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rapid degradation of halogenated hydrocarbons by soluble methane monooxygenase
Patent Survey
306 l/
I/
,/4 Fig. 2. inlet (15) and, optionally, recycle wastewater via a gravity feed conduit (16). A wet well (18) is positioned intermediate to the filter unit (2) and the combined sump (14) to receive treated wastewater from the filter unit (2) via a gravity feed conduit (19). The wet well (18) includes an outlet (20) which can optionally be connected to a clarifier, other treatment means or disposal means as required. Feed apparatus in the form of a pipe (22) and pump (not shown) extends between the combined sump (14) and filter unit liquid inlet (4). The infeed apparatus (22), oxygen meter (9), flow meter (10) and oxygen analyser (11) are all connected to a central control unit (not shown). In use, the untreated wastewater requiring treatment is fed through the inlet (15) into the combined sump (14) where it is mixed with filtered recycled fluid via gravity feed (16) which has already been at least partially treated in the biological filter unit (2). The mixture is then pumped to the top of the filter unit (2) via feed means (22) where it is distributed over the packing material (12) by any suitable known distribution means. The wastewater trickles down over the packing material (12) and accumulated supported biomass. During this contact, part of the pollution is removed from the wastewater and converted into products of respiration including carbon dioxide, water and excess biomass. The flow of partly treated water (plus any sloughed biomass) passes by gravity through the first gravity feed (19) to the wet wall (18), whereby a smaller flow of liquid is optionally pumped through the outlet (20) into a clarifier or other disposal or purification means. The rest of the treated waste overflows through gravity feed (16) into the combined sump (14) for recycling around the process. The recycle rate is predetermined
and is based on the oxygen consumption rate. During the filtration process, substantially pure oxygen is fed into the filter unit via the oxygen meter (9) which then directs the oxygen through inlet (8). This enhances the aerobic digestion process and excess oxygen exits the filter unit via the exhaust gas vent (5) along with stripped gases such as carbon dioxide and nitrogen. Typical vent gas purities are expected to be in the range 40-50% oxygen. Real time control of both the oxygen supply and demand is achieved by measuring the oxygen consumption rate across the biological filter unit (2). The main control loop uses the ratio of oxygen consumption to liquid flow through the feed apparatus (22) (a controlled variable) to determine the filter plant biological demand (BODy) of the infeed/recycle liquid. The feed rate through feed pipe (22) is adjusted until the BODy is at its set point. The BOD s is determined empirically for each unit and for the particular type of wastewater that is to be processed, since this is decided in part by the reaction constants for each wastewater composition. Two subsidiary control loops adjust the oxygen feed rate into inlet (8) to maintain a predetermined level of oxygen in the exhaust gas from the filter unit that passes through vent (5) and control the flow rate through the vent opening to protect the filter unit from being exposed to excessive pressure. The oxygen consumption rate is calculated by the control unit and is derived from the oxygen feed rate, and the product of the vent gas flow rate and its oxygen mole fraction. If oxygen from a source known to deviate significantly from 100% purity is used, its composition may be assumed (if steady) or measured (if known to be varying). One such source is oxygen derived from air by the pressure swing absorption process.
References Oases is Oxygen in Wastewater Treatment, Air Products and Chemicals Brochure, 1973. 9 US patent publications. 15 other patent publications.
assignors of the aforementioned University, have developed a method for the degradation of halogenated hydrocarbon compounds such as trichloroethylene (TCE) which utilizes Methylosinus trichosporium OB3b, a soluble methane monooxygenase-producing bacterium. The bacterium is exposed to a continuous-flow gas mixture of air and methane in a ratio of about 25:1-1:20, respectively. Methylosinus trichosporium (Mt) OB3b and its methane monooxygenase are capable of degrading TCE at rates from about 500-10000 ,umoles h -~ per gram of cells. Their method is useful to degrade halogenated hydrocarbon compounds at initial concentrations up to 10 000/~moles. A continuous culture of Mt OB3b was performed in which the bacteria grew at a rate of approximately 10 h per generation on Higgins medium. The continuous culture was grown in a chemostat growth chamber with a volume of 0.185 litres. The apparatus consisted of a water-jacketed growth chamber supplied with sterile, warm, moist air and a constant supply of medium; oxygen was supplied from the atmosphere. Figure 3 shows the apparatus in detail where the alphabetic references describe specific features: (A) chemostat growth flask; (B) medium pump; (C) medium reservoir; (D) constant temperature bath; (E) constant temperature heater pump; (F) air humidifying chamber; (G) air sterilizing chamber; (H) air pump; and (I) culture collection flask. As a laboratory example of Hanson and his colleague's method a TCE degradation test by head-space assay was carried out. Head-space assays of TCE degradation were conducted by adding bacterial cells from the continuous culture described earlier or cells diluted with spent Higgins medium (2 ml) into assay vials (10 ml volume). Dilution of cells in spent Higgins medium had no effect on the rate of TCE oxidation, therefore, there does not seem to be protective compounds in the medium. Heat-killed controls indicated that no TCE was lost from the vials.
Rapid degradation of halogenated hydrocarbons by soluble methane monooxygenase
(US patent 5 441 887 to the Regents of the University of Minnesota and to BioTrol Inc., both of MN, USA) CHEMOSTAT ~SE MSI.y
Richard S. Hanson together with his cobiochemists Lipscomb and Fox, all
Fig. 3.
Patent Survey
qable 8. TCE degradation in a two-phase a~say t ell density Initial TCE in assay concentration 'ials (g/l) (pmolar) 0.695 0.521 0.347 0.173 0.070
22 22 22 23 22
Rate (pmoles) TCE oxidized (g/cells/h) 281 308 465 461 808
The rates of TCE utilization in twot hase head-space assays are shown in "fable 8. Rates were calculated from l e a k heights of recorder tracings from ~ gas chromatograph equipped with an electron capture detector. An extensive background of the i wention and a detailed description of t i e characteristics of methanotrophic I acteria and 10 examples of laboratory I :sts are described. l",~eferences t 'olby et al., Resolution of the methane J lono-oxygenase of M. capsulatae I Bath) into three compounds. Biochem. ; , 171 (1978) 461-8. l)epamphilis, J. & Hansen, R. J. Bacterl.d. 98 (1969) 222-5. t,gli et al., Anaerobic dechlorination of t ~ztrachlorination of tetrachloromeI lane. FEMS Microbiol. Lett., 43 (1987) ~57-61. f ox et al., Complex formation between I~e protein components of methane Jlonooxygenase from Methylosinus tric hosporium OB3b. J. Bio. Chem., 266 q 1991) 540-50. ~lossbauer et al., Evidence for a M-Oxoiridged bin-clear iron cluster. Vol. 263, 988, pp. 10553-6. Nakajima et al., Purification properties c f soluble methane monooxygenase. iqosci. Biotech. Biochem., 56 (1992) ' 36-40. ~4elson et al., Aerobic metabolism of "'CE by a bacterial isolate. App. i "nviron. Microbiol., (1986) 383-4. ~'ilkington et al., Purification and haracterization of the soluble methane lonooxygenase from M. Sporium. I'EMS Microbiol. Lett., 78 (1991) 103-8. Wilson et al. Appl. Environ. Microbiol., ,~9 (1985) 242. US patent publications. European patent publication. i ~ purification plant for removing r~utrients from sewage
World patent application 95/07861 to t.'arsten Andersen, Virum, Denmark) .~,ndersen has designed a purification .wstem of the activated sludge type for
the removal of organics and nutrients including a process tank. The process tank has a partition wall dividing the tank into anoxic and oxic chambers, into both of which one or more mixers are placed. Aeration equipment is also provided in the oxic chamber. The novel component in his design is that between the oxic and anoxic chamber a recirculation takes place which is effected by turbulence in a hydraulic boundary layer in one or more openings in the partition wall. According to Andersen's proposal, the reduction of nitrate into free oxygen occurs by using the organic matter in the sewage as a reductor. The sewage flows either directly into the chamber or indirectly after passing a central tank. The microorganisms (the activated sludge) are mixed by one or more mixers, which are designed as propellor-mixers. While the anoxic chamber is always unaerated, the organic matter in the sewage is used optimally for denitrification. From the anoxic chamber the sewage flows through one or more openings in the partition wall into the aerated (oxic) chamber. In the oxic chamber the sewage is aerated, whereby ammonia is oxidized into nitrate. The aerators can either be surface-aerators or submerged aerators. From the oxic chamber the nitrate is reeirculated by hydraulic dispersion into the anoxie chamber through the openings in the partition wall. In the anoxic chamber the nitrate is reduced into free nitrogen, which escapes into the atmosphere. Each nitrogen molecules passes through the openings in the partition wall in and out of the oxic and anoxic chamber (more than 20 times). Oxidation of ammonia and reduction of nitrate occurs alternately until all ammonia is almost converted into free nitrogen. In this way a high degree of recirculation and thereby purification is achieved without pumping. In the traditional recirculation plant, pumping is necessary to recirculate and this restricts the degree of reeirculation because of hydraulic limitations. The transport of ammonia and nitrate between the oxic and anoxic chamber through the two openings takes place by turbulent mixing in the boundary layer between the flow into the oxic and anoxic chambers. The nitrate is transported naturally by hydraulic dispersion from water with a high concentration of nitrate (oxic chamber) into water with a low concentration of nitrate (anoxic chamber) and ammonia is conveyed naturally by hydraulic dispersion from water with a high concentration of ammonia (anoxic chamber) into water with a low concen-
307
tration of ammonia (oxic chamber). This is astonishing, as a designer will usually install pumps to recireulate water from the oxic chamber back into the anoxic chamber. A mass-balance across the oxic chamber results in the normal expression for the recirculation rate R: R=TotN[in]/NO3[out]- l=Qr/O
(1)
where TotN[in]=total nitrogen in the inlet (g/m3); NO3[out]=nitrate in the outlet (g/m~); Qr=the recirculation flow (m3/s); Q=the inlet flow (m3/s). If A (m 2) is the area of the openings in the partition wall, the area A can be calculated as: A =R x Q/v=(TotN[inl/NO3[out] -1)xQ/v (m 3) (2) where v=the velocity of recirculation flow (m/s). The sludge load SL is defined as: SL=Q × BOD/V×X
(g BOD/g SS day)
(3) where B O D = t h e concentration of BOD in the inlet (g/m3); V=the volume of the process tank (m"); X=the sludge concentration (g/m3). Substituting eqn (3) into eqn (2) results in the surface area for the openings as: A = (TotN[in]/NO3[out]- 1) x (SL x X / B O D x v) x V(m 2)
(4) Inserting different realistic figures into eqn (4) one will find that appropriate surface areas of the openings will be in the range 0"05-0"25 times the area of the partition wall including the openings. If the openings are too large, too much oxygen will be transported from the oxic chamber into the anoxic chamber. If the openings are too small, the recirculation of nitrate will be too small. From eqn (2) is is seen that the appropriate area of the openings depends on the degree of purification and the wastewater flow. If the geometry of the process tank is cylindrical the relation between the area of the openings and the area of the partition wall (Ap) including the openings is: A/Ap = (TotN[in]/NO~[out] - 1) x (SL x X/BOD x v) x 0'7R
(5) where R=the radius of the process tank. From eqn (5) it can be seen that a larger process tank requires a larger relative opening in the partition wall. From the oxic chamber the mixture of purified water and activated sludge flows into one or more sedimentation tanks or separators where the purified
306 l/
I/
,/4 Fig. 2. inlet (15) and, optionally, recycle wastewater via a gravity feed conduit (16). A wet well (18) is positioned intermediate to the filter unit (2) and the combined sump (14) to receive treated wastewater from the filter unit (2) via a gravity feed conduit (19). The wet well (18) includes an outlet (20) which can optionally be connected to a clarifier, other treatment means or disposal means as required. Feed apparatus in the form of a pipe (22) and pump (not shown) extends between the combined sump (14) and filter unit liquid inlet (4). The infeed apparatus (22), oxygen meter (9), flow meter (10) and oxygen analyser (11) are all connected to a central control unit (not shown). In use, the untreated wastewater requiring treatment is fed through the inlet (15) into the combined sump (14) where it is mixed with filtered recycled fluid via gravity feed (16) which has already been at least partially treated in the biological filter unit (2). The mixture is then pumped to the top of the filter unit (2) via feed means (22) where it is distributed over the packing material (12) by any suitable known distribution means. The wastewater trickles down over the packing material (12) and accumulated supported biomass. During this contact, part of the pollution is removed from the wastewater and converted into products of respiration including carbon dioxide, water and excess biomass. The flow of partly treated water (plus any sloughed biomass) passes by gravity through the first gravity feed (19) to the wet wall (18), whereby a smaller flow of liquid is optionally pumped through the outlet (20) into a clarifier or other disposal or purification means. The rest of the treated waste overflows through gravity feed (16) into the combined sump (14) for recycling around the process. The recycle rate is predetermined
and is based on the oxygen consumption rate. During the filtration process, substantially pure oxygen is fed into the filter unit via the oxygen meter (9) which then directs the oxygen through inlet (8). This enhances the aerobic digestion process and excess oxygen exits the filter unit via the exhaust gas vent (5) along with stripped gases such as carbon dioxide and nitrogen. Typical vent gas purities are expected to be in the range 40-50% oxygen. Real time control of both the oxygen supply and demand is achieved by measuring the oxygen consumption rate across the biological filter unit (2). The main control loop uses the ratio of oxygen consumption to liquid flow through the feed apparatus (22) (a controlled variable) to determine the filter plant biological demand (BODy) of the infeed/recycle liquid. The feed rate through feed pipe (22) is adjusted until the BODy is at its set point. The BOD s is determined empirically for each unit and for the particular type of wastewater that is to be processed, since this is decided in part by the reaction constants for each wastewater composition. Two subsidiary control loops adjust the oxygen feed rate into inlet (8) to maintain a predetermined level of oxygen in the exhaust gas from the filter unit that passes through vent (5) and control the flow rate through the vent opening to protect the filter unit from being exposed to excessive pressure. The oxygen consumption rate is calculated by the control unit and is derived from the oxygen feed rate, and the product of the vent gas flow rate and its oxygen mole fraction. If oxygen from a source known to deviate significantly from 100% purity is used, its composition may be assumed (if steady) or measured (if known to be varying). One such source is oxygen derived from air by the pressure swing absorption process.
References Oases is Oxygen in Wastewater Treatment, Air Products and Chemicals Brochure, 1973. 9 US patent publications. 15 other patent publications.
assignors of the aforementioned University, have developed a method for the degradation of halogenated hydrocarbon compounds such as trichloroethylene (TCE) which utilizes Methylosinus trichosporium OB3b, a soluble methane monooxygenase-producing bacterium. The bacterium is exposed to a continuous-flow gas mixture of air and methane in a ratio of about 25:1-1:20, respectively. Methylosinus trichosporium (Mt) OB3b and its methane monooxygenase are capable of degrading TCE at rates from about 500-10000 ,umoles h -~ per gram of cells. Their method is useful to degrade halogenated hydrocarbon compounds at initial concentrations up to 10 000/~moles. A continuous culture of Mt OB3b was performed in which the bacteria grew at a rate of approximately 10 h per generation on Higgins medium. The continuous culture was grown in a chemostat growth chamber with a volume of 0.185 litres. The apparatus consisted of a water-jacketed growth chamber supplied with sterile, warm, moist air and a constant supply of medium; oxygen was supplied from the atmosphere. Figure 3 shows the apparatus in detail where the alphabetic references describe specific features: (A) chemostat growth flask; (B) medium pump; (C) medium reservoir; (D) constant temperature bath; (E) constant temperature heater pump; (F) air humidifying chamber; (G) air sterilizing chamber; (H) air pump; and (I) culture collection flask. As a laboratory example of Hanson and his colleague's method a TCE degradation test by head-space assay was carried out. Head-space assays of TCE degradation were conducted by adding bacterial cells from the continuous culture described earlier or cells diluted with spent Higgins medium (2 ml) into assay vials (10 ml volume). Dilution of cells in spent Higgins medium had no effect on the rate of TCE oxidation, therefore, there does not seem to be protective compounds in the medium. Heat-killed controls indicated that no TCE was lost from the vials.
Rapid degradation of halogenated hydrocarbons by soluble methane monooxygenase
(US patent 5 441 887 to the Regents of the University of Minnesota and to BioTrol Inc., both of MN, USA) CHEMOSTAT ~SE MSI.y
Richard S. Hanson together with his cobiochemists Lipscomb and Fox, all
Fig. 3.
Patent Survey
qable 8. TCE degradation in a two-phase a~say t ell density Initial TCE in assay concentration 'ials (g/l) (pmolar) 0.695 0.521 0.347 0.173 0.070
22 22 22 23 22
Rate (pmoles) TCE oxidized (g/cells/h) 281 308 465 461 808
The rates of TCE utilization in twot hase head-space assays are shown in "fable 8. Rates were calculated from l e a k heights of recorder tracings from ~ gas chromatograph equipped with an electron capture detector. An extensive background of the i wention and a detailed description of t i e characteristics of methanotrophic I acteria and 10 examples of laboratory I :sts are described. l",~eferences t 'olby et al., Resolution of the methane J lono-oxygenase of M. capsulatae I Bath) into three compounds. Biochem. ; , 171 (1978) 461-8. l)epamphilis, J. & Hansen, R. J. Bacterl.d. 98 (1969) 222-5. t,gli et al., Anaerobic dechlorination of t ~ztrachlorination of tetrachloromeI lane. FEMS Microbiol. Lett., 43 (1987) ~57-61. f ox et al., Complex formation between I~e protein components of methane Jlonooxygenase from Methylosinus tric hosporium OB3b. J. Bio. Chem., 266 q 1991) 540-50. ~lossbauer et al., Evidence for a M-Oxoiridged bin-clear iron cluster. Vol. 263, 988, pp. 10553-6. Nakajima et al., Purification properties c f soluble methane monooxygenase. iqosci. Biotech. Biochem., 56 (1992) ' 36-40. ~4elson et al., Aerobic metabolism of "'CE by a bacterial isolate. App. i "nviron. Microbiol., (1986) 383-4. ~'ilkington et al., Purification and haracterization of the soluble methane lonooxygenase from M. Sporium. I'EMS Microbiol. Lett., 78 (1991) 103-8. Wilson et al. Appl. Environ. Microbiol., ,~9 (1985) 242. US patent publications. European patent publication. i ~ purification plant for removing r~utrients from sewage
World patent application 95/07861 to t.'arsten Andersen, Virum, Denmark) .~,ndersen has designed a purification .wstem of the activated sludge type for
the removal of organics and nutrients including a process tank. The process tank has a partition wall dividing the tank into anoxic and oxic chambers, into both of which one or more mixers are placed. Aeration equipment is also provided in the oxic chamber. The novel component in his design is that between the oxic and anoxic chamber a recirculation takes place which is effected by turbulence in a hydraulic boundary layer in one or more openings in the partition wall. According to Andersen's proposal, the reduction of nitrate into free oxygen occurs by using the organic matter in the sewage as a reductor. The sewage flows either directly into the chamber or indirectly after passing a central tank. The microorganisms (the activated sludge) are mixed by one or more mixers, which are designed as propellor-mixers. While the anoxic chamber is always unaerated, the organic matter in the sewage is used optimally for denitrification. From the anoxic chamber the sewage flows through one or more openings in the partition wall into the aerated (oxic) chamber. In the oxic chamber the sewage is aerated, whereby ammonia is oxidized into nitrate. The aerators can either be surface-aerators or submerged aerators. From the oxic chamber the nitrate is reeirculated by hydraulic dispersion into the anoxie chamber through the openings in the partition wall. In the anoxic chamber the nitrate is reduced into free nitrogen, which escapes into the atmosphere. Each nitrogen molecules passes through the openings in the partition wall in and out of the oxic and anoxic chamber (more than 20 times). Oxidation of ammonia and reduction of nitrate occurs alternately until all ammonia is almost converted into free nitrogen. In this way a high degree of recirculation and thereby purification is achieved without pumping. In the traditional recirculation plant, pumping is necessary to recirculate and this restricts the degree of reeirculation because of hydraulic limitations. The transport of ammonia and nitrate between the oxic and anoxic chamber through the two openings takes place by turbulent mixing in the boundary layer between the flow into the oxic and anoxic chambers. The nitrate is transported naturally by hydraulic dispersion from water with a high concentration of nitrate (oxic chamber) into water with a low concentration of nitrate (anoxic chamber) and ammonia is conveyed naturally by hydraulic dispersion from water with a high concentration of ammonia (anoxic chamber) into water with a low concen-
307
tration of ammonia (oxic chamber). This is astonishing, as a designer will usually install pumps to recireulate water from the oxic chamber back into the anoxic chamber. A mass-balance across the oxic chamber results in the normal expression for the recirculation rate R: R=TotN[in]/NO3[out]- l=Qr/O
(1)
where TotN[in]=total nitrogen in the inlet (g/m3); NO3[out]=nitrate in the outlet (g/m~); Qr=the recirculation flow (m3/s); Q=the inlet flow (m3/s). If A (m 2) is the area of the openings in the partition wall, the area A can be calculated as: A =R x Q/v=(TotN[inl/NO3[out] -1)xQ/v (m 3) (2) where v=the velocity of recirculation flow (m/s). The sludge load SL is defined as: SL=Q × BOD/V×X
(g BOD/g SS day)
(3) where B O D = t h e concentration of BOD in the inlet (g/m3); V=the volume of the process tank (m"); X=the sludge concentration (g/m3). Substituting eqn (3) into eqn (2) results in the surface area for the openings as: A = (TotN[in]/NO3[out]- 1) x (SL x X / B O D x v) x V(m 2)
(4) Inserting different realistic figures into eqn (4) one will find that appropriate surface areas of the openings will be in the range 0"05-0"25 times the area of the partition wall including the openings. If the openings are too large, too much oxygen will be transported from the oxic chamber into the anoxic chamber. If the openings are too small, the recirculation of nitrate will be too small. From eqn (2) is is seen that the appropriate area of the openings depends on the degree of purification and the wastewater flow. If the geometry of the process tank is cylindrical the relation between the area of the openings and the area of the partition wall (Ap) including the openings is: A/Ap = (TotN[in]/NO~[out] - 1) x (SL x X/BOD x v) x 0'7R
(5) where R=the radius of the process tank. From eqn (5) it can be seen that a larger process tank requires a larger relative opening in the partition wall. From the oxic chamber the mixture of purified water and activated sludge flows into one or more sedimentation tanks or separators where the purified