Journal ofApplied Bacteriology 1978,45,147-15 1
SHORT COMMUNICATION Microbial Metabolism of Methanol in a Model Activated Sludge System H. M. SWAIN
AND
H. J. SOMERVILLE
Shell Research Limited, Shell Biosciences Laboratory, Sittingbourne Research Centre, Sittingbourne, Kent, England Received 29 June 19 77 and accepted 13 January 19 7 8
1. Methanol is not broken down when added transiently (0.23% v/v) to a model activated sludge system operating with a retention time of 11 h. Measurement of methanol in the effluent agreed closely with calculated values. 2. Adaptation of the sludge in such a system to 0.1% (v/v) methanol occurs over a period of several days. More than 80%of the methanol is then metabolized.
IT IS GENERALLY ACCEPTED that methanol is readily degraded by micro-organisms. Indeed the addition of methanol to promote denitritkation has been suggested in situations where nitrate accumulates (Barth et al. 1968), and methanol has been added as an economic exogenous organic carbon source to increase denitrification (Stensel et al. 1973). However, at high concentrations, methanol is inhibitory to micro-organisms (Dawson 8z Jenkins 1950). Although methanol has been in large-scale use as a chemical feedstock for many years, interest has recently arisen in the possibility of using methanol as an alternative to natural gas as a transportable energy source. Such a development would result in a massive increase in the amounts of methanol transported and consequently there would be increased risks of accidental release of large quantitities of methanol into the environment. Although many micro-organisms can use methanol as a carbon and energy source, and the biodegradation of methanol by activated sludge has been demonstrated, no work appears to have been done on the effect of substantial amounts of methanol when added transiently to sewage systems. The present work was undertaken to investigate the effect of methanol on oxygen uptake by activated sludge and on the ability of a model activated sludge system to metabolize methanol.
Materials and Methods Media
A synthetic sewage medium, ‘Newcastle medium’, was used for the experiments with the porous pot model system. This contained (per litre of distilled water): nutrient broth, 0.2 g; soluble starch, 0.2 g; NaCl, 30 mg; CaCl,, 4.1 mg; MgCl,, 2.5 mg; Al,(SO,),, 5.0 mg; Na,HPO,, 25.0 mg;-NaHCO, (0.03 mg/l) and urea (0.168 mg/l) were filter sterilized and added separately. Sterilization was carried out by autoclaving 30 litre batches at 121 OC for 15 h. 002 1-8847/78/045 1 - 0 147%01.00/0
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0 1978 The Society for Applied Bacteriology
148
H. M. SWAIN AND H. J. SOMERVILLE
Porous pot apparatus Cylindrical porous pots (Anon. 1974), were constructed from Viyon F of 2.5 mm thickness with a maximum pore size of 90 pm (Porvair Ltd, King's Lynn, Norfolk). The working volume was 2.0 1. The pot was placed in a Pyrex glass cylinder (155 mm diam. x 245 mm), fitted with a glass head plate. Air was sparged into the medium through a sealed glass spiral punched with holes (a total of 30, 0.5 mm diam.) and this was the only means of agitation. A dissolved oxygen electrode of the Mackereth type (Western Biological, Sherborne, Dorset) was calibrated in Newcastle medium or distilled water equilibrated with air: nitrogen mixtures, assuming that 100% air saturation was equivalent to a dissolved oxygen tension (DOT) of 160 mm Hg. The medium and effluent flow were controlled with a peristaltic pump. The outlet gas was passed through a condenser before samples were taken with a gas syringe for methanol analyses. At the commencement of each experiment, the pot was inoculated with 2.0 1 fresh activated sludge obtained from the Milton Regis sewage treatment works (Sittingbourne, Kent). Efluent medium and gas analyses Effluent medium samples were filtered immediately and stored in gas tight vials at 4 OC. Total organic carbon was estimated in a Tocsin total carbon analyser (Phase Separation Ltd, Queensferry, Clwyd, Wales) and nitrate, nitrite and ammonia were estimated in a Technicon autoanalyser (Technicon Instrument Co. Ltd, Basingstoke) by standard methods. Methanol in the effluent medium was estimated using a flame ionization detector fitted to a Pye Unicam 104 with a Hewlett Packard 7670 automatic sampler. A glass column (1260 x 3 mm) containing Porapak Q was maintained at 160OC. For estimation of methanol in the effluent gas, samples (2.0 ml) were taken with a gas-tight syringe and injected into a Hewlett Packard 402 gas chromatograph fitted with a flame ionization detector and using a glass column (1260 x 3 mm) containing Chromosorb 102 with 10% carbowax 20 M, maintained at 8 O O C . In both cases the carrier gas was nitrogen and the flow rate was adjusted to give a retention time of ca. 2 min. Assuming that methanol was not metabolized, the concentration of this compound in the effluent medium (c,) was calculated:
D , , dilution rate in the main compartment; D,, dilution rate in the space between the porous pot and the glass retaining vessel; and c,, concentration of methanol in the working compartment, which changes according to
By substitution of eqn 2, eqn 1 becomes
dc2 dt
-- + D,c, = D2coe-D1t
which gives
(3)
MICROBIAL METABOLISM OF METHANOL
149
Warburg manometry Q,, values [pl 0, consumed (mg dry wt sludge)-' h-'I were determined manometrically. Each flask contained approx. 3 mg dry wt of sludge suspension [prepared by centrifugation at 10000 g, followed by washing and suspension in 50 mM-Na+/K+ phosphate buffer (pH 7-4)1 and &lo% (v/v) methanol as required, in a total volume of 3 ml. The centre wells contained 0.2 ml 0.5 M-KOH. After equilibration at 30°C for 10 min, methanol was added from the side arm to start the reaction. The shaking speed (74 strokes min-') of the water bath was slow so that the sludge flocs were not broken up.
Results Transient addition of methanol Methanol was added directly, to a final concentration of 0.23% (vlv), to the working compartment of a model activated sludge system (porous pot) operating with a retention time of 11 h and which had been adapted to the synthetic sewage for four days. There was no change in the dissolved oxygen tension (142 +_ 2 mm Hg) or in the organic carbon content of the effluent, excluding methanol, over the period of measurement. The concentration of methanol in the effluent closely followed that predicted on the basis of wash out (Fig. 1). The best fit was obtained assuming no metabolism of
1
0.00,
,
I
2
4
, 6
I
8
I
1
"''\o
_______J
0
22
Time ( h )
Fig. 1. Metabolism of methanol. Methanol was added (0.23% v/v) to the working compartment of the model sewage system and effluent samples were analysed as described in Materials and Methods. Retention time, 1 1 h; aeration, 1.05 I min-*; temperature, 21.5 k 1 OC; pH, 7.8; dissolved oxygen tension, 142 mm Hg. 0 , theoretical value of residual methanol; and 0, measured methanol concentration.
methanol and for a hold-up volume of 75 ml between the porous pot and the retaining vessel. This volume (75 ml) is slightly smaller than that measured (G. J. Thijsse, pers. comm.); the methanol presumably reaches the outer space partly by diffusion thus reducing the effective hold-up volume. No appreciable methanol was recovered in the gas phase.
Adaptation to methanol The results of the above experiment suggested that a period of adaptation would be required for sludge to utilize methanol. Accordingly, methanol (0-1% v/v) was added to
150
H. M. SWAIN AND H. J. SOMERVILLE
the feed medium after a two-day adaptation of fresh activated sludge to synthetic sewage as described above. The results (Table 1) show that after a two-day period ca.
TABLE1 Adaptation of activated sludge to methanol metabolism Methanol Time
(4
in inlet medium (g h-I)
in spent medium (g h-l)
in efffuent gas (g h - 9
utilized (%) ~
0 2 6 10
0.25
0.23 0.123 0.025
U.0 19
0.25
0.049
0.017
0.25 0.25
ND ND
8 50
90 80.4
ND, not determined. Methanol was added in the feed medium to sludge which had previously been adapted to synthetic sewage. Retention time, 6.2 h; temperature, 20-25OC.
60% of the methanol was degraded and after six days this rose to at least 80%. A steady state appeared to be reached at which approximately 80% of the methanol was metabolized. No nitrification was observed before or after addition of methanol and the dissolved oxygen tension dropped slightly over the period of the experiment (129 f 5 to 91 k 5 mmHg) as would be expected from the increased biological oxygen demand. In a separate experiment, nitrification was observed before methanol addition and was sustained subsequently. The initial DOT was lower than in the previous experiment and was probably caused by the higher sludge dry weight; some variation in the DOT was accounted for by temperature change. Inhibition of sludge respiration The effect of different concentrations of methanol on the respiration of sludge was studied using the Warburg respirometer. Three types of sludge were used: (a) fresh from the sewage treatment works; (b) sludge from the porous pot, adapted to synthetic sewage; and (c) sludge from the porous pot, adapted to synthetic sewage + O . 1% (v/v) methanol. In each case the adaptation period was at least four days and sludge samples were washed before testing (respiration was measured without the addition of a substrate other than methanol). Respiration of fresh, adapted and methanol-adapted sludge was not affected at the concentrations used in the porous pot experiments. Concentrations up to 5% (v/v) were not toxic but complete inhibition of respiration was observed at 10% (v/v) methanol. Complete suppression of oxygen uptake by activated sludge with 20% methanol has been previously reported (Dawson & Jenkins 1950).
Discussion The results from the porous pot experiments show that methanol is not metabolized by activated sludge when it is added as a single pulse over a short period. The lack of effect on either dissolved oxygen tension or the utilization of non-methanol organic carbon indicates that methanol added in this way has no inhibitory effect on sludge metabolism. When methanol was added as a continuous feed to the porous pot, however, at
MICROBIAL METABOLISM OF METHANOL
151
least 80% of the methanol was metabolized after an adaptation period of six days. There were no apparent toxic effects caused by the addition of methanol (0.1% v/v) to the sludge prior to and after adaptation to methanol. It can be concluded that the concentration of methanol which reaches the activated sludge in the sewage works and the period of time in which it is retained in the system will determine the effect of methanol on the activated sludge system in a sewage treatment works. We thank B. Khosrovi and G. Paterson for useful discussions.
References ANON.1974 In: The Tenth Progress Report of the Standing Technical Committee on Synthetic Detergents Department of the Environment, London: H.M.S.O. BARTH,E. F., BRENNER,R. C. & LEWIS,R. F. 1968 Chemical and biological control of nitrogen and phosphorus in wastewater effluent. Journal of the Water Pollution Control Federation 40,2040-2054.
DAWSON, P. S. S. & JENKINS,S. H. 1950 The oxygen requirements of activated sludge determined by manometric methods. Sewage and Industrial Wastes 22,490-507. STENSEL, H. D., LOEHR,R. C. & LAWRENCE, A. W. 1973 Biological kinetics of suspended growth denitrification.Journal of the Water Pollution Control Federation 45,249-26 1.
SHORT COMMUNICATION Microbial Metabolism of Methanol in a Model Activated Sludge System H. M. SWAIN
AND
H. J. SOMERVILLE
Shell Research Limited, Shell Biosciences Laboratory, Sittingbourne Research Centre, Sittingbourne, Kent, England Received 29 June 19 77 and accepted 13 January 19 7 8
1. Methanol is not broken down when added transiently (0.23% v/v) to a model activated sludge system operating with a retention time of 11 h. Measurement of methanol in the effluent agreed closely with calculated values. 2. Adaptation of the sludge in such a system to 0.1% (v/v) methanol occurs over a period of several days. More than 80%of the methanol is then metabolized.
IT IS GENERALLY ACCEPTED that methanol is readily degraded by micro-organisms. Indeed the addition of methanol to promote denitritkation has been suggested in situations where nitrate accumulates (Barth et al. 1968), and methanol has been added as an economic exogenous organic carbon source to increase denitrification (Stensel et al. 1973). However, at high concentrations, methanol is inhibitory to micro-organisms (Dawson 8z Jenkins 1950). Although methanol has been in large-scale use as a chemical feedstock for many years, interest has recently arisen in the possibility of using methanol as an alternative to natural gas as a transportable energy source. Such a development would result in a massive increase in the amounts of methanol transported and consequently there would be increased risks of accidental release of large quantitities of methanol into the environment. Although many micro-organisms can use methanol as a carbon and energy source, and the biodegradation of methanol by activated sludge has been demonstrated, no work appears to have been done on the effect of substantial amounts of methanol when added transiently to sewage systems. The present work was undertaken to investigate the effect of methanol on oxygen uptake by activated sludge and on the ability of a model activated sludge system to metabolize methanol.
Materials and Methods Media
A synthetic sewage medium, ‘Newcastle medium’, was used for the experiments with the porous pot model system. This contained (per litre of distilled water): nutrient broth, 0.2 g; soluble starch, 0.2 g; NaCl, 30 mg; CaCl,, 4.1 mg; MgCl,, 2.5 mg; Al,(SO,),, 5.0 mg; Na,HPO,, 25.0 mg;-NaHCO, (0.03 mg/l) and urea (0.168 mg/l) were filter sterilized and added separately. Sterilization was carried out by autoclaving 30 litre batches at 121 OC for 15 h. 002 1-8847/78/045 1 - 0 147%01.00/0
11471
0 1978 The Society for Applied Bacteriology
148
H. M. SWAIN AND H. J. SOMERVILLE
Porous pot apparatus Cylindrical porous pots (Anon. 1974), were constructed from Viyon F of 2.5 mm thickness with a maximum pore size of 90 pm (Porvair Ltd, King's Lynn, Norfolk). The working volume was 2.0 1. The pot was placed in a Pyrex glass cylinder (155 mm diam. x 245 mm), fitted with a glass head plate. Air was sparged into the medium through a sealed glass spiral punched with holes (a total of 30, 0.5 mm diam.) and this was the only means of agitation. A dissolved oxygen electrode of the Mackereth type (Western Biological, Sherborne, Dorset) was calibrated in Newcastle medium or distilled water equilibrated with air: nitrogen mixtures, assuming that 100% air saturation was equivalent to a dissolved oxygen tension (DOT) of 160 mm Hg. The medium and effluent flow were controlled with a peristaltic pump. The outlet gas was passed through a condenser before samples were taken with a gas syringe for methanol analyses. At the commencement of each experiment, the pot was inoculated with 2.0 1 fresh activated sludge obtained from the Milton Regis sewage treatment works (Sittingbourne, Kent). Efluent medium and gas analyses Effluent medium samples were filtered immediately and stored in gas tight vials at 4 OC. Total organic carbon was estimated in a Tocsin total carbon analyser (Phase Separation Ltd, Queensferry, Clwyd, Wales) and nitrate, nitrite and ammonia were estimated in a Technicon autoanalyser (Technicon Instrument Co. Ltd, Basingstoke) by standard methods. Methanol in the effluent medium was estimated using a flame ionization detector fitted to a Pye Unicam 104 with a Hewlett Packard 7670 automatic sampler. A glass column (1260 x 3 mm) containing Porapak Q was maintained at 160OC. For estimation of methanol in the effluent gas, samples (2.0 ml) were taken with a gas-tight syringe and injected into a Hewlett Packard 402 gas chromatograph fitted with a flame ionization detector and using a glass column (1260 x 3 mm) containing Chromosorb 102 with 10% carbowax 20 M, maintained at 8 O O C . In both cases the carrier gas was nitrogen and the flow rate was adjusted to give a retention time of ca. 2 min. Assuming that methanol was not metabolized, the concentration of this compound in the effluent medium (c,) was calculated:
D , , dilution rate in the main compartment; D,, dilution rate in the space between the porous pot and the glass retaining vessel; and c,, concentration of methanol in the working compartment, which changes according to
By substitution of eqn 2, eqn 1 becomes
dc2 dt
-- + D,c, = D2coe-D1t
which gives
(3)
MICROBIAL METABOLISM OF METHANOL
149
Warburg manometry Q,, values [pl 0, consumed (mg dry wt sludge)-' h-'I were determined manometrically. Each flask contained approx. 3 mg dry wt of sludge suspension [prepared by centrifugation at 10000 g, followed by washing and suspension in 50 mM-Na+/K+ phosphate buffer (pH 7-4)1 and &lo% (v/v) methanol as required, in a total volume of 3 ml. The centre wells contained 0.2 ml 0.5 M-KOH. After equilibration at 30°C for 10 min, methanol was added from the side arm to start the reaction. The shaking speed (74 strokes min-') of the water bath was slow so that the sludge flocs were not broken up.
Results Transient addition of methanol Methanol was added directly, to a final concentration of 0.23% (vlv), to the working compartment of a model activated sludge system (porous pot) operating with a retention time of 11 h and which had been adapted to the synthetic sewage for four days. There was no change in the dissolved oxygen tension (142 +_ 2 mm Hg) or in the organic carbon content of the effluent, excluding methanol, over the period of measurement. The concentration of methanol in the effluent closely followed that predicted on the basis of wash out (Fig. 1). The best fit was obtained assuming no metabolism of
1
0.00,
,
I
2
4
, 6
I
8
I
1
"''\o
_______J
0
22
Time ( h )
Fig. 1. Metabolism of methanol. Methanol was added (0.23% v/v) to the working compartment of the model sewage system and effluent samples were analysed as described in Materials and Methods. Retention time, 1 1 h; aeration, 1.05 I min-*; temperature, 21.5 k 1 OC; pH, 7.8; dissolved oxygen tension, 142 mm Hg. 0 , theoretical value of residual methanol; and 0, measured methanol concentration.
methanol and for a hold-up volume of 75 ml between the porous pot and the retaining vessel. This volume (75 ml) is slightly smaller than that measured (G. J. Thijsse, pers. comm.); the methanol presumably reaches the outer space partly by diffusion thus reducing the effective hold-up volume. No appreciable methanol was recovered in the gas phase.
Adaptation to methanol The results of the above experiment suggested that a period of adaptation would be required for sludge to utilize methanol. Accordingly, methanol (0-1% v/v) was added to
150
H. M. SWAIN AND H. J. SOMERVILLE
the feed medium after a two-day adaptation of fresh activated sludge to synthetic sewage as described above. The results (Table 1) show that after a two-day period ca.
TABLE1 Adaptation of activated sludge to methanol metabolism Methanol Time
(4
in inlet medium (g h-I)
in spent medium (g h-l)
in efffuent gas (g h - 9
utilized (%) ~
0 2 6 10
0.25
0.23 0.123 0.025
U.0 19
0.25
0.049
0.017
0.25 0.25
ND ND
8 50
90 80.4
ND, not determined. Methanol was added in the feed medium to sludge which had previously been adapted to synthetic sewage. Retention time, 6.2 h; temperature, 20-25OC.
60% of the methanol was degraded and after six days this rose to at least 80%. A steady state appeared to be reached at which approximately 80% of the methanol was metabolized. No nitrification was observed before or after addition of methanol and the dissolved oxygen tension dropped slightly over the period of the experiment (129 f 5 to 91 k 5 mmHg) as would be expected from the increased biological oxygen demand. In a separate experiment, nitrification was observed before methanol addition and was sustained subsequently. The initial DOT was lower than in the previous experiment and was probably caused by the higher sludge dry weight; some variation in the DOT was accounted for by temperature change. Inhibition of sludge respiration The effect of different concentrations of methanol on the respiration of sludge was studied using the Warburg respirometer. Three types of sludge were used: (a) fresh from the sewage treatment works; (b) sludge from the porous pot, adapted to synthetic sewage; and (c) sludge from the porous pot, adapted to synthetic sewage + O . 1% (v/v) methanol. In each case the adaptation period was at least four days and sludge samples were washed before testing (respiration was measured without the addition of a substrate other than methanol). Respiration of fresh, adapted and methanol-adapted sludge was not affected at the concentrations used in the porous pot experiments. Concentrations up to 5% (v/v) were not toxic but complete inhibition of respiration was observed at 10% (v/v) methanol. Complete suppression of oxygen uptake by activated sludge with 20% methanol has been previously reported (Dawson & Jenkins 1950).
Discussion The results from the porous pot experiments show that methanol is not metabolized by activated sludge when it is added as a single pulse over a short period. The lack of effect on either dissolved oxygen tension or the utilization of non-methanol organic carbon indicates that methanol added in this way has no inhibitory effect on sludge metabolism. When methanol was added as a continuous feed to the porous pot, however, at
MICROBIAL METABOLISM OF METHANOL
151
least 80% of the methanol was metabolized after an adaptation period of six days. There were no apparent toxic effects caused by the addition of methanol (0.1% v/v) to the sludge prior to and after adaptation to methanol. It can be concluded that the concentration of methanol which reaches the activated sludge in the sewage works and the period of time in which it is retained in the system will determine the effect of methanol on the activated sludge system in a sewage treatment works. We thank B. Khosrovi and G. Paterson for useful discussions.
References ANON.1974 In: The Tenth Progress Report of the Standing Technical Committee on Synthetic Detergents Department of the Environment, London: H.M.S.O. BARTH,E. F., BRENNER,R. C. & LEWIS,R. F. 1968 Chemical and biological control of nitrogen and phosphorus in wastewater effluent. Journal of the Water Pollution Control Federation 40,2040-2054.
DAWSON, P. S. S. & JENKINS,S. H. 1950 The oxygen requirements of activated sludge determined by manometric methods. Sewage and Industrial Wastes 22,490-507. STENSEL, H. D., LOEHR,R. C. & LAWRENCE, A. W. 1973 Biological kinetics of suspended growth denitrification.Journal of the Water Pollution Control Federation 45,249-26 1.
