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Production of biogas from Azolla pinnata R.Br and Lemna minor L.: Effect of heavy metal contamination
Bioresource Technology' 41 (1992) 273-277
Production of Biogas from Azolla pinnata R.Br and Lemna minor L.: Effect of Heavy Metal Contamination S. K. Jain," G.. S. Gujral," N. K. Jha b & p. Vasudevan "Centre for Rural Development and Appropriate Technology, ~'Departmentof Chemistry, Indian Institute of Technology,Hauz Khas, New Delhi-110 016, India (Received 15 June 1991 ; revised version received 14 November 1991 ; accepted 24 November 1991 )
Abstract
The ahsorption of iron, copper, cadmium, nickel, lead, zinc, manganese and cobalt by Azolla pinnata R.Br and Lemna minor L., and subsequent utilization of this biomass for production of biogas (methane), have been investigated. Iron or manganese did not have any toxic effect on the anaerobic fermentation of Azolla and Lemna, while copper, cobalt, lead and zinc showed toxicity. At low concentrations cadmium and nickel showed a favourable effect on the rate ofbiogas production and its methane content, but with increase in concentrations rate of biogas production and methane content decreased. However, although there was this decrease in biogas production and methane content, the methane content of biogas was still higher than that which was obtained from non-c~ mtarninated biomass. Key words: Azolla pinnata, Lernna minor, heavy metals, anaerobic fermentation, biogas.
INTRODUCTION The rising cost of fossil fuels and high capital cost of transmission of electricity to rural areas suggest that local energy sources should be used wherever feasible. The greatest potential lies with biomass. In this context, cultivation of biomass for use in wastewater treatment and subsequent utilization for fuel production is economically appealing (Benemann, 1978). Since this biomass is a byproduct of wastewater treatment, this will not be a competitor of food- or fiber-producing plants. National Space Technology Laboratories, USA,
have investigated a variety of plants which have the common characteristic of high growth potential (Wolverton & McKown, 1976; Wolverton & McDonald, 1979, 1981). It is also important to utilize biomass which is locally available in sufficient quantities but with lower rates of growth. For example, aquatic weeds like Azolla pinnata R.Br (water velvet) and Lemna minor L. (duckweed), which grow commonly in the natural aquatic systems in India, are not uncontrollable invaders like water hyacinth. They will grow in wastewater and have a tendency to absorb and incorporate the dissolved materials from the water (NAS, 1976). Aquatic plants currently being considered for wastewater treatment could also be a good substrate for methane production. However, concern has often been expressed that some heavy metals absorbed from wastewaters may adversely affect the metabolic activity of microorganisms involved in the biodegradation of the biomass. These effects may vary from a lowering of the rate of biodegradation, thus increasing the time necessary for biological treatment, to complete cessation of the process (Ahring & Westermann, 1983; Dar & Tandon, 1987a, b; Stanogias & Pearce, 1987). The present paper discusses the effects of heavy metal contamination in Azolla pinnata and Lernna minor on their anaerobic fermentation to biogas.
METHODS Experimental conditions for absorption of heavy metals Adult Azolla and Lemna plants were obtained from a permanent pond in a village 20 km from
273 Bioresource Technolo~,~ 0960-8524/92/S05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain
274
S. K. Jain, G. S. Gujral, N. K. Jha, P. Vasudevan
Delhi and were kept in plastic tanks containing tap water for one week prior to starting the experiments. The 6.0 kg plant samples (fresh weight) were then transferred and allowed to float in 100 litres of experimental solution contained in a 200 litre plastic pot for 24 h (kept in a phytotron house). The plants were harvested, dried at 48°C and used as substrates for production of biogas. For each metal concentration four replicate samples were taken. The plants were exposed to the individual metals iron, copper, cadmium, nickel, lead, zinc, manganese and cobalt at nominal concentrations of 1-0, 4.0 and 8.0 mg/litre by pipetting appropriate amounts of 10 000 mg/litre metal solutions into a known volume of tap water. The stock solutions of the metals were prepared by dissolving nitrate or sulphate salts in deionized water. The plants were also exposed to solutions of mixtures of these metal ions: iron+ copper, cadmium + nickel, lead + zinc and manganese + cobalt, each at 1.0, 4-0 and 8.0 mg/litre under conditions as described above. The tap water contained (mg/litre): Ca 15.6, Mg 2.8, Na 1-2, K 0.23, C1 9.7, Fe 0.16, Mn 0.21, Cu 0.005 and total dissolved solids 89"2. The other heavy metals under study were not present in the tap water. The water pH was 7.2-7.4. The basic control group used the samples without addition of any metals.
Experimental set-up for biogas production The experiments for biogas production were carried out in a batch system (Maramba, 1978). A 2 litre capacity digester bottle was connected to a water-displacement gas-measuring bottle. The digester was charged with prepared experimental digester slurry which was a mixture of dried plant, unchlorinated water and inoculum. The inoculum was 15-day-old wet slurry from digested cattle manure and was added at 10% level. The total solids concentration in the feed was approximately 8%. The experiments were carried out in a fermentation chamber at 37°C. The digester bottle was vigorously shaken once a day so as to loosen the gas bubbles in the feed and allow the homogeneous mixing of the aquatic plants with inoculum as the former float on the surface. The digestion was carried out until the biogas production fell to zero. The gas samples for analysis were taken out with a syringe from the pressure tubing connecting the digester to the gas bottle. As a precaution to prevent escape of gas, the site was immediately sealed with Teflon tape.
Analysis The plant samples were ashed by heating for 8-10 h in a furnace at 800°C and the ash digested with concentrated nitric acid and filtered into a volumetric flask. The final volume was made up with deionized water and the solutions analysed for heavy metal content (Vogel, 1978) by atomic absorption spectroscopy using a Pye Unicam, Model Sp-191 single-beam atomic absorption spectrophotometer. The biogas was analysed using a Nucon-5700 gas chromatograph fitted with thermal conductivity detector and molecular sieve, stainless-steel column of 1.8 m length and 2 mm inner diameter. Working regimes were: temperature of the detector, injector and oven 40°C; current 150-155 A; and hydrogen as carrier gas at a flow rate of 30 ml per minute. Individual gases were identified on the basis of retention intervals compared to standard gases.
RESULTS AND DISCUSSION
Absorption of heavy metals by Azoila and Lemna The concentrations of heavy metals in Azolla pinnata R.Br and Lemna minor L. exposed to the metals (both single and mixed groups) are shown in Tables 1 and 2, respectively. A comparison of the metal contents of untreated Azolla and Lemna plants with those treated with metal ions shows that with increase in metal concentration in the initial solutions the percentage increase in metal content of Azolla and Lemna is maximum for cadmium and lead, the trend being Cd > Pb > Co > Ni > Cu > Zn > Mn > Fe. The percentage uptake of heavy metals by these two plants was highest when their initial concentration in solution was 1.0 mg/litre. The presence of one metal ion in solutions decreased the uptake rate of the other. Similar results have also been earlier reported (Jain etal., 1988, 1989a, b, 1990).
Effect of heavy metals contained in biomass on the production of biogas The amount of biogas produced from Azolla pinnata and Lemna minor at different concentrations of metal ions and its methane content are shown in Tables 1 and 2, respectively. Biogas data for non-contaminated biomass are shown in the first row. Comparison of data obtained from contaminated biomass with those obtained by digestion of non-contaminated biomass shows that iron and
275
Effects of heavy metals on digestion Table
1.
Effect of heavy metals contained in Azolla pinnata on its fermentation to biogas"
Metal
Group 1 (1"0mg/litre)
Group 111 ¢8"0mg/litre)
Group 11 (4"0 mg/litre)
% Metal Digestion Volume % Metal Digestion Volume % Metal Digestion Volume Methane content period of Methane content period of Methane content period of (/~g/g ( d a y s ) biogas &gig ( d a y s ) biogas (/~g/g ( d a y s ) biogas dry (litre/ kg) dry (litre/ kg) d 0' flitre/ kg) matte 0 matter) matter) 42 42 42 42
189 189 188 188
62 62 62 62
. 686 708 510 541
165 133 138 109
38 37 36
183 183 183
70 72 83
Pb Zn Pb+ Zn
167 130 158 122
42 42 42
180 184 180
Mn Co Mn+ Co
170 120 142 108
42 42 42
188 185 179
'
--
Fc Cu Fe+ Cu
174 169 143 148
Cd Ni Cd + Ni
. 42 42 42
.
. 186 185 185
614 442 497 352
38 37 36
58 60 60
558 481 525 424
62 60 60
612 468 565 410
.
.
.
62 62 62
1308 1498 963 829
183 183 183
70 72 79
1112 865 948 540
42 42 42
176 170 162
58 60 60
42 42 42
186 183 176
62 60 60
. 42 42 42
. 188 163 1711
62 59 60
42 42 40
1711 1711 170
65 66 62
1076 967 975 858
42 42 42
150 150 132
48 46 45
1112 791 1006 735
42 42 42
186 176 I65
62 59 58
"Average of four replicate samples w i t h r e l a t i v e mean deviations of _+5 - 0 % . ~'Non-contaminated Azolla; metal content (/~g/g): Fe 36"0, Cu 10-8, Cd 0'01, Ni 9.4, Pb 0-(I I. Zn 18"8. Mn 26'4 and Co 7"8.
"fable 2. Effect of heavy metals contained in Lemna minor on its fermentation to biogas"
Metal
Group 1 (1.0 mg/litre)
Group 11 (4"0mg/litre)
Group I11 (8"0mg/litre)
% Metal Digestion Vohone % Metal Digestion Vohtme % Metal Digestion Volume content period of Methane content period of Methane content period o] Methane (/~g/g ( d a y s ) biogas (ktg/g ( d a y s ) biogas (/~g/g ( d a y s ) biogas d O, (litre/kg) dr>, (litre/kg) dry (litre/kg) matter) matter) matter) 42 42 42 42
176 176 176 175
60 60 60 60
. 609 601 459 369
159 1119 127 96
38 37 36
176 174 172
71 73 85
Pb Zn Pb + Zn
155 117 141 112
42 42 42
172 170 172
Mn Co Mn + Co
140 104 120 103
42 42 42
176 174 172
r
--
Fe Cu F~+ Cu
156 152 142 126
Cd Ni Cd + Ni
. 42 42 42
. 174 170 172
.
. 61) 60 59
. 1158 1184 801 798
593 409 443 361
38 37 36
176 174 172
71 73 80
55 58 57
550 421 488 413
42 42 42
164 166 166
60 60 58
523 386 467 349
42 42 42
176 174 170
.
. 42 42 42
174 158 164
60 59 59
1070 7211 608 534
42 42 4{)
164 164 164
66 68 68
55 58 57
1062 734 853 698
42 42 42
142 142 132
45 44 43
611 58 58
865 706 796 593
42 42 42
176 164 167
61/ 54 56
"Average of four replicate samples w i t h r e l a t i v e m e a n d e v i a t i o n s of _+5 . 0 % . /'Non-contaminated Lemna; metal content (/~g/g): Fe 31-6, Cu 10.4, Cd I).3, Ni 9-8, Pb 0-3, Z n 18.6, Mn 23.6 and Co 6-9.
276
S. K. Jain, G. S. Gujral, N. K. Jha, P. Vasudevan
manganese did not affect biogas production at any of the concentrations studied. The presence of copper or cobalt at low concentration in the biomass slightly decreased the amount of biogas produced, but with increasing copper or cobalt concentration there was no significant change in the digestion period or in the methane content. Other reports have also shown that iron is not toxic even at extremely high concentration (Lawrence & McCarty, 1965). However, very little information is available on the effect of manganese and cobalt on anaerobic digestion of biomass (Fisher et al., 1973; Schonheit et al., 1979; Perry & Silver, 1982). Taiganides et al. (1963) reported that a copper concentration of 60-85 mg/litre in a digester resulted in biogas failure. The presence of iron together with copper in contaminated biomass decreased the toxic effect of the copper. Although manganese increased the toxic effect of cobalt on biogas production, the presence of Mn plus Co did not alter the biogas methane content. Lead or zinc contained in the biomass tended to decrease biogas production, and this toxicity effect increased with increase in concentration of the metals. At low concentrations there was no large difference in methane content of biogas compared with non-contaminated biomass (Tables 1 and 2). However, higher concentrations sharply decreased the methane content. The combined effects of lead and zinc at low concentration (group I and group II) have similar effects to that of lead or zinc alone. However, higher concentrations (group III) result in a low value of both biogas produced and its methane content. The lead and zinc content (alone or mixed) do not affect the digestion period. Similar results for lead and zinc toxicity on anaerobic digestion of domestic waste were earlier reported by Regan and Peters (1970), Mosey (1976), Hayes and Theis (1978), Lawrence and McCarty (1965), Mosey and Hughes ( 1975) and Mosey et al. ( 1971 ). The presence of cadmium or nickel at low concentration in biomass decreased the digestion period and the methane content of the biogas produced was much higher than that obtained with non-contaminated biomass. However, with increase in cadmium or nickel content in biomass (e.g. group III) there is a decrease in both the amount of biogas produced and its methane content. However, the methane content of the biogas was still higher than that obtained from non-contaminated biomass.
The presence of Cd + Ni at low concentrations did not have any adverse effect on the rate of biogas produced per day or on its percentage methane content. Cd + Ni-contaminated biomass produced biogas containing a much higher percentage of methane than gases obtained with cadmium or nickel alone or with non-contaminated biomass. The total volume of biogas produced from Cd + Ni-contaminated biomass was comparable with that from non-contaminated biomass. However, overall the Cd + Ni-contaminated biomass yielded more methane than non-contaminated biomass. As the biomass content of Cd + Ni increased, the total volume of biogas and its methane content decreased. Similar data on the effect of cadmium and nickel on the digestion of other substrates have also been reported earlier (Regan & Peters, 1970; Wolverton et al., 1975; Murry & Van den Berg, 1981; Capone et al., 1983; Srivastava, 1983 ). In summary, it is seen that iron or manganese does not have any toxic effect on the fermentation of Azolla and Lemna, but copper, cobalt, lead and zinc show toxicity. Interestingly, cadmium and nickel at low concentration show a favourable effect on the rate of biogas production and its methane content. With increase in concentration, both the rate of biogas production and its methane content decrease. ACKNOWLEDGEMENT One of the authors (S.K.J.) thanks the University Grants Commission (UGS), New Delhi, India for financial assistance. REFERENCES Ahring, B. K. & Westermann, R (1983). Toxicity of heavy metals to thermophilic anaerobic digestion. Eur. J. Appl. Microbiol. Biotechnol., 17,365-70. Benemann, J. R. (1978). Bio fuels: A survey. EPE1 Report No. ER-746-SR. Electric Power Research Institute, Palo Alto, CA. Capone, D. G., Reese, D. D. & Kiene, R. R (1983), Effect of metals on methanogenesis, SO4-reduction, CO, evolution and microbial biomass in anoxic salt marsh sediments. Appl. Environ. Microbiol., 45, 1986-91. Dar, G. H. & Tandon. S. M. (1987a). Biogas production from pretreated wheat straw, lantana residue, apple and peach leaf litter with cattle dung. Biol. Wastes, 21, 75-83. Dar, G. H. & Tandon, S. M. (1987b). Response of a cattle dung methane fermentation to nickel. Biol. Wastes', 22, 261-8. Fisher, F., Bauxbaum, L., Toth, K., Eisenstadt, E. & Silver, S. (1973). Regulation of manganese accumulation and exchange in Bacillus subtilis W23. J. Bacteriol., 113, 1373-80.
Effects o f h e m y metals on digestion Hayes, J. D. & Theis, T. L. (1978). The distribution of heavy metals in anaerobic digestion. J. Water Pollut. Control Fed., 50, 61-72. Jain, S. K., Guiral, G. S., Jha, N. K. & Vasudevan, P. (1988). Heavv metal uptake by Pleurotus sajor-caju from metal enriched duckweed substrate. Biol. Wastes, 24,275-82. Jain, S. K.. Vasudevan, R & Jha, N. K. (1989a). Removal of some heavy metals from polluted water by aquatic plants: Studies on duckweed and water velvet. Biol. Wastes, 28, 115-26. Jain, S. K., Gujral, G. S., Vasudevan, P. & Jha, N. K. (1989b). Studies on uptake of heavy metals by Azolla pinnata and their translocation into the fruit bodies of l'leurotus sajorcaju. J. fi'rmentation & Bioengineering, 68, 64-7. ,lain, S. K., Vasudevan., P. & Jha, N. K. (1990). Azollapinnata R.Br. and Lemna minor L. for removal of lead and zinc from polluted water. Water Res., 24, 177-83. Lawrence, A. W. & McCarty, P. L. (1965). The role of sulphide in preveming heavy metal toxicity in anaerobic treatment. J. Water Pollut. Control Fed., 37,392-506. Maramba, F. D. (ed.)(1978). Laboratory and pilot plant experiment. In Biogas and Waste Recycfing." The Philippines Experience. Maya Farm Division, Liberty Flour Mills, Inc., Manila, Philippines, pp. 21-36. Mosey, t-. E. (1976). Assessments of the maximum concentration of heavy metals in crude sewage which will not inhibit the anaerobic digestion of sludge. Water Polha. Control, 75, 10-20. Mosey, F E. & Hughes, D. A. (1975). The toxicity of heavy metal ions to anaerobic digestion. Water Polha. Control, 74, 18-39. Mosey, f. E., Swanwick, J. D. & Hughes, D. A. (1971.~. Factors affecting the availability of heavy metals to inhibit anaerobic digestion. Water Pollut. Control, 70, 668-80. Murry, \V. D. & Van den Berg, L. (1981). Effects of nickel, cobalt and molybdenum on performance of methanogenic fixed film reactors. Appl. Environ. Microbiol., 42,502-5. NAS (1~)76). Making aquatic weeds useful: some perspectives f~)r developing countries. Report of the National
277
Academy of Science, National Academy Press, Washington DC. Perry, R. D. & Silver, S. (19821). Cadmium and manganese transport in Staphylococcus aureus membrane vesicles. ,/. Bacteriol., 150, 973-6. Regan, T. N. & Peters, M. M. (1970). Heavy metals in digesters: Failure and care. J. Water l'ollut. ('ontrol Fed., 42, 1832-9. Schonheit, P., Moll, J. & Yhaucr, R. K., 1979). Nickel, cobah and molybdenum requirement for growth of Medtanobacterium therrnoautotrophicum. .4 rch. 'Ilicrobiol., 123, 105-7. Srivastava, A. K. (1983). Potential of biogas production by anaerobic digestion of cowdung supplemented with poultry litter and garbage materials. MSc thesis, G. B. Pant University of Agriculture and Technology, Panmagar. India. Stanogias, G. B. & Pearcc, G. R. (1987i. l h e effect of high concentration of minerals in pig diets on the convertibility to methane and in vitro digestibility of the resulting pig faeces. Biol. Wastes, 21. 111-32. Taiganides, E. P., Baumann. E. R., Johnson, 1t. P. &Mazcn, T. E. (1963). Anaerobic digestion of hog wastes. J. Agric. Eng. Res., 8, "~'~-' "" Vogel, A. 1. (1978). A Textbook O/ ()mmtitative husrganic Analysis (4th edn). Longman, New York. Wolverton, B. C. & McDonald, R. C. (1979). The water hyacinth: From prolific pest to potential provider. Ambio, 8.2-9. Wolverton, B. C. & McDonald, R. ( . 1981 ,,. [-nergy from vascular plant waste water treatment systems. Econ. Bot., 35,224-32. Wolverton, B. ('., McDonakl, R. C. & Gordon, J. (1975,. Bioconversion of water hyacinths into melhane gas: Part 1, NASA Tcch. Memo. TM-X-72725 Bay lxmis, MO, 13 pp. Wolverton, B. C. & McKown. M. M. i 1976). Water hyacinth for removal of phenols from polluted aatcrs. Aquatic Bot., 2, 191-201.
Production of Biogas from Azolla pinnata R.Br and Lemna minor L.: Effect of Heavy Metal Contamination S. K. Jain," G.. S. Gujral," N. K. Jha b & p. Vasudevan "Centre for Rural Development and Appropriate Technology, ~'Departmentof Chemistry, Indian Institute of Technology,Hauz Khas, New Delhi-110 016, India (Received 15 June 1991 ; revised version received 14 November 1991 ; accepted 24 November 1991 )
Abstract
The ahsorption of iron, copper, cadmium, nickel, lead, zinc, manganese and cobalt by Azolla pinnata R.Br and Lemna minor L., and subsequent utilization of this biomass for production of biogas (methane), have been investigated. Iron or manganese did not have any toxic effect on the anaerobic fermentation of Azolla and Lemna, while copper, cobalt, lead and zinc showed toxicity. At low concentrations cadmium and nickel showed a favourable effect on the rate ofbiogas production and its methane content, but with increase in concentrations rate of biogas production and methane content decreased. However, although there was this decrease in biogas production and methane content, the methane content of biogas was still higher than that which was obtained from non-c~ mtarninated biomass. Key words: Azolla pinnata, Lernna minor, heavy metals, anaerobic fermentation, biogas.
INTRODUCTION The rising cost of fossil fuels and high capital cost of transmission of electricity to rural areas suggest that local energy sources should be used wherever feasible. The greatest potential lies with biomass. In this context, cultivation of biomass for use in wastewater treatment and subsequent utilization for fuel production is economically appealing (Benemann, 1978). Since this biomass is a byproduct of wastewater treatment, this will not be a competitor of food- or fiber-producing plants. National Space Technology Laboratories, USA,
have investigated a variety of plants which have the common characteristic of high growth potential (Wolverton & McKown, 1976; Wolverton & McDonald, 1979, 1981). It is also important to utilize biomass which is locally available in sufficient quantities but with lower rates of growth. For example, aquatic weeds like Azolla pinnata R.Br (water velvet) and Lemna minor L. (duckweed), which grow commonly in the natural aquatic systems in India, are not uncontrollable invaders like water hyacinth. They will grow in wastewater and have a tendency to absorb and incorporate the dissolved materials from the water (NAS, 1976). Aquatic plants currently being considered for wastewater treatment could also be a good substrate for methane production. However, concern has often been expressed that some heavy metals absorbed from wastewaters may adversely affect the metabolic activity of microorganisms involved in the biodegradation of the biomass. These effects may vary from a lowering of the rate of biodegradation, thus increasing the time necessary for biological treatment, to complete cessation of the process (Ahring & Westermann, 1983; Dar & Tandon, 1987a, b; Stanogias & Pearce, 1987). The present paper discusses the effects of heavy metal contamination in Azolla pinnata and Lernna minor on their anaerobic fermentation to biogas.
METHODS Experimental conditions for absorption of heavy metals Adult Azolla and Lemna plants were obtained from a permanent pond in a village 20 km from
273 Bioresource Technolo~,~ 0960-8524/92/S05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain
274
S. K. Jain, G. S. Gujral, N. K. Jha, P. Vasudevan
Delhi and were kept in plastic tanks containing tap water for one week prior to starting the experiments. The 6.0 kg plant samples (fresh weight) were then transferred and allowed to float in 100 litres of experimental solution contained in a 200 litre plastic pot for 24 h (kept in a phytotron house). The plants were harvested, dried at 48°C and used as substrates for production of biogas. For each metal concentration four replicate samples were taken. The plants were exposed to the individual metals iron, copper, cadmium, nickel, lead, zinc, manganese and cobalt at nominal concentrations of 1-0, 4.0 and 8.0 mg/litre by pipetting appropriate amounts of 10 000 mg/litre metal solutions into a known volume of tap water. The stock solutions of the metals were prepared by dissolving nitrate or sulphate salts in deionized water. The plants were also exposed to solutions of mixtures of these metal ions: iron+ copper, cadmium + nickel, lead + zinc and manganese + cobalt, each at 1.0, 4-0 and 8.0 mg/litre under conditions as described above. The tap water contained (mg/litre): Ca 15.6, Mg 2.8, Na 1-2, K 0.23, C1 9.7, Fe 0.16, Mn 0.21, Cu 0.005 and total dissolved solids 89"2. The other heavy metals under study were not present in the tap water. The water pH was 7.2-7.4. The basic control group used the samples without addition of any metals.
Experimental set-up for biogas production The experiments for biogas production were carried out in a batch system (Maramba, 1978). A 2 litre capacity digester bottle was connected to a water-displacement gas-measuring bottle. The digester was charged with prepared experimental digester slurry which was a mixture of dried plant, unchlorinated water and inoculum. The inoculum was 15-day-old wet slurry from digested cattle manure and was added at 10% level. The total solids concentration in the feed was approximately 8%. The experiments were carried out in a fermentation chamber at 37°C. The digester bottle was vigorously shaken once a day so as to loosen the gas bubbles in the feed and allow the homogeneous mixing of the aquatic plants with inoculum as the former float on the surface. The digestion was carried out until the biogas production fell to zero. The gas samples for analysis were taken out with a syringe from the pressure tubing connecting the digester to the gas bottle. As a precaution to prevent escape of gas, the site was immediately sealed with Teflon tape.
Analysis The plant samples were ashed by heating for 8-10 h in a furnace at 800°C and the ash digested with concentrated nitric acid and filtered into a volumetric flask. The final volume was made up with deionized water and the solutions analysed for heavy metal content (Vogel, 1978) by atomic absorption spectroscopy using a Pye Unicam, Model Sp-191 single-beam atomic absorption spectrophotometer. The biogas was analysed using a Nucon-5700 gas chromatograph fitted with thermal conductivity detector and molecular sieve, stainless-steel column of 1.8 m length and 2 mm inner diameter. Working regimes were: temperature of the detector, injector and oven 40°C; current 150-155 A; and hydrogen as carrier gas at a flow rate of 30 ml per minute. Individual gases were identified on the basis of retention intervals compared to standard gases.
RESULTS AND DISCUSSION
Absorption of heavy metals by Azoila and Lemna The concentrations of heavy metals in Azolla pinnata R.Br and Lemna minor L. exposed to the metals (both single and mixed groups) are shown in Tables 1 and 2, respectively. A comparison of the metal contents of untreated Azolla and Lemna plants with those treated with metal ions shows that with increase in metal concentration in the initial solutions the percentage increase in metal content of Azolla and Lemna is maximum for cadmium and lead, the trend being Cd > Pb > Co > Ni > Cu > Zn > Mn > Fe. The percentage uptake of heavy metals by these two plants was highest when their initial concentration in solution was 1.0 mg/litre. The presence of one metal ion in solutions decreased the uptake rate of the other. Similar results have also been earlier reported (Jain etal., 1988, 1989a, b, 1990).
Effect of heavy metals contained in biomass on the production of biogas The amount of biogas produced from Azolla pinnata and Lemna minor at different concentrations of metal ions and its methane content are shown in Tables 1 and 2, respectively. Biogas data for non-contaminated biomass are shown in the first row. Comparison of data obtained from contaminated biomass with those obtained by digestion of non-contaminated biomass shows that iron and
275
Effects of heavy metals on digestion Table
1.
Effect of heavy metals contained in Azolla pinnata on its fermentation to biogas"
Metal
Group 1 (1"0mg/litre)
Group 111 ¢8"0mg/litre)
Group 11 (4"0 mg/litre)
% Metal Digestion Volume % Metal Digestion Volume % Metal Digestion Volume Methane content period of Methane content period of Methane content period of (/~g/g ( d a y s ) biogas &gig ( d a y s ) biogas (/~g/g ( d a y s ) biogas dry (litre/ kg) dry (litre/ kg) d 0' flitre/ kg) matte 0 matter) matter) 42 42 42 42
189 189 188 188
62 62 62 62
. 686 708 510 541
165 133 138 109
38 37 36
183 183 183
70 72 83
Pb Zn Pb+ Zn
167 130 158 122
42 42 42
180 184 180
Mn Co Mn+ Co
170 120 142 108
42 42 42
188 185 179
'
--
Fc Cu Fe+ Cu
174 169 143 148
Cd Ni Cd + Ni
. 42 42 42
.
. 186 185 185
614 442 497 352
38 37 36
58 60 60
558 481 525 424
62 60 60
612 468 565 410
.
.
.
62 62 62
1308 1498 963 829
183 183 183
70 72 79
1112 865 948 540
42 42 42
176 170 162
58 60 60
42 42 42
186 183 176
62 60 60
. 42 42 42
. 188 163 1711
62 59 60
42 42 40
1711 1711 170
65 66 62
1076 967 975 858
42 42 42
150 150 132
48 46 45
1112 791 1006 735
42 42 42
186 176 I65
62 59 58
"Average of four replicate samples w i t h r e l a t i v e mean deviations of _+5 - 0 % . ~'Non-contaminated Azolla; metal content (/~g/g): Fe 36"0, Cu 10-8, Cd 0'01, Ni 9.4, Pb 0-(I I. Zn 18"8. Mn 26'4 and Co 7"8.
"fable 2. Effect of heavy metals contained in Lemna minor on its fermentation to biogas"
Metal
Group 1 (1.0 mg/litre)
Group 11 (4"0mg/litre)
Group I11 (8"0mg/litre)
% Metal Digestion Vohone % Metal Digestion Vohtme % Metal Digestion Volume content period of Methane content period of Methane content period o] Methane (/~g/g ( d a y s ) biogas (ktg/g ( d a y s ) biogas (/~g/g ( d a y s ) biogas d O, (litre/kg) dr>, (litre/kg) dry (litre/kg) matter) matter) matter) 42 42 42 42
176 176 176 175
60 60 60 60
. 609 601 459 369
159 1119 127 96
38 37 36
176 174 172
71 73 85
Pb Zn Pb + Zn
155 117 141 112
42 42 42
172 170 172
Mn Co Mn + Co
140 104 120 103
42 42 42
176 174 172
r
--
Fe Cu F~+ Cu
156 152 142 126
Cd Ni Cd + Ni
. 42 42 42
. 174 170 172
.
. 61) 60 59
. 1158 1184 801 798
593 409 443 361
38 37 36
176 174 172
71 73 80
55 58 57
550 421 488 413
42 42 42
164 166 166
60 60 58
523 386 467 349
42 42 42
176 174 170
.
. 42 42 42
174 158 164
60 59 59
1070 7211 608 534
42 42 4{)
164 164 164
66 68 68
55 58 57
1062 734 853 698
42 42 42
142 142 132
45 44 43
611 58 58
865 706 796 593
42 42 42
176 164 167
61/ 54 56
"Average of four replicate samples w i t h r e l a t i v e m e a n d e v i a t i o n s of _+5 . 0 % . /'Non-contaminated Lemna; metal content (/~g/g): Fe 31-6, Cu 10.4, Cd I).3, Ni 9-8, Pb 0-3, Z n 18.6, Mn 23.6 and Co 6-9.
276
S. K. Jain, G. S. Gujral, N. K. Jha, P. Vasudevan
manganese did not affect biogas production at any of the concentrations studied. The presence of copper or cobalt at low concentration in the biomass slightly decreased the amount of biogas produced, but with increasing copper or cobalt concentration there was no significant change in the digestion period or in the methane content. Other reports have also shown that iron is not toxic even at extremely high concentration (Lawrence & McCarty, 1965). However, very little information is available on the effect of manganese and cobalt on anaerobic digestion of biomass (Fisher et al., 1973; Schonheit et al., 1979; Perry & Silver, 1982). Taiganides et al. (1963) reported that a copper concentration of 60-85 mg/litre in a digester resulted in biogas failure. The presence of iron together with copper in contaminated biomass decreased the toxic effect of the copper. Although manganese increased the toxic effect of cobalt on biogas production, the presence of Mn plus Co did not alter the biogas methane content. Lead or zinc contained in the biomass tended to decrease biogas production, and this toxicity effect increased with increase in concentration of the metals. At low concentrations there was no large difference in methane content of biogas compared with non-contaminated biomass (Tables 1 and 2). However, higher concentrations sharply decreased the methane content. The combined effects of lead and zinc at low concentration (group I and group II) have similar effects to that of lead or zinc alone. However, higher concentrations (group III) result in a low value of both biogas produced and its methane content. The lead and zinc content (alone or mixed) do not affect the digestion period. Similar results for lead and zinc toxicity on anaerobic digestion of domestic waste were earlier reported by Regan and Peters (1970), Mosey (1976), Hayes and Theis (1978), Lawrence and McCarty (1965), Mosey and Hughes ( 1975) and Mosey et al. ( 1971 ). The presence of cadmium or nickel at low concentration in biomass decreased the digestion period and the methane content of the biogas produced was much higher than that obtained with non-contaminated biomass. However, with increase in cadmium or nickel content in biomass (e.g. group III) there is a decrease in both the amount of biogas produced and its methane content. However, the methane content of the biogas was still higher than that obtained from non-contaminated biomass.
The presence of Cd + Ni at low concentrations did not have any adverse effect on the rate of biogas produced per day or on its percentage methane content. Cd + Ni-contaminated biomass produced biogas containing a much higher percentage of methane than gases obtained with cadmium or nickel alone or with non-contaminated biomass. The total volume of biogas produced from Cd + Ni-contaminated biomass was comparable with that from non-contaminated biomass. However, overall the Cd + Ni-contaminated biomass yielded more methane than non-contaminated biomass. As the biomass content of Cd + Ni increased, the total volume of biogas and its methane content decreased. Similar data on the effect of cadmium and nickel on the digestion of other substrates have also been reported earlier (Regan & Peters, 1970; Wolverton et al., 1975; Murry & Van den Berg, 1981; Capone et al., 1983; Srivastava, 1983 ). In summary, it is seen that iron or manganese does not have any toxic effect on the fermentation of Azolla and Lemna, but copper, cobalt, lead and zinc show toxicity. Interestingly, cadmium and nickel at low concentration show a favourable effect on the rate of biogas production and its methane content. With increase in concentration, both the rate of biogas production and its methane content decrease. ACKNOWLEDGEMENT One of the authors (S.K.J.) thanks the University Grants Commission (UGS), New Delhi, India for financial assistance. REFERENCES Ahring, B. K. & Westermann, R (1983). Toxicity of heavy metals to thermophilic anaerobic digestion. Eur. J. Appl. Microbiol. Biotechnol., 17,365-70. Benemann, J. R. (1978). Bio fuels: A survey. EPE1 Report No. ER-746-SR. Electric Power Research Institute, Palo Alto, CA. Capone, D. G., Reese, D. D. & Kiene, R. R (1983), Effect of metals on methanogenesis, SO4-reduction, CO, evolution and microbial biomass in anoxic salt marsh sediments. Appl. Environ. Microbiol., 45, 1986-91. Dar, G. H. & Tandon. S. M. (1987a). Biogas production from pretreated wheat straw, lantana residue, apple and peach leaf litter with cattle dung. Biol. Wastes, 21, 75-83. Dar, G. H. & Tandon, S. M. (1987b). Response of a cattle dung methane fermentation to nickel. Biol. Wastes', 22, 261-8. Fisher, F., Bauxbaum, L., Toth, K., Eisenstadt, E. & Silver, S. (1973). Regulation of manganese accumulation and exchange in Bacillus subtilis W23. J. Bacteriol., 113, 1373-80.
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