Selection of Xenobiotic-Degrading Microorganisms in a Biphasic Aqueous-Organic System

Microbial selection on mixtures of chlorinated and nonchlorinated compounds that are poorly soluble in water and/or toxic to growing microbial cells was examined in both biphasic aqueous-organic and monophasic aqueous systems. A biphasic system in which silicone oil was used as the organic phase permitted the acceleration of acclimation, leading to rapid selection and to an increase in xenobiotic compound degradation. In contrast, acclimation, selection, and degradation were very slow in the monophasic aqueous system. The variation in microbial growth rate with the degree of dispersion (i.e., dispersion at different silicone oil concentrations and agitation rates), and cell adhesion to the silicone oil indicate that the performance of the biphasic aqueous-organic system is dependent on the interfacial area between the two phases and that microbial activity is important at this interface. Therefore, the biphasic water-silicone oil system could be used for microbial selection in the presence of xenobiotic compounds that are toxic and have low water solubility.     

Plasmid as a measure of microbial degradation capacity for 2,4-dichlorophenoxyacetic acid

The purpose of this research was to pursuit the quantification of microbial degradation capacity for 2,4-dichlorophenoxyacetic acid (2,4-D) by detecting and quantifying a prominent 2,4-D degradation encoding plasmid. Batch reactor acclimation, de-acclimation, and re-acclimation tests were conducted during which periods the courses of 2,4-D dissipation and plasmid evolution were quantitatively measured. Pure cultures of bacterial strains were detected to give rise to a plasmid approximately the size of 90 kb after acclimation. The 90 kb plasmid content of Arthrobacter sp. increased when degradation occurred after acclimation, with a rate that corresponded closely to the degradation rate. During de-acclimation, plasmid content declined exponentially at a half-life of approximately 3.5 days. Re-acclimation saw a renewed induction of plasmid, but substrate consumption limited the rise of plasmid to a level much lower than after the first acclimation. This research recommends a method for measuring the microbial degradation capability for a xenobiotic.     

Long-term dynamics of catabolic plasmids introduced to a microbial community in a polluted environment: a mathematical model

The long-term dynamics of mobile plasmids in natural environments are unclear. This is the first study of the long-term dynamics of introduced plasmids with xenobiotic degradation abilities using a mathematical model that describes the horizontal gene transfer (HGT) of plasmids into indigenous bacteria via conjugation. We focussed on negative feedback between the spread of plasmids and their selective advantage, i.e. the severe competition between plasmid-bearing and plasmid-free bacteria resulting from a decrease in xenobiotic concentration caused by the gene expression of plasmids, favoring plasmid-free bacteria. Two types of HGT enhanced the persistence of plasmids and the degradation of the xenobiotic in different conditions: a relatively low rate of 'intergeneric HGT' from introduced to indigenous bacteria and a high rate of 'intraindigenous HGT' from indigenous to indigenous bacteria. In addition, when the indigenous resource supply rate was high and when the cost of bearing plasmids was low, both types of HGT made large contributions to xenobiotic degradation compared to the contribution of vertical transfer via plasmid replication within the introduced host population. Initial conditions were also important; a higher initial density of introduced plasmid-bearing bacteria led to a lower degradation rate over a long time scale.     

Loss of degradation capacity of activated sludge for a xenobiotic after a period without its influent

Xenobiotic shock experiments were conducted on lab-scale continuous-flow activated sludge systems to examine activated sludge treatment performance and to determine the xenobiotic degrader loss after periods of xenobiotic absence. The systems were operated with normal influent of a xenobiotic and a biogenic substrate until steady state, and were then artificially disturbed by removing and re-adding the xenobiotic in the influent. Substantial xenobiotic leaks were found when xenobiotic absent time was approximately one mean cell residence time (theta(c)), and the system failed when xenobiotic absent time was longer than a theta(c). Amount of degrader at the time of dual substrate steady state was estimated to be approximately 6% of the total sludge. As the xenobiotic absence time was lengthened, degrader amount in the system was reduced exponentially at a half life of approximately three days. The loss rate could be attributed mainly to the rate of displacement by theta(c) operation, followed by endogenous decay and de-acclimation loss.     

Biodegradation of BTE in soil by indigenous microbial populations with and without biogenic substrates

Degradation of benzene, toluene, and ethylbenzene (BTE) by microbial populations indigenous to the soil and populations proliferated from the indigenous using biogenic substrates were compared. The reaction system consisted of aerobic microcosms representing an unsaturated soil. Microcosms supplemented with glucose and citrate, when compared to the unsupplemented microcosms, showed increases in bacterial counts, but the overall degradation rates for B, T, or E were reduced in spite of shorter lag times. Both biogenic substrate supplements were non-beneficial for BTE degradation due largely to the preferential and healthy growth of the indigenous populations on the biogenic substrates, and thus the urgency of developing a favorable amount of BTE degraders was reduced.     

Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils.

We studied the mineralization of pyrene, carbazole, and benzo[a]pyrene in soils obtained from three abandoned coal gasification plants in southern Illinois. The soils had different histories of past exposure to hydrocarbon contamination and different amounts of total organic carbon, microbial biomass, and microbial activity. Mineralization was measured by using serum bottle radiorespirometry. The levels of indigenous mineralization of 14C-labeled compounds ranged from 10 to 48% for pyrene, from undetectable to 46% for carbazole, and from undetectable to 25% for benzo[a]pyrene following long-term (greater than 180-day) incubations. Pyrene and carbazole were degraded with short or no lag periods in all soils, but benzo[a]pyrene mineralization occurred after a 28-day lag period. Mineralization was not dependent on high levels of microbial biomass and activity in the soils. Bacterial cultures that were capable of degrading pyrene and carbazole were isolated by enrichment, grown in pure culture, and reintroduced into soils. Reintroduction of a pyrene-degrading bacterium enhanced mineralization to a level of 55% within 2 days, compared with a level of 1% for the indigenous population. The carbazole degrader enhanced mineralization to a level of 45% after 7 days in a soil that showed little indigenous carbazole mineralization. The pyrene and carbazole degraders which we isolated were identified as a Mycobacterium sp. and a Xanthamonas sp., respectively. Our results indicated that mineralization of aromatic hydrocarbons can be significantly enhanced by reintroducing isolated polycyclic aromatic hydrocarbon-degrading bacteria.     

Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils.

We studied the mineralization of pyrene, carbazole, and benzo[a]pyrene in soils obtained from three abandoned coal gasification plants in southern Illinois. The soils had different histories of past exposure to hydrocarbon contamination and different amounts of total organic carbon, microbial biomass, and microbial activity. Mineralization was measured by using serum bottle radiorespirometry. The levels of indigenous mineralization of 14C-labeled compounds ranged from 10 to 48% for pyrene, from undetectable to 46% for carbazole, and from undetectable to 25% for benzo[a]pyrene following long-term (greater than 180-day) incubations. Pyrene and carbazole were degraded with short or no lag periods in all soils, but benzo[a]pyrene mineralization occurred after a 28-day lag period. Mineralization was not dependent on high levels of microbial biomass and activity in the soils. Bacterial cultures that were capable of degrading pyrene and carbazole were isolated by enrichment, grown in pure culture, and reintroduced into soils. Reintroduction of a pyrene-degrading bacterium enhanced mineralization to a level of 55% within 2 days, compared with a level of 1% for the indigenous population. The carbazole degrader enhanced mineralization to a level of 45% after 7 days in a soil that showed little indigenous carbazole mineralization. The pyrene and carbazole degraders which we isolated were identified as a Mycobacterium sp. and a Xanthamonas sp., respectively. Our results indicated that mineralization of aromatic hydrocarbons can be significantly enhanced by reintroducing isolated polycyclic aromatic hydrocarbon-degrading bacteria.     

Biomonitoring of continuous microbial community adaptation towards more efficient phenol-degradation in a fed-batch bioreactor

The anaerobic degradation of phenol was studied in a fed-batch culture. Nitrate was added as electron acceptor and phenol was provided three times, to a final concentration of 200 mg/l. Randomly amplified polymorphic DNA (RAPD) and terminal fraction fragment length polymorphism (T-RFLP) were used and compared in order to monitor the microbial succession in the reactor. Phenol degradation started after an initial lag phase of 14 days and was then completed within a few days. In addition, the duration of the lag phase was shortened and the degradation rate was increased after each phenol amendment. Nitrate reduction correlated with microbial growth and phenol depletion, confirming that the degradation was carried out anaerobically. Results from the DNA analysis showed that the structure of the microbial community changed after each phenol amendment. This study confirms the potential for anaerobic degradation of environmental pollutants and also confirms that microbial acclimation towards faster degradation rates occurred upon repeated substrate amendments. Furthermore, both of the DNA-based techniques described the phenol degradation-linked community shifts with similar general results. RAPD is a faster, simpler technique that gives a higher resolution and consequently reflects the shifts in the microbial community structure better, whereas T-RFLP is more suitable for phylogenetic studies.     

Microbial dynamics in a continuous stirred tank bioreactor exposed to an alternating sequence of organic compounds

Microbial dynamics during aerobic biodegradation of an alternating mixture of organic compounds was investigated experimentally in a continuous stirred tank bioreactor (CSTB). A mathematical model describing this system was developed and tested using the experimental results. A model microbial culture consisting of Pseudomonas sp. JS150, a monochlorobenzene (MCB) degrader, and Xanthobacter autotrophicus GJ10, a 1,2-dichloroethane (DCE) degrader, each with exclusive degradation capabilities, was used. The CSTB was inoculated with both microbial strains and exposed to an alternating sequence of the two compounds at noninhibitory concentrations. Concentrations of each microbial strain, of each organic compound, and of degradation product evolved, as well as specific microbial activities via oxygen uptake tests, were monitored. Reduction of the residual DCE discharged from the bioreactor after an MCB to DCE transition was successfully achieved by continuously feeding a low flow of a concentrated solution of both compounds.     

Biodegradation of phenoxyacetic acid in soil by Pseudomonas putida PP0301(pR0103), a constitutive degrader of 2, 4–dichlorophenoxyacetate

The efficacy of using genetically engineered microbes (GEMs) to degrade recalcitrant environmental toxicants was demonstrated by the application of Pseudomonas putida PP0301(pR0103) to an Oregon agricultural soil amended with 500 micrograms/g of a model xenobiotic, phenoxyacetic acid (PAA). P. putida PP0301(pR0103) is a constitutive degrader of 2,4-dichlorophenoxyacetate (2,4-D) and is also active on the non-inducing substrate, PAA. PAA is the parental compound of 2,4-dichlorophenoxyacetic acid (2,4-D) and whilst the indigenous soil microbiota degraded 500 micrograms/g 2,4-D to less than 10 micrograms/g, PAA degradation was insignificant during a 40-day period. No significant degradation of PAA occurred in soil inoculated with the parental strain P. putida PP0301 or the inducible 2,4-D degrader P. putida PP0301(pR0101). Moreover, co-amendment of soil with 2,4-D and PAA induced the microbiota to degrade 2,4-D; PAA was not degraded. P. putida PP0301-(pR0103) mineralized 500-micrograms/g PAA to trace levels within 13 days and relieved phytotoxicity of PAA to Raphanus sativus (radish) seeds with 100% germination in the presence of the GEM and 7% germination in its absence. In unamended soil, survival of the plasmid-free parental strain P. putida PP0301 was similar to the survival of the GEM strain P. putida PP0301(pR0103). However, in PAA amended soil, survival of the parent strain was over 10,000-fold lower (< 3 colony forming units per gram of soil) than survival of the GEM strain after 39 days.     

采油微生物在微驱过程中的生长、运移及分布规律

[目的]深入了解现场微生物驱油机理、效果评价标准及影响因素.[方法]结合现场微生物驱油过程产出液的跟踪监测及室内物模实验对微生物在地层中的生长繁殖、运移及分布规律进行研究.[结果]结果表明,通过从水井注入的外源微生物在油藏中能够有效生长繁殖,而且注入的营养液也能够激活内源微生物,但由于地层渗透率及营养液浓度的影响,产出液菌浓要比注入菌浓低1-2个数量级;葡萄糖的快速降解以及地层对微生物的过滤及吸附作用使大量的微生物停留在近井地带,仅有部分微生物能够从生产井采出,而且其运移速度要比营养液慢.[结论]地层渗透率和产出液中营养物浓度是影响微生物数量及分布的两个关键因素,现场微生物驱油产出液中的菌浓一般很难达到106个/mL以上,该研究结果对微生物驱油技术的发展和应用具有重要意义.     

Key players and team play: anaerobic microbial communities in hydrocarbon-contaminated aquifers

Biodegradation of anthropogenic pollutants in shallow aquifers is an important microbial ecosystem service which is mainly brought about by indigenous anaerobic microorganisms. For the management of contaminated sites, risk assessment and control of natural attenuation, the assessment of in situ biodegradation and the underlying microbial processes is essential. The development of novel molecular methods, “omics” approaches, and high-throughput techniques has revealed new insight into complex microbial communities and their functions in anoxic environmental systems. This review summarizes recent advances in the application of molecular methods to study anaerobic microbial communities in contaminated terrestrial subsurface ecosystems. We focus on current approaches to analyze composition, dynamics, and functional diversity of subsurface communities, to link identity to activity and metabolic function, and to identify the ecophysiological role of not yet cultured microbes and syntrophic consortia. We discuss recent molecular surveys of contaminated sites from an ecological viewpoint regarding degrader ecotypes, abiotic factors shaping anaerobic communities, and biotic interactions underpinning the importance of microbial cooperation for microbial ecosystem services such as contaminant degradation.     

Coupled reductive and oxidative degradation of 4-chloro-2-nitrophenol by a co-immobilized mixed culture system

Summary The restriction of oxygen transfer in Ca-alginate beads used for the immobilization of microbial cells was applied to a coupled reductive and oxidative microbial degradation of the xenobiotic 4-chloro-2-nitrophenol (CNP). The conversion of CNP by Enterobacter cloacae under anaerobic conditions led to the formation of 4-chloro-2-aminophenol (CAP, 81%) and 4-chloro-2-acetaminophenol (CAAP, 16%) after 50 h incubation. CAP, the main reduction product, was further degraded under aerobic conditions by Alcaligenes sp. TK-2, a hybrid strain isolated by conjugative in-vivo gene transfer. Whereas both degradation steps excluded one another in homogeneous systems with free cells, a coupled reductive and oxidative degradation of CNP was observed in one aerated reactor system after co-immobilization of both strains in Ca alginate. The diameter of the alginate beads used for immobilization was recognized as one main factor determining the properties of this mixed culture system. Offprint requests to: H.-J. Rehm     

Unveiling metabolic characteristics of an uncultured Gammaproteobacterium responsible for in situ PAH biodegradation in petroleum polluted soil

Exploring the metabolic characteristics of indigenous PAH degraders is critical to understanding the PAH bioremediation mechanism in the natural environment. While stable-isotopic probing (SIP) is a viable method to identify functional microorganisms in complex environments, the metabolic characteristics of uncultured degraders are still elusive. Here, we investigated the naphthalene (NAP) biodegradation of petroleum polluted soils by combining SIP, amplicon sequencing and metagenome binning. Based on the SIP and amplicon sequencing results, an uncultured Gammaproteobacterium sp. was identified as the key NAP degrader. Additionally, the assembled genome of this uncultured degrader was successfully obtained from the ¹³C-DNA metagenomes by matching its 16S rRNA gene with the SIP identified OTU sequence. Meanwhile, a number of NAP degrading genes encoding naphthalene/PAH dioxygenases were identified in this genome, further confirming the direct involvement of this indigenous degrader in the NAP degradation. The degrader contained genes related to the metabolisms of several carbon sources, energy substances and vitamins, illuminating potential reasons for why microorganisms cannot be cultivated and finally realize their cultivation. Our findings provide novel information on the mechanisms of in situ PAH biodegradation and add to our current knowledge on the cultivation of non-culturable microorganisms by combining both SIP and metagenome binning.     

The role of cytochrome P450 monooxygenases in microbial fatty acid metabolism

Cytochrome P450 monooxygenases (P450s) are a diverse collection of enzymes acting on various endogenous and xenobiotic molecules. Most of them catalyse hydroxylation reactions and one group of possible substrates are fatty acids and their related structures. In this minireview, the significance of P450s in microbial fatty acid conversion is described. Bacteria and yeasts possess various P450 systems involved in alkane and fatty acid degradation, and often several enzymes with different activities and specificities are retrieved in one organism. Furthermore, P450s take part in the formation of fatty acid-based secondary metabolites. Finally, there are a substantial number of microbial P450s displaying activity towards fatty acids, but to which no biological role could be assigned despite the often quite intense research.     

Biotransformation of D-limonene to (+) trans-carveol by toluene-grown Rhodococcus opacus PWD4 cells

The toluene-degrading strain Rhodococcus opacus PWD4 was found to hydroxylate D-limonene exclusively in the 6-position, yielding enantiomerically pure (+) trans-carveol and traces of (+) carvone. This biotransformation was studied using cells cultivated in chemostat culture with toluene as a carbon and energy source. The maximal specific activity of (+) trans-carveol formation was 14.7 U (g of cells [dry weight])(-1), and the final yield was 94 to 97%. Toluene was found to be a strong competitive inhibitor of the D-limonene conversion. Glucose-grown cells did not form any trans-carveol from D-limonene. These results suggest that one of the enzymes involved in toluene degradation is responsible for this allylic monohydroxylation. Another toluene degrader (Rhodococcus globerulus PWD8) had a lower specific activity but was found to oxidize most of the formed trans-carveol to (+) carvone, allowing for the biocatalytic production of this flavor compound.     

Soil microbial population dynamics following bioaugmentation with a 3-chlorobenzoate-degrading bacterial culture

Changes in microbial populations were evaluated following inoculation of contaminated soil with a 3-chlorobenzoate degrader. Madera sandy loam was amended with 0, 500, or 1000 g 3-chlorobenzoate g^-1 dry soil. Selected microcosms were inoculated with the degrader Comamonas testosteroni BR60. Culturable bacterial degraderswere enumerated on minimal salts media containing 3-chlorobenzoate. Culturableheterotrophic bacteria were enumerated on R2A. Isolated degraders were grouped by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction fingerprints and identified based on 16S ribosomal-DNA sequences. Bioaugmentation increased the rate of degradation at both levels of 3-chlorobenzoate. In both the 500 and 1000 g 3-chlorobenzoate g^-1 dry soil inoculated microcosms, degradersincreased from the initial inoculum and decreased following degradation of 3-CB.Inoculation delayed the development of indigenous 3-chlorobenzoate degrading populations. It is unclear if inoculation altered the composition of indigenous degrader populations. In the uninoculated soil, degraders increased from undetectable levels to 6.6 × 10⁷ colony-forming-units g^-1 dry soil in the 500 g 3-chlorobenzoate g^-1 dry soil microcosms, but none were detected in the 1000 g 3-chlorobenzoate g^-1 dry soil microcosms. Degraders isolated from uninoculated soil were identified as one of two distinct Burkholderia species.In the uninoculated soil, numbers of culturable heterotrophic bacteria initially decreased following addition of 1000 g 3-chlorobenzoate g^-1 dry soil. Inoculation with C. testosteroni reduced this negative impact on culturable bacterial numbers. The results indicate that bioaugmentation may not only increase the rate of 3-chlorobenzoate degradation but also reduce the deleterious effects of 3-chlorbenzoate on indigenous soil microbial populations.     

Anaerobic mineralization of pentachlorophenol (PCP) by combining PCP-dechlorinating and phenol-degrading cultures

The dechlorination and mineralization of pentachlorophenol (PCP) was investigated by simultaneously or sequentially combining two different anaerobic microbial populations, a PCP-dechlorinating culture capable of the reductive dechlorination of PCP to phenol and phenol- degrading cultures able to mineralize phenol under sulfate- or iron-reducing conditions. In the simultaneously combined mixture, PCP (about 35 microM) was mostly dechlorinated to phenol after incubation for 17 days under sulfate-reducing conditions or for 22 days under iron-reducing conditions. Thereafter, the complete removal of phenol occurred within 40 days under both conditions. In the sequentially combined mixture, most of the phenol, the end product of PCP dechlorination, was degraded within 12 days of inoculation with the phenol degrader, without a lag phase, under both sulfate- and iron-reducing conditions. In a radioactivity experiment, [14C-U]-PCP was mineralized to 14CO2 and 14CH4 by the combined anaerobic microbial activities. Analysis of electron donor and acceptor utilization and of the production and consumption of H2, CO2, and CH4 suggested that the dechlorinating and degrading microorganisms compete with other microorganisms to perform PCP dechlorination and part of the phenol degradation in complex anoxic environments in the presence of electron donors and acceptors. The presence of a small amount of autoclaved soil slurry in the medium was possibly another advantageous factor in the successful dechlorination and mineralization of PCP by the combined mixtures. This anaerobic-anaerobic combination technology holds great promise as a cost-effective strategy for complete PCP bioremediation in situ.     

Shift from growth to nutrient-limited maintenance kinetics during biofilter acclimation

During long-term operation of a biofilter, the mandatory absence of net cell growth forces the cells into maintenance metabolism, which is of relatively low rate compared to substrate consumption during the active growth of the acclimation phase. A model based on this shift in metabolism can explain the postacclimation decrease in activity sometimes reported for biofilters. The cessation of growth can be caused by nutrient depletion in the bed. Postacclimation nutrient addition increases activity primarily by allowing a return to the high substrate consumption rate of active growth, and only secondarily helps raise bed activity because of the ultimately higher amount of biomass in the bed. Simulations incorporating the acclimation period and the role of maintenance metabolism predict about 4 logarithms of growth during acclimation of a hexane biofilter, which was confirmed experimentally. Changes in a biofilter's biomass during the acclimation phase can be estimated from substrate conversion data using two approximate methods. The first follows the cumulative amount of substrate converted and uses the estimated yield of cells from substrate during active growth to estimate the total biomass created. The second method follows a rate constant for conversion of substrate in the bed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 330-339, 1997.