Bioresource Technology 58 (1996) 323-325 Copyright 1997 Elsevier Science Limited Printed in Great Britain.

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ELSEVIER PII:S0960-8524(96)O0119-8

Short Communication
Effect of Brackish Water on Biogas Production from Cattle Dung and Methanogens
Abstract

which are commonly found in anaerobic digesters (Ranade et al., 1980).

METHODS

Semicontinuous anaerobic digestion of cattle dung using brackish waters with conductivity from 1000-15000 pS/cm was carried out in 25 l biogas plants of floating-dome design. Water with a conductivity higher than 5000 pS/cm was inhibitory to the process. Experiments using three cultures of methanogens: Methanobacterium bryantii, Methanobacterium formicicum and Methanosarcina barkeri, confirmed these findings. Methanobacterium bryantii was found to tolerate water of the highest conductivity. Copyright 1997 Elsevier Sc&nce Ltd. Key words: Cattle dung, biogas, brackish water, conductivity, methanogens, salts.
INTRODUCTION

In India biogas technology has become popular, particularly in rural areas, to obtain cooking fuel. There are over 1.98 million family-size biogas plants and most of these are run on cattle dung (CD) (Anon, 1994). Biogas-plant owners use ground-water to prepare the slurry for the daily feed. In many parts of the country ground-water has become brackish, with high conductivity and high salt content. The quality of ground-water varies from place to place (Minhas & Gupta, 1992). Therefore, even under otherwise identical environmental conditions, the yield of biogas per unit mass of CD could vary from place to place. The effect of different environmental factors on biogas production from CD have been studied (Singh et al., 1982; Malik et al., 1984; Ranade et al., 1990). McCarty and McKinney (1961) studied salt toxicity in anaerobic digestion of substrates other than CD. A few authors have reported the inhibitory effect of pure salts on methanogens (Patel & Roth, 1977; Zinder, 1993). However, the effects of brackish waters on biogas production from CD and pure cultures of methanogens have not been reported. To determine any effects, experiments were carried out on anaerobic digestion of CD and cultures of three methanogens: Methanobacterium bryantii, Methanobacterium formicicum and Methanosarcina barkeri, 323

Brackish water from a well about 60 km away from Pune showed the highest conductivity (15000 /~S/ cm) amongst a number of wells tested. Brackish and tap waters (conductivity _<50 /~S/cm)were analysed for CI-, N O ; , S O z - , S - - and Ca ++, K +, Mg ++, Na by standard methods (APHA, 1992). Conductivity was measured by a conductivity meter (Toshniwal, Bombay). Brackish water was suitably diluted with tap water to have conductivities from 1000 to 15000 pS/cm. Seven biogas plants (25 1) were charged initially with CD slurry (8% TS), prepared using equal quantity of CD and the water under test (1000, 5000, 7500, 10000, 12500, 15000 pS/cm) and tap water which served as the control. After stabilization of these plants daily addition of 830 ml CD slurry (8% TS) prepared with 415 g CD and 415 ml of the respective water was then commenced to give a 30 day hydraulic retention time (HRT). The experiment was run at ambient temperature (26-30C) over a period of 100 days. Daily gas produced from each plant was measured with a wet gas-flow meter (Toshniwal, Madras) and analysed daily for methane by gas chromatography (Ranade et al., 1987). Volatile fatty acids (VFA) in the effluent from each plant were estimated twice a week by gas chromatography (Yeole et al., 1989). Different dilutions of the brackish water under test, as prepared earlier, were used for media preparation along with tap water, which served as the control. The medium described by Touzel and Albagnac (1983) with H2:CO2 (80:20) as gas phase was used for M. bryantii (MCMB 606) and M. formicicum (MCMB 607). The same medium, but amended by the addition of acetate (50 mM) as the substrate and nitrogen as the gas phase, was used for Ms. barkeri (MCMB 701). These cultures were obtained from the 'MACS Collection of Microorganisms' established at this Institute. The medium was dispensed (20 ml aliquots) anaerobically (Miller & Wolin, 1974) in 65 ml serum vials. These vials were inoculated in duplicate with test organisms (2 ml log phase culture) and incubated at 35C for 7 days. Inoculated vials with Hz:CO2 were pressur-

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Short communication
Table 1. Analysis of the brackish water*

Anion & cations (mM) Tap water < 50 C1NOZ SO4 Ca Mg Na t *Averages of four samples. ized daily with H2"CO2 ( = 1 atm) to ensure availability of the substrate. At the end of the incubation period a 0.5 ml gas sample from each vial was analysed for methane production. 0.10 0.00 0.08 0-05 0-45 0.00 1000 7.2 1.0 1.9 1-8 5.8 1.4

Conductivity (pm/cm) Brackish water 5000 36.8 4.9 9-8 9.5 27.5 6.8 7500 55.5 8.1 14.8 13.6 39-2 11.8 15000 112.8 15.5 30.5 28.0 80.5 22-5

RESULTS AND DISCUSSION

The analyses given in Table 1 showed a very high content of anions and cations in the brackish water compared to tap water. Chlorides and Mg + were in highest concentration; S - and K + were not detected in any waters. The results presented in Table 2 indicated that brackish water up to 5000 pS/cm was not inhibitory to the process, and in fact there was 18.5 and 6.5% stimulation in gas production due to the use of 1000 and 5000 pS/cm conductivity water when compared to the control. However, there was a linear decrease in gas production with the increase in conductivity above 5000 /~S/cm. There was not much difference in methane content of the gas and pH of the fermenting slurries of all the plants (50-53% CH4 and pH 6.5-7). The VFA of the fermenting slurry showed a slight increase with an increase in conductivity. The culture studies showed that M. formicicum and Ms. barked produced more methane than the control when grown in the media with low conduc-

tivities, up to 5000 /~S/cm. At higher conductivities methane production was progressively inhibited, being less than 50% of the control at 15 000 #S/cm. M. bryantii produced maximum methane at 7500/~S/ cm conductivity and it tolerated waters of higher conductivities, upto 10000 pS/cm, after which methane production fell below the control. Perski et al. (1982) have reported that a low concentration of Na is required for the formation of methane from H2+CO2, acetate and methanol. However, the presence of Na in a higher concentration than the optimum (10 mM) lowers the specific growth rate and yield of acetoclastic methanogens (Kugelman & Chin, 1971). In another study, Patel and Roth (1977) have stated that CI-, though in a minute quantity, is required for maximum methanogenic activity. In the present study, also, low conductivity water ( < 5000 #S/cm) containing a low concentration of these ions proved stimulatory to methane production (Table 2). These findings are in agreement with the quoted authors. With respect to N O 3 concentration, Belay et al. (1990) and Sai Ram et al. (1993) have reported that N O 3 above 5 mM affects methanogenesis; however, SO4 is not inhibitory even at 30 mM concentration. In our study high conductivity water (5000/~S/cm) contained Na 11.8, N O 3 8.2 and CI- 55.5 mM or more. All these ions must have contributed to the inhibition of methanogenesis. Among these ions N O 3 is the most toxic.

Table 2. Production of biogas from cattle dung using brackish water

Plant no.

Conductivity of water (#S/cm)

Biogas" (l/day)

Methane

Effluent pH VFA (mg/l) 395 450 465 550 665 705 780

% (v/v)

1 (control) 2 3 4 5 6 7

<50 1000 5000 7500 10000 12500 15 000

9-5 11-25 10.15 6"90 6-10 5.75 4.50

51-5 53-4 52"5 52.0 51.5 50.2 50-0

6-80 7.0 7-20 7.15 7.10 6.85 6.80

Results are the average of the last 60 days of the experiment. a Gas volume corrected to STP.

Short communication Considering the anaerobic nature of methanogens, the more toxic effect of N O 3 compared to other ions could be explained on the basis of oxidation level, as N O y is a stronger oxidizing agent than C I and SO4. Hajarnis (1993) in his study reported that amongst the cations Na , Ca + and Mg + studied, Na was more inhibitory than Ca + and Mg +. In our study, though the brackish water contained a high concentration of Ca + , Mg ++ and SO4 , these ions were presumably less toxic than Na to methanogens. The methanogens used in the present study were mesophilic and non-halophilic. The brackish water was deficient in K but contained a high concentration of Na , hence the K transport system in methanogens would be affected (Sprott et al., 1985), and this also could have affected the methanogenic activity. Along with the other factors impeding methane production in the field one should also suspect high conductivity in the water being used. Hence, it is advisable to carry out the analysis of water for its conductivity and specific ions like Na , CI-, N O 3 , etc., before predicting gas yield in the particular area. Use of bacterial cultures, especially methanogens adapted to grow in water of high conductivity or high concentrations of specific ions, could help in alleviating the salt toxicity and, in turn, the poor yield of methane.

325

ACKNOWLEDGEMENT The authors are thankful to the Ministry of NonConventional Energy Sources, Government of India, New Delhi, for the financial support for the investigations.

Kugelman, I. J. & Chin, K. K. (1971). Toxicity, synergism and antagonism in anaerobic waste treatment processes. Adv. Chem. Ser., 105, 55-90. Malik, R. K., Singh, R. & Tauro, P. (1984). Optimization of biogas production from cattle waste. Urja June 1984, p. 429. McCarty, P. L. & McKinney, E. (1961). Salt toxicity in anaerobic digestion. JWPCF, 33, 399-415. Miller, T. L. & Wolin, M. J. (1974). A serum bottle modification of Hungate technique for cultivating obligate anaerobes. Appl. Microbiol., 27, 985-987. Minhas, P. S. & Gupta, R. K. (1992). Quality of Irrigation Water, Assessment and Management. Published by Publication and Information Division, Indian Council of Agricultural Research, Krishi Anusandhan Bhavan, Pusa, New Delhi, India. Patel, G. B. & Roth, L. A. (1977). Effect of sodium chloride on growth and methane production of methanogens. Can. J. Microbiol., 23, 893-897. Perski, H. J., Schonheit, P. & Thauer, R. K. (1982). FEBS Lett., 14, 323-326. Ranade, D. R., Gore, J. A. & Godbole, S. H. (1980). Methanogenic organisms from fermenting slurry of the gobar gas plant. Current Sci., 49, 395-397. Ranade, D. R., Yeole, T. Y. & Godbole, S. H. (1987). Production of biogas from market waste. Biomass, 13, 147-153. Ranade, D. R., Nagarwala, N. N., Dudhbhate, J. A., Gadre, R. V. & Godbole, S. H. (1990). Production of biogas at different total solid content in cattle dung. Indian J. Environ. Health, 32, 63-65. Sai Ram, M., Singh, L. & Alam, S. I. (1993). Effect of sulphate and nitrate on anaerobic degradation of night soil. Biores. Technol., 45, 229-232. Singh, R., Jain, M. K. & Tauro, P. (1982). Rate of anaerobic digestion of cattle waste. Agric. Waste, 4, 267-272. Sprott, G. D., Shaw, K. M. & Jarrell, K. F. (1985). Methanogenesis and the K transport system are activated by divalent cations in ammonia-treated cells of Methanospirillum hungatei. J. Biol. Chem., 260, 9244-9250. Touzel, J. P. & Albagnac, G. (1983). Isolation and characterization of Methanococcus mazei strain MC3. FEMS Microbiol. Lett., 16, 241-245. Yeole, T. Y., Ranade, D. R. & Gadre, R. V. (1989). Biogas from liquid waste arising in liver and beef extract production. RERIC Int. Energy J., 11, 35-39. Zinder, S. H. (1993). Physiological ecology of methanogens. In Methanogenesis, ed. J. G. Ferry. Chapman & Hall, New York.

REFERENCES

Anon (1994). Annual Report 1994-95. Ministry of NonConventional Energy Sources, Government of India, New Delhi, India. APHA (1992). Standard Methods for the Examination of Water and Wastewater, 18th edn. American Public Health Association, Washington, DC. Belay, N., Jung, K. Y., Rajagopal, B. S., Kremer, J. D. & Daniel, L. (1990). Nitrate as a sole nitrogen source for Methanococcus thermolithotrophicus and its effect on growth of several methanogenic bacteria. Current Microbiol., 21, 193-198. Hajarnis, S. R. (1993). Inhibition of some methanogenic bacteria by certain chemicals, toxicants and their reaction. Ph.D. Thesis, University of Poona, Poona, India.

T. Y. Yeole,* S. Gokhale, S. R. Hajarnis* & D. R. Ranade

Agharkar Research Institute, Agarkar Road, Pune 411 004, India

(Received 25 July 1996; revised version received 26 August 1996; accepted 30 August 1996 *Author to whom correspondence should be addressed. *Present address: Applied Microbiology Department, Lund University, Lund, Sweden.