attained a steady-state, the one running at 20 day HRT produced the lowest quantity of biogas (1.8 l/day) with the lowest methane content (49%). The digester at 25 day HRT produced a good amount of biogas with 73.0% methane content.

From the above study it is concluded that anaero- bic digestion of night soil can be carried out at 10°C using an adapted inoculum. It is desirable to operate the digesters in semi-continuous mode with 25 day HRT using 1:1 diluted night soil.

ACKNOWLEDGEMENTS

The authors are thankful to Dr R. V. Swamy, Direc- tor, and Shri K. M. Rao, Joint Director, D.R.D.E., Gwalior, for their encouragement and for providing facilities to carry out this work.

REFERENCES

Balch, W. E., Fox, G. E., Magrum, C. R., Woese, C. R. & Wolfe, R. S. (1979). Methanogens: reevaluation of a unique biological group. Microbiol. Rev., 43, 260-96.

Hashimoto, S., Fujita, M. & Baccay, R. A. (1982a). Effect of temperature and sludge retention time on the anaer- obic digestion of night soil. J. Ferment. Technol., 60, 139-48.

Hashimoto, S., Fujita, M. & Baccay, R. A. (1982b). Kinet- ics of the effect of temperature on the anaerobic digestion of night soil. J. Ferment. Technol., 60, 149-55.

Malathi, S. & Chakraborty, R. (1991). Production of alka- line protease by a new Aspergillus flavus isolate under solid-substrate fermentation conditions for use as a depilation agent. Appl. Environ. Microbiol., 57, 712-6.

Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem., 31, 426-8.

Pfennig, N., Widdel, F. & Truper, H. G. (1981). The dissimilatory sulfate reducing bacteria. In The Prokar- yotes, ed. M. P. Starr, H. Stolp, H. G. Truper, A. Balows & M. P. Schlegel. Springer, Berlin, Germany, pp. 926-40.

Ranade, D. R., Yeole, T. Y. & Godbole, S. H. (1987). Production of biogas from market waste. Biomass, 13, 147-53.

Sai Ram, M., Singh, L. & Alam, S. I. (1993). Effect of sulfate and nitrate on anaerobic degradation of night soil. Biores. Technol., 45, 229-32.

Singh, L., Maurya, M. S., Sai Ram, M. & Alam, S. I. (1993). Biogas production from night s o i l - effects of loading and temperature. Biores. Technol., 45, 59-61.

Sztajer, H. & Maliszewska, I. (1988). Production of exoge- nous lipases by bacteria, fungi and actinomycetes. Enzym. Microbiol. Technol., 10, 492-7.