- Email: [email protected]
Ultramicrobacteria and their biotechnological applications
Ultramicrobacteria and their biotechnological applications Hilary M. Lappin-Scott and I. William Costerton University of Exeter, UK, and the University of Calgary, Alberta, Canada Bacteria survive periods of low-nutrient conditions by adopting a series of starvation-survival responsesto produce ultramicrobacteria. These small cells are better able to penetrate many different environments than vegetative cells, which helps them reach subterranean environments. They may therefore be used to enhance oil recovery or for in situ bioremedies. Current Opinion in Biotechnology 1992, 3:283-285
Introduction Bacteria that do not form spores or cysts survive lownutrient conditions by adopting a series of starvationsurvival responses, including a reduction in cell size, the use of cell storage products, a reduction in the e n d o g e n o u s respiration rate, the degradation of proteins, a reduction in RNA and the production of specific starvation proteins. These responses have been discussed in two recent reviews [1,2]. The decrease in cell size from vegetative ceils to starved cells is very obvious by microscopy. These small, starved cells, 0.3 g m in diameter, are usually termed ultramicrobacteria (UMB). UMB are able to resuscitate and grow w h e n growth nutrients are available, and the cells produce larger vegetative ceils once more, so the condition is reversible. Starvation-survival appears to be a widespread response, as bacteria from many different environments, including soils, estuarine waters, seawater and underground rocks have been demonstrated to survive the absence of growth nutrients and form UMB. In laboratories at the University of Calgary and the University of Exeter we have examined biotechnological applications of the changes in cell size and shape that occur during starvation and nutrient resuscitation [3,4]. Although these changes are well documented, little w o r k has been undertaken on the biotechnological applications of this p h e n o m e n o n except in our laboratories. To date, these investigations have included using UMB to increase oil recovery rates, to build subterranean barriers (termed biobarriers) to physically separate pollutants from underground aquifers or other water bodies, and for i n s i t u bioremedies. This review discusses each of these topics in turn, focusing particularly o n recent publications in each area.
U M B and oil recovery Conventional oil recovery methods are not able to extract more than 40 % of the oil in place in a reservoir,
largely because the oil is trapped in pockets of low permeability rock or is too viscous to move. Agents, such as chemicals, fibres, heat or other energy sources, or water-flooding, are a d d e d to move the oil and to help to push the oil to the surface. Bacteria have been used to assist oil recovery as they produce exopolysaccharides, biosurfactant acids or solvents, which thicken injection waters and help push oil out or assist oil flow by thinning it [5"']. However, in a new biotechnological application of bacteria, currently being researched, bacteria are used to physically block off highly permeable underground strata, so that water which is flooded into the formation to increase oil recovery rates is prevented from entering highly permeable areas and is diverted into the low permeability areas that still contain oil [5"]. This application is termed selective plugging, as the bacteria physically prevent the flood of water from entering areas of the reservoir already drained of oil. Normal-size vegetative bacteria tend to stick to rock surfaces; UMB are useful in selective plugging because they are smaller and do not stick, and are therefore able to penetrate deeply into a porous matrix such as sandstone rock [6",7"]. The technology involves a number of steps beginning with collection of water from the oil-water mix produced at the oil well. Bacteria able to survive the environmental conditions in the reservoir and able to produce UMB are isolated by laboratory screening programmes. The cultures are starved to produce the UMB which can then be injected into the reservoir to be carried by the waterflood to the areas of the highest permeability. Specific growth nutrients, of which the UMB have been starved, are injected into the reservoir so the UMB grow, produce exopolysaccharide and return to full-size vegetative cells. A dense physical barrier of bacteria is formed which effectively blocks off the high-permeability zone and diverts subsequent flooding from the high- to the low-permeability zone, thus increasing oil recovery. Much of the experimental programme to develop this technology has b e e n undertaken at the Univer-
Abbreviation UMB--ultramicrobacteria. © Current Biology Ltd ISSN 0958-1669
283
184 Environmentalbiotechnology sity of Calgary, the Alberta Research Council and the Petroleum Recovery Institute in Alberta, Canada [5"',6"]. The research included initial small-scale modelling to compare the ability of UMB and of vegetative Klebsiella p n e u m o n i a e cells to penetrate porous matrices [3,4]. Scanning electron microscopy was u s e d to observe the distribution of the cells after injection through the matrices. The micrographs s h o w e d that the UMB were evenly distributed throughout the cores whereas the vegetative ceils were mostly located in a sticky biofilm of ceils around the core inlet [4]. The modelling was then further d e v e l o p e d to include the use of reservoir rock and three-dimensional reservoir simulators to monitor the distribution of UMB and resuscitated bacteria during nutrient injections [5"',6"]. These simulations s h o w e d that the UMB of p s e u d o m o n a d s penetrated evenly throughout a h o m o g e n e o u s sand pack and then resuscitated w h e n nutrients w e r e injected into the sand. The experimental run was repeated with a heterogeneous simulator, incorporating a 22 cm long, 7 cm diameter, sandstone core surrounded by a sandpack of higher permeability, to simulate reservoirs of differing permeabilities. The UMB w e r e injected into the heterogeneous simulator and the injection of nutrients to resuscitate the starved cells followed. The viable cell counts, levels of exopolysaccharide production and scanning electron micrographs s h o w e d that the high-permeability sand had b e e n preferentially plugged by the resuscitation of the UMB. Other applications of the use of UMB in oil recovery include using them to stop coning. Coning can occur during primary oil recovery operations w h e n an aquifer lies below the oil reservoir. The water is of lower viscocity than oil, so the water is p u m p e d to the surface instead of the oil [7"]. UMB can be injected between the oil and water layers, and subsequent nutrient injections stimulate growth and resuscitation, forming a physical barrier that holds the aquifer in place so that the oil can be recovered.
UMB and biobarriers In a manner similar to that described above for coning control, UMB can also be used to produce a physical barrier, termed a biobarrier, to separate adjacent areas of the subterranean environment [7"]. These biobarriers are particularly important in minimizing environmental damage after spills of hazardous materials, such as chemicals or oil. The spills m a y seep into underground locations and may spread and contaminate bodies of water. The injection of UMB into the vicinity of the spill followed by the injection of nutrients to resuscitate the starved cells can form a physical barrier to separate the pollutant from the aquifers or lakes. Other potential uses of UMB technology are indicated by investigations into methods to cap acidogenic mine tailings, in order to generate anaerobic conditions and increase the pH of the tailings. One timely study has described the application of bacteria to uranium mine tailings together with growth nutrients; growth of the
bacteria uses up oxygen, thus generating anaerobic conditions, which reduce environmental d a m a g e b y the acidogenic railings [8].
UMB and in situ bioremedies for subterranean environments Bioremedies include the use of microorganisms to degrade environmental pollutants. Many pollutants s e e p from soils into subterranean environments and can enter and contaminate groundwater. Microbial processes can be harnessed to degrade or transform the pollutants and produce harmless by-products in situ [9"',10"]. This technology is very appealing b e c a u s e it involves the use of indigenous microorganisms in subterranean environments, but bacteria may also b e introduced to underground polluted sites to degrade the pollutants. The review b y Crawford [9"'] s u m m a rizes recent developments in this field and highlights the versatility of the technology. A detailed investigation with a similar approach has b e e n described by Madsen et al. [11"']. This study used in situ degradation of naphthalene and phenanthrene in an aquifer contaminated with polyaromatic hydrocarbons. Our research has focused on further developing this technology of in situ bioremedies b y using UMB [12]. UMB, b y virtue of their small size, m a y be injected or introduced into subterranean environments and then allowed to resuscitate and grow on pollutants in situ. Preliminary w o r k has demonstrated that bacteria that are able to degrade xenobiotic compounds can form UMB and remain in a viable state. More importantly, these UMB of xenobiotic degraders can be resuscitated o n the recalcitrant c o m p o u n d and can fully degrade it. A study b y Tett and Lappin-Scott (VA Tett and HM Lappin-Scott, abstract Ql18, 91st General Meeting of the American Society for Microbiology, Dallas, USA, May 1991) involved starving mixed cultures of bacteria that w e r e able to degrade the chlorinated p h e n o x y alkanoate herbicide m e c o p r o p [2(2-methyl 4-chloro phenoxyproprionic acid)]. The UMB remained viable throughout the 6 w e e k starvation period and did not lose their degradative capabilities. The mixed culture grew and resuscitated on m e c o p r o p and thus d e m o n strated that UMB can be used to penetrate soils and rocks to reach d e e p contaminated sites and degrade pollutants in situ.
Conclusions The technology described in this review has focused on the morphological changes that occur w h e n bacteria are deprived of growth nutrients. Novel biotechnological applications have b e e n described that harness this change in cell size b e t w e e n starved UMB and resuscitated vegetative cells and these are currently being licensed for field testing to enhance oil recovery and to prevent coning. Further uses of this exciting technology are n o w being developed and may b e c o m e commercially available in the near future.
Ultramicrobacteria and their biotechnological applications Lappin-Scott, Costerton
References and recommended reading Papers of particular interest, published within the annual period o f review, have b e e n highlighted as: of special interest °. of outstanding interest 1.
LAPPIN-SCO'ITHM, COSTERTONJW: S t a r v a t i o n a n d Penetration o f B a c t e r i a i n R o c k s a n d Soils. Experientia 1990, 46:807-821.
2.
MOPaTARY: T h e S t a r v a t i o n - s u r v i v a l State o f Microorgani s m s i n N a t u r e a n d its R e l a t i o n s h i p to t h e Bioavailabili t y E n e r g y . Experientta 1990, 46:813-817.
3.
MACLEODFA, LAPPIN-SCOTT HM, COSTERTON JW: P l u g g i n g of a Model Rock System by Using Starved Bacteria. Appl Environ Microbiol 1988, 54:1365-1372.
4.
LAPPIN-SCOTTHM, CUSACK F, COSTERTON J'W: N u t r i e n t Res u s c i t a t i o n and G r o w t h o f Starved Cells i n Sandstone Cores: a N o v e l Approach to E n h a n c e d Oil R e c o v e r y . Appl Environ Microbiol 1988, 54:1373-1382.
5. °.
CUSACKF, SINGH S, MCCARTHY C, GRIECO J, DE ROCCO M, NGUYEND, LAPPIN-SCOTT HM, COSTERTON J~W: E n h a n c e d O i l Recovery: Three Dimensional Sandpack S i m u l a t i o n o f U l t r a m i c r o b a c t e r i a R e s u s c i t a t i o n i n Reservoir Formation. J Gen Microbiol 1992, 138:457466. Describes the experimental details of the injection of UMB into three-dimensional simulators a n d their s u b s e q u e n t resuscitation to produce dense plugs within the high-permeability zones in the simulator. CUSACKF, COSTERTON JW, NOVOSAD J: Ultralnierobaacter i a E n h a n c e o i l R e c o v e r y . In Proceedings of the 4th International IGT Symposium on Gas, Oil and Environmental Biotechnology, Dec 9-11, 1992, Colorado Springs, USA. Chicago: Institute of Gas Technology; in press. Reports the injection of UMB of m i x e d cultures of bacteria into reservoir rock cores containing oil. The UMB penetrated the core, g r e w w h e n supplied with growth nutrients, and subsequently 90 % of t h e residual oil was recovered from the reservoir core.
7. BLENKINSOPPSA, COSTERTONJ'W: U n d e r s t a n d i n g Bacterial .. B i o f t l m s . Trends Biotechnol 1991, 9:138-143. Describes the m a n y uses of bacterial biofilms a n d gives details of the u s e of UMB for off recovery and biobarrier formation. 8.
BLENtONSOPP SA, HERMAN DC, MCCREADY RGL, COSTERTON jl~: A c i d o g e n i c M i n e Tailings: the Use o f B i o f i l m B a c t e r i a to E x c l u d e O x y g e n . Appl Biochem Biotechnol 1992, 34/35:801-809.
9. CRAWFORDRL: B i o r e m e d i a t i o n o f G r o u n d w a t e r Pollu•. t i o n . Curr Opin Biotechnol 1991, 2:436439. A timely review of bioremediation, including the use of pure and m i x e d subsurface microbial cultures, methanotrophic microbes, aquifer bioremediation studies a n d deep sub-surface microbes. 10. •,
FREDRICKSONJ, BALKWILLD, ZACHARAJ, LI S-M, BROCKMAN F, SIMMONS M: Physiological Diversity a n d D i s t r i b u t i o n s o f H e t e r o t r o p h i c B a c t e r i a i n Deep Cretaceous Sediments of the A t l a n t i c C o a s t a l Plain. Appl Environ Microbiol 1991, 57:402-411. The microflora of intact core s e g m e n t s retrieved from deep sub-surface formations was described a n d the possibility of using these diverse populations for in situ bioremediation w a s discussed. 11. •.
MADSENEL, SINCLAIRJL, GHIORSE w e : I n Sit'~ B i o d e g r a d a t i o n : Microbiological Patterns i n a C o n t a m i n a t e d Aquifer. Science 1991, 252:830-833. Highlights the limitations of conventional cleanup m e t h o d s for polluted aquifers and describes in situ bioremediation of a polyaromatic contaminated site.
6.
HM Lappin-Scott, Department of Biological Sciences, University of Exeter, Exeter EX4 4PS, UK. JW Costerton, Department of Biological Sciences, University of Calgary, Alberta, Canada T2N 1N4.
285
Introduction Bacteria that do not form spores or cysts survive lownutrient conditions by adopting a series of starvationsurvival responses, including a reduction in cell size, the use of cell storage products, a reduction in the e n d o g e n o u s respiration rate, the degradation of proteins, a reduction in RNA and the production of specific starvation proteins. These responses have been discussed in two recent reviews [1,2]. The decrease in cell size from vegetative ceils to starved cells is very obvious by microscopy. These small, starved cells, 0.3 g m in diameter, are usually termed ultramicrobacteria (UMB). UMB are able to resuscitate and grow w h e n growth nutrients are available, and the cells produce larger vegetative ceils once more, so the condition is reversible. Starvation-survival appears to be a widespread response, as bacteria from many different environments, including soils, estuarine waters, seawater and underground rocks have been demonstrated to survive the absence of growth nutrients and form UMB. In laboratories at the University of Calgary and the University of Exeter we have examined biotechnological applications of the changes in cell size and shape that occur during starvation and nutrient resuscitation [3,4]. Although these changes are well documented, little w o r k has been undertaken on the biotechnological applications of this p h e n o m e n o n except in our laboratories. To date, these investigations have included using UMB to increase oil recovery rates, to build subterranean barriers (termed biobarriers) to physically separate pollutants from underground aquifers or other water bodies, and for i n s i t u bioremedies. This review discusses each of these topics in turn, focusing particularly o n recent publications in each area.
U M B and oil recovery Conventional oil recovery methods are not able to extract more than 40 % of the oil in place in a reservoir,
largely because the oil is trapped in pockets of low permeability rock or is too viscous to move. Agents, such as chemicals, fibres, heat or other energy sources, or water-flooding, are a d d e d to move the oil and to help to push the oil to the surface. Bacteria have been used to assist oil recovery as they produce exopolysaccharides, biosurfactant acids or solvents, which thicken injection waters and help push oil out or assist oil flow by thinning it [5"']. However, in a new biotechnological application of bacteria, currently being researched, bacteria are used to physically block off highly permeable underground strata, so that water which is flooded into the formation to increase oil recovery rates is prevented from entering highly permeable areas and is diverted into the low permeability areas that still contain oil [5"]. This application is termed selective plugging, as the bacteria physically prevent the flood of water from entering areas of the reservoir already drained of oil. Normal-size vegetative bacteria tend to stick to rock surfaces; UMB are useful in selective plugging because they are smaller and do not stick, and are therefore able to penetrate deeply into a porous matrix such as sandstone rock [6",7"]. The technology involves a number of steps beginning with collection of water from the oil-water mix produced at the oil well. Bacteria able to survive the environmental conditions in the reservoir and able to produce UMB are isolated by laboratory screening programmes. The cultures are starved to produce the UMB which can then be injected into the reservoir to be carried by the waterflood to the areas of the highest permeability. Specific growth nutrients, of which the UMB have been starved, are injected into the reservoir so the UMB grow, produce exopolysaccharide and return to full-size vegetative cells. A dense physical barrier of bacteria is formed which effectively blocks off the high-permeability zone and diverts subsequent flooding from the high- to the low-permeability zone, thus increasing oil recovery. Much of the experimental programme to develop this technology has b e e n undertaken at the Univer-
Abbreviation UMB--ultramicrobacteria. © Current Biology Ltd ISSN 0958-1669
283
184 Environmentalbiotechnology sity of Calgary, the Alberta Research Council and the Petroleum Recovery Institute in Alberta, Canada [5"',6"]. The research included initial small-scale modelling to compare the ability of UMB and of vegetative Klebsiella p n e u m o n i a e cells to penetrate porous matrices [3,4]. Scanning electron microscopy was u s e d to observe the distribution of the cells after injection through the matrices. The micrographs s h o w e d that the UMB were evenly distributed throughout the cores whereas the vegetative ceils were mostly located in a sticky biofilm of ceils around the core inlet [4]. The modelling was then further d e v e l o p e d to include the use of reservoir rock and three-dimensional reservoir simulators to monitor the distribution of UMB and resuscitated bacteria during nutrient injections [5"',6"]. These simulations s h o w e d that the UMB of p s e u d o m o n a d s penetrated evenly throughout a h o m o g e n e o u s sand pack and then resuscitated w h e n nutrients w e r e injected into the sand. The experimental run was repeated with a heterogeneous simulator, incorporating a 22 cm long, 7 cm diameter, sandstone core surrounded by a sandpack of higher permeability, to simulate reservoirs of differing permeabilities. The UMB w e r e injected into the heterogeneous simulator and the injection of nutrients to resuscitate the starved cells followed. The viable cell counts, levels of exopolysaccharide production and scanning electron micrographs s h o w e d that the high-permeability sand had b e e n preferentially plugged by the resuscitation of the UMB. Other applications of the use of UMB in oil recovery include using them to stop coning. Coning can occur during primary oil recovery operations w h e n an aquifer lies below the oil reservoir. The water is of lower viscocity than oil, so the water is p u m p e d to the surface instead of the oil [7"]. UMB can be injected between the oil and water layers, and subsequent nutrient injections stimulate growth and resuscitation, forming a physical barrier that holds the aquifer in place so that the oil can be recovered.
UMB and biobarriers In a manner similar to that described above for coning control, UMB can also be used to produce a physical barrier, termed a biobarrier, to separate adjacent areas of the subterranean environment [7"]. These biobarriers are particularly important in minimizing environmental damage after spills of hazardous materials, such as chemicals or oil. The spills m a y seep into underground locations and may spread and contaminate bodies of water. The injection of UMB into the vicinity of the spill followed by the injection of nutrients to resuscitate the starved cells can form a physical barrier to separate the pollutant from the aquifers or lakes. Other potential uses of UMB technology are indicated by investigations into methods to cap acidogenic mine tailings, in order to generate anaerobic conditions and increase the pH of the tailings. One timely study has described the application of bacteria to uranium mine tailings together with growth nutrients; growth of the
bacteria uses up oxygen, thus generating anaerobic conditions, which reduce environmental d a m a g e b y the acidogenic railings [8].
UMB and in situ bioremedies for subterranean environments Bioremedies include the use of microorganisms to degrade environmental pollutants. Many pollutants s e e p from soils into subterranean environments and can enter and contaminate groundwater. Microbial processes can be harnessed to degrade or transform the pollutants and produce harmless by-products in situ [9"',10"]. This technology is very appealing b e c a u s e it involves the use of indigenous microorganisms in subterranean environments, but bacteria may also b e introduced to underground polluted sites to degrade the pollutants. The review b y Crawford [9"'] s u m m a rizes recent developments in this field and highlights the versatility of the technology. A detailed investigation with a similar approach has b e e n described by Madsen et al. [11"']. This study used in situ degradation of naphthalene and phenanthrene in an aquifer contaminated with polyaromatic hydrocarbons. Our research has focused on further developing this technology of in situ bioremedies b y using UMB [12]. UMB, b y virtue of their small size, m a y be injected or introduced into subterranean environments and then allowed to resuscitate and grow on pollutants in situ. Preliminary w o r k has demonstrated that bacteria that are able to degrade xenobiotic compounds can form UMB and remain in a viable state. More importantly, these UMB of xenobiotic degraders can be resuscitated o n the recalcitrant c o m p o u n d and can fully degrade it. A study b y Tett and Lappin-Scott (VA Tett and HM Lappin-Scott, abstract Ql18, 91st General Meeting of the American Society for Microbiology, Dallas, USA, May 1991) involved starving mixed cultures of bacteria that w e r e able to degrade the chlorinated p h e n o x y alkanoate herbicide m e c o p r o p [2(2-methyl 4-chloro phenoxyproprionic acid)]. The UMB remained viable throughout the 6 w e e k starvation period and did not lose their degradative capabilities. The mixed culture grew and resuscitated on m e c o p r o p and thus d e m o n strated that UMB can be used to penetrate soils and rocks to reach d e e p contaminated sites and degrade pollutants in situ.
Conclusions The technology described in this review has focused on the morphological changes that occur w h e n bacteria are deprived of growth nutrients. Novel biotechnological applications have b e e n described that harness this change in cell size b e t w e e n starved UMB and resuscitated vegetative cells and these are currently being licensed for field testing to enhance oil recovery and to prevent coning. Further uses of this exciting technology are n o w being developed and may b e c o m e commercially available in the near future.
Ultramicrobacteria and their biotechnological applications Lappin-Scott, Costerton
References and recommended reading Papers of particular interest, published within the annual period o f review, have b e e n highlighted as: of special interest °. of outstanding interest 1.
LAPPIN-SCO'ITHM, COSTERTONJW: S t a r v a t i o n a n d Penetration o f B a c t e r i a i n R o c k s a n d Soils. Experientia 1990, 46:807-821.
2.
MOPaTARY: T h e S t a r v a t i o n - s u r v i v a l State o f Microorgani s m s i n N a t u r e a n d its R e l a t i o n s h i p to t h e Bioavailabili t y E n e r g y . Experientta 1990, 46:813-817.
3.
MACLEODFA, LAPPIN-SCOTT HM, COSTERTON JW: P l u g g i n g of a Model Rock System by Using Starved Bacteria. Appl Environ Microbiol 1988, 54:1365-1372.
4.
LAPPIN-SCOTTHM, CUSACK F, COSTERTON J'W: N u t r i e n t Res u s c i t a t i o n and G r o w t h o f Starved Cells i n Sandstone Cores: a N o v e l Approach to E n h a n c e d Oil R e c o v e r y . Appl Environ Microbiol 1988, 54:1373-1382.
5. °.
CUSACKF, SINGH S, MCCARTHY C, GRIECO J, DE ROCCO M, NGUYEND, LAPPIN-SCOTT HM, COSTERTON J~W: E n h a n c e d O i l Recovery: Three Dimensional Sandpack S i m u l a t i o n o f U l t r a m i c r o b a c t e r i a R e s u s c i t a t i o n i n Reservoir Formation. J Gen Microbiol 1992, 138:457466. Describes the experimental details of the injection of UMB into three-dimensional simulators a n d their s u b s e q u e n t resuscitation to produce dense plugs within the high-permeability zones in the simulator. CUSACKF, COSTERTON JW, NOVOSAD J: Ultralnierobaacter i a E n h a n c e o i l R e c o v e r y . In Proceedings of the 4th International IGT Symposium on Gas, Oil and Environmental Biotechnology, Dec 9-11, 1992, Colorado Springs, USA. Chicago: Institute of Gas Technology; in press. Reports the injection of UMB of m i x e d cultures of bacteria into reservoir rock cores containing oil. The UMB penetrated the core, g r e w w h e n supplied with growth nutrients, and subsequently 90 % of t h e residual oil was recovered from the reservoir core.
7. BLENKINSOPPSA, COSTERTONJ'W: U n d e r s t a n d i n g Bacterial .. B i o f t l m s . Trends Biotechnol 1991, 9:138-143. Describes the m a n y uses of bacterial biofilms a n d gives details of the u s e of UMB for off recovery and biobarrier formation. 8.
BLENtONSOPP SA, HERMAN DC, MCCREADY RGL, COSTERTON jl~: A c i d o g e n i c M i n e Tailings: the Use o f B i o f i l m B a c t e r i a to E x c l u d e O x y g e n . Appl Biochem Biotechnol 1992, 34/35:801-809.
9. CRAWFORDRL: B i o r e m e d i a t i o n o f G r o u n d w a t e r Pollu•. t i o n . Curr Opin Biotechnol 1991, 2:436439. A timely review of bioremediation, including the use of pure and m i x e d subsurface microbial cultures, methanotrophic microbes, aquifer bioremediation studies a n d deep sub-surface microbes. 10. •,
FREDRICKSONJ, BALKWILLD, ZACHARAJ, LI S-M, BROCKMAN F, SIMMONS M: Physiological Diversity a n d D i s t r i b u t i o n s o f H e t e r o t r o p h i c B a c t e r i a i n Deep Cretaceous Sediments of the A t l a n t i c C o a s t a l Plain. Appl Environ Microbiol 1991, 57:402-411. The microflora of intact core s e g m e n t s retrieved from deep sub-surface formations was described a n d the possibility of using these diverse populations for in situ bioremediation w a s discussed. 11. •.
MADSENEL, SINCLAIRJL, GHIORSE w e : I n Sit'~ B i o d e g r a d a t i o n : Microbiological Patterns i n a C o n t a m i n a t e d Aquifer. Science 1991, 252:830-833. Highlights the limitations of conventional cleanup m e t h o d s for polluted aquifers and describes in situ bioremediation of a polyaromatic contaminated site.
6.
HM Lappin-Scott, Department of Biological Sciences, University of Exeter, Exeter EX4 4PS, UK. JW Costerton, Department of Biological Sciences, University of Calgary, Alberta, Canada T2N 1N4.
285