Retrospective on microbial transformations of halogenated organics.

Prior to the 1960s, knowledge of biological transformations of highly halogenated aliphatic compounds was limited, except in mammalian organisms where enzymatic transformations occurred to rid the body of ingested harmful chemicals. Limited abiotic transformation of such compounds had also been observed, with half-lives varying from days to centuries. Commonly believed was that aerobic transformation might occur by cometabolism rather than to conserve energy for respiration, while anaerobic transformations were in general thought not to occur. However, in the late 1960s anaerobic transformation of chlorinated pesticides was noted, and then in the early 1980s, partial microbial dehalogenation of chlorinated solvents such as tetrachlorethene, trichloroethene, trichlorethane, and carbon tetrachloride was also found to occur. With only partial dechlorination, complete detoxification was not achieved. And at the time, dehalogenation reactions were not believed to yield energy for growth to the degrading microorganisms. However, in the 1990s bacteria began to be found that obtain energy from anaerobic transformations, often enabling complete dechlorination and detoxification. Since then such ability has been found among several bacterial species, many of which use molecular hydrogen as a donor substrate and halogenated organics as electron acceptors, thus conserving energy through reductive dehalogenation. Growth of knowledge in this field has grown rapidly since the 1960s. Broad usages of such microorganisms are now underway to rid contaminated groundwater of hazardous halogenated chemicals.

Effect of temperature on the treatment of domestic wastewater with a staged anaerobic fluidized membrane bioreactor.

A laboratory staged anaerobic fluidized membrane bioreactor (SAF-MBR) system was applied to the treatment of primary clarifier effluent from a domestic wastewater treatment plant with temperature decreasing from 25 to 10 °C. At all temperatures and with a total hydraulic retention time of 2.3 h, overall chemical oxygen demand (COD) and biochemical oxygen demand (BOD5) removals were 89% and 94% or higher, with permeate COD and BOD5 of 30 and 7 mg/L or lower, respectively. No noticeable negative effects of low temperature on organic removal were found, although a slight increase to 3 mg/L in volatile fatty acids concentrations in the effluent was observed. Biosolids production was 0.01-0.03 kg volatile suspended solids/kg COD, which is far less than that with aerobic processes. Although the rate of trans-membrane pressure at the membrane flux of 9 L/m(2)/h increased as temperature decreased, the SAF-MBR was operated for longer than 200 d before chemical cleaning was needed. Electrical energy potential from combustion of the total methane production (gaseous and dissolved) was more than that required for system operation.

Two-stage anaerobic fluidized-bed membrane bioreactor treatment of settled domestic wastewater.

A two-stage anaerobic fluidized-bed membrane bioreactor (SAF-MBR) system was applied for the treatment of primary-settled domestic wastewater that was further pre-treated by either 10 µm filtration or 1 mm screening. While the different pre-treatment options resulted in different influent qualities, the effluent qualities were quite similar. In both cases at a total hydraulic retention time of 2.3 h and 25 °C, chemical oxygen demand and biochemical oxygen demand (BOD5) removals were 84-91% and 92-94%, with effluent concentrations lower than 25 and 7 mg/L, respectively. With a membrane flux of 6-12 L/m(2)/h, trans-membrane pressure remained below 0.2 bar during 310 d of continuous operation without need for membrane chemical cleaning or backwashing. Biosolids production was estimated to be 0.028-0.049 g volatile suspended solids/g BOD5, which is far less than that with comparable aerobic processes. Electrical energy production from combined heat and power utilization of the total methane produced (gaseous and dissolved) was estimated to be more than sufficient for total system operation.

Effects of biomass accumulation on microbially enhanced dissolution of a PCE pool: a numerical simulation.

Recent studies have shown that dechlorinating bacteria can accelerate the dissolution rate of dense, nonaqueous phase liquids (DNAPLs) containing tetrachloroethene (PCE). We present an advection-dispersion-reaction model for a two-dimensional domain, with groundwater flowing over a pool of free-product PCE. PCE is converted to cis-1,2-dichloroethene (cDCE) and toxicity due to PCE or cDCE is neglected. We adopt previously published correlations relating biomass concentrations and hydraulic conductivity, accounting for biofilm growth and plug-like growth. The system of coupled equations is solved numerically. The high biotransformation rate of PCE increases the concentration gradient of PCE at the water-DNAPL interface, enhancing dissolution. The higher the electron donor (ED) concentration, the larger the dissolution enhancement. Based on the values of maximum specific rate we used, when the electron donor is unlimited, the active biomass accumulates adjacent to the water-NAPL interface and microbial reactions can significantly enhance the pool dissolution. The resulting steady-state dissolution rate can be approximated by a half-order solution when zero-order kinetics are suitable for representing the microbial reaction. However, bioclogging may significantly reduce local hydraulic conductivity; thus, it decreases the flow near the water-DNAPL interface, decreasing dissolution. When the ED is the limiting factor, active biomass accumulates away from the interface. This creates a no-flow zone between the active biomass and the interface. The enlargement of the no-flow zone, due to the donor limitation, diminishes the concentration gradient and the flushing around the water-DNAPL interface. Such adverse impacts may significantly decrease the enhancement predicted by models that do not consider the effects of bioclogging.

The development of anaerobic treatment and its future.

Anaerobic treatment is well over 100 years old. Its initial development was for the treatment of domestic wastewaters, using anaerobic filters and hybrid processes that are still of interest today. It then progressed in application to separate sludge digestion, then to treatment of dilute industrial wastewaters. Several processes have been developed that accomplish efficient treatment of wastewaters at short detention times. Major contributions to the broad application of anaerobic treatment and the better understanding of this process has come from efforts by G. Lettinga and his colleagues. They have emphasized its importance for meeting the need for sustainable development in the future. Greater efforts are now needed for broad application of anaerobic treatment for ridding the environment of unwanted organic materials by converting them into methane, a renewable energy source.

Laboratory evaluation of a two-stage treatment system for TCE cometabolism by a methane-oxidizing mixed culture.

The objective of this research was to evaluate several factors affecting the performance of a two-stage treatment system employing methane-oxidizing bacteria for trichloroethylene (TCE) biodegradation. The system consists of a completely mixed growth reactor and a plug-flow transformation reactor in which the TCE is cometabolized. Laboratory studies were conducted with continuous growth reactors and batch experiments simulating transformation reactor conditions. Performance was characterized in terms of TCE transformation capacity (T(C), g TCE/g cells), transformation yield (T(Y), g TCE/g CH(4)), and the rate coefficient ratio k(TCE)/K(S,TCE) (L/mg-d). The growth reactor variables studied were solids retention time (SRT) and nutrient nitrogen (N) concentration. Formate and methane were evaluated as potential transformation reactor amendments. Comparison of cultures from 2- and 8-day SRT (nitrogen-limited) growth reactors indicated that there was no significant effect of growth reactor SRT or nitrogen availability on T(C) or T(Y), but N-limited conditions yielded higher k(TCE)/K(S,TCE). The TCE cometabolic activity of the 8-day SRT, N-limited growth reactor culture varied significantly during a 7-year period of operation. The T(C) and T(Y) of the resting cells increased gradually to levels a factor of 2 higher than the initial values. The reasons for this increase are unknown. Formate addition to the transformation reactor gave higher T(C) and T(Y) for 2-day SRT growth reactor conditions and significantly lower T(C), T(Y), and k(TCE)/K(S,TCE) for 8-day SRT N-limited conditions. Methane addition to the transformation reactor inhibited TCE cometabolism at low TCE concentrations and enhanced TCE cometabolism at high TCE concentrations, indicating that the TCE cometabolism in the presence of methane does not follow simple competitive inhibition kinetics. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 650-659, 1997.

A novel means to develop strain-specific DNA probes for detecting bacteria in the environment.

A simple means to develop strain-specific DNA probes for use in monitoring the movement and survival of bacteria in natural and laboratory ecosystems was developed. The method employed amplification of genomic DNA via repetitive sequence-based PCR (rep-PCR) using primers specific for repetitive extragenic palindromic (REP) elements, followed by cloning of the amplified fragments. The cloned fragments were screened to identify those which were strain specific, and these were used as probes for total genomic DNA isolated from microbial communities and subjected to rep-PCR. To evaluate the utility of the approach, we developed probes specific for Burkholderia cepacia G4 and used them to determine the persistence of the strain in aquifer sediment microcosms following bioaugmentation. Two of four probes tested were found to specifically hybridize to DNA fragments of the expected sizes in the rep-PCR fingerprint of B. cepacia G4 but not to 64 genetically distinct bacteria previously isolated from the aquifer. One of these probes, a 650-bp fragment, produced a hybridization signal when as few as 10 CFU of B. cepacia G4 were present in a mixture with 10(6) CFU nontarget strains, indicating that the sensitivity of these probes was comparable to those of other PCR-based detection methods. The probes were used to discriminate groundwater and microcosm samples that contained B. cepacia G4 from those which did not. False-positive results were obtained with a few samples, but these were readily identified by using hybridization to the second probe as a confirmation step. The general applicability of the method was demonstrated by constructing probes specific to three other environmental isolates.

Microbial Succession during a Field Evaluation of Phenol and Toluene as the Primary Substrates for Trichloroethene Cometabolism.

Microbial community composition and succession were studied in an aquifer that was amended with phenol, toluene, and chlorinated aliphatic hydrocarbons to evaluate the effectiveness of these aromatic substrates for stimulating trichloroethene (TCE) bioremediation. Samples were taken after the previous year's field studies, which used phenol as the primary substrate, and after three successive monthly treatments of phenol plus 1,1-dichloroethene (1,1-DCE) plus TCE, phenol plus TCE, and toluene plus TCE. Dominant eubacteria in the community were assessed after each of the four treatments by characterizing isolates from the most dilute most-probable-number tubes and by extracting DNA from aquifer samples. The succession of dominant phenol- and toluene-degrading strains was evaluated by genomic fingerprinting, cellular fatty acid methyl ester (FAME) analysis, and amplified ribosomal DNA restriction analysis (ARDRA). 1,1-DCE was found to drastically reduce microbial growth and species richness, which corresponded to the reduction in bioremediation effectiveness noted previously for this treatment (G. D. Hopkins and P. L. McCarty, Environ. Sci. Technol. 29:1628-1637, 1995). Only a few gram-positive isolates could be obtained after treatment with 1,1-DCE, and these were not seen after any other treatments. Microbial densities returned to their original levels following the subsequent phenol-TCE treatment, but the original species richness was not restored until after the subsequent toluene-TCE treatment. Genomic fingerprinting and FAME analysis indicated that six of the seven originally dominant microbial groups were still dominant after the last treatment, indicating that the community is quite resilient to toxic disturbance by 1,1-DCE. FAME analysis indicated that six microbial taxa were dominant: three members of the (beta) subclass of the class Proteobacteria (Comamonas-Variovorax, Azoarcus, and Burkholderia) and three gram-positive groups (Bacillus, Nocardia, and an unidentified group). ARDRA revealed that the dominant community members were stable during the three nontoxic treatments and that virtually all of the bands could be accounted for by isolates from five of the dominant taxa, indicating that the isolation protocol used likely recovered most of the dominant members of this community.

Transformation yields of chlorinated ethenes by a methanotrophic mixed culture expressing particulate methane monooxygenase.

Transformation yields for the aerobic cometabolic degradation of five chlorinated ethenes were determined by using a methanotrophic mixed culture expressing particulate methane monooxygenase (pMMO). Transformation yields (expressed as moles of chlorinated ethene degraded per mole of methane consumed) were 0.57, 0.25, 0.058, 0.0019, and 0.00022 for trans-1,2-dichloroethylene (t-DCE), vinyl chloride (VC), cis-1,2-dichloroethylene (c-DCE), trichloroethylene (TCE), and 1,1-dichloroethylene (1,1-DCE), respectively. Degradation of t-DCE and VC was observed only in the presence of formate or methane, sources of reducing energy necessary for cometabolism. The t-DCE and VC transformation yields represented 35 and 15%, respectively, of the theoretical maximum yields, based on reducing-energy availability from methane dissimilation to carbon dioxide, exclusive of all other processes that require reducing energy. The yields for t-DCE and VC were 20 times greater than the yields reported by others for cells expressing soluble methane monooxygenase (sMMO). Transformation yields for c-DCE, TCE, and 1,1-DCE were similar to or less than those for cultures expressing sMMO. Although methanotrophic biotreatment systems have typically been designed to incorporate cultures expressing sMMO, these results suggest that pMMO expression may be highly advantageous for degradation of t-DCE or VC. It may also be much easier to maintain pMMO expression in treatment systems, because pMMO is expressed by all methanotrophs whereas sMMO is expressed only by type II methanotrophs under copper-limited conditions.

Numerical modeling and uncertainties in rate coefficients for methane utilization and TCE cometabolism by a methane-oxidizing mixed culture.

The rates of methane utilization and trichloroethylene (TCE) cometabolism by a methanotrophic mixed culture were characterized in batch and pseudo-steady-state studies. Procedures for determination of the rate coefficients and their uncertainties by fitting a numerical model to experimental data are described. The model consisted of a system of differential equations for the rates of Monod kinetics, cell growth on methane and inactivation due to TCE transformation product toxicity, gas/liquid mass transfer of methane and TCE, and the rate of passive losses of TCE. The maximum specific rate of methane utilization (k(CH(4) )) was determined by fitting the numerical model to batch experimental data, with the initial concentration of active methane-oxidizing cells (X(0) (a)) also used as a model fitting parameter. The best estimate of k(CH(4) ) was 2.2 g CH(4)/g cells-d with excess copper available, with a single-parameter 95% confidence interval of 2.0-2.4 mg/mg-d. The joint 95% confidence region for k(CH(4) ) and X(0) (a) is presented graphically. The half-velocity coefficient (K(S,CH(4) )) was 0.07 mg CH(4)/L with excess copper available and 0.47 mg CH(4)/L under copper limitation, with 95% confidence intervals of 0.02-0.11 and 0.35-0.59 mg/L, respectively. Unique values of the TCE rate coefficients k(TCE) and K(S,TCE) could not be determined because they were found to be highly correlated in the model fitting analysis. However, the ratio k(TCE)/K(S,TCE) and the TCE transformation capacity (T(C)) were well defined, with values of 0.35 L/mg-day and 0.21 g TCE/g active cells, respectively, for cells transforming TCE in the absence of methane or supplemental formate. The single-parameter 95% confidence intervals for k(TCE)/K(S,TCE) and T(C) were 0.27-0.43 L/mg-d and 0.18-0.24 g TCE/g active cells, respectively. The joint 95% confidence regions for k(TCE)/K(S,TCE) and T(C) are presented graphically.

In vitro studies on reductive vinyl chloride dehalogenation by an anaerobic mixed culture.

Reductive dehalogenation of vinyl chloride (VC) was studied in an anaerobic mixed bacterial culture. In growth experiments, ethene formation from VC increased exponentially at a rate of about 0.019 h(sup-1). Reductive VC dehalogenation was measured in vitro by using cell extracts of the mixed culture. The apparent K(infm) for VC was determined to be about 76 (mu)M; the V(infmax) was about 28 nmol (middot) min(sup-1) (middot) mg of protein(sup-1). The VC-dehalogenating activity was membrane associated. Propyl iodide had an inhibitory effect on the VC-dehalogenating activity in the in vitro assay. However, this inhibition could not be reversed by illumination. Cell extracts also catalyzed the reductive dehalogenation of cis-1,2-dichloroethene (cis-DCE) and, at a lower rate, of trichloroethene (TCE). Tetrachloroethene (PCE) was not transformed. The results indicate that the reductive dehalogenation of VC and cis-DCE described here is different from previously reported reductive dehalogenation of PCE and TCE.

Isolation and Characterization of a Facultatively Aerobic Bacterium That Reductively Dehalogenates Tetrachloroethene to cis-1,2-Dichloroethene.

A rapidly-growing facultatively aerobic bacterium that transforms tetrachloroethene (PCE) via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-1,2-DCE) at high rates in a defined medium was isolated from a contaminated site. Metabolic characterization, cellular fatty acid analysis, and partial sequence analysis of 16S rRNA showed that the new isolate, strain MS-1, has characteristics matching those of the members of the family Enterobacteriaceae. Strain MS-1 can oxidize about 58 substrates including many carbohydrates, short-chain fatty acids, amino acids, purines, and pyrimidines. It can transform up to 1 mM PCE (aqueous) at a rate of about 0.5 (mu)mol of PCE(middot) h(sup-1)(middot)mg (dry weight) of cell(sup-1). PCE transformation occurs following growth on or with the addition of single carbon sources such as glucose, pyruvate, formate, lactate, or acetate or with complex nutrient sources such as yeast extract or a mixture of amino acids. PCE dehalogenation requires the absence of oxygen, nitrate, and high concentrations of fermentable compounds such as glucose. Enterobacter agglomerans biogroup 5 (ATCC 27993), a known facultative bacterium that is closely related to strain MS-1, also reductively dehalogenated PCE to cis-1,2-DCE. To our knowledge, this is the first report on isolation of a facultative bacterium that can reductively transform PCE to cis-1,2-DCE under defined physiological conditions. Also, this is the first report of the ability of E. agglomerans to dehalogenate PCE.

Microcosm and in situ field studies of enhanced biotransformation of trichloroethylene by phenol-utilizing microorganisms.

The ability of different aerobic groundwater microorganisms to cometabolically degrade trichloroethylene (TCE), 1,2-cis-dichloroethylene (c-DCE), and 1,2-trans-dichloroethylene (t-DCE) was evaluated both in groundwater-fed microcosms and in situ in a shallow aquifer. Microcosms amended with phenol or toulene were equally effective in removing c-DCE (> 90%) followed by TCE (60 to 70%), while the microcosm fed methane was most effective in removing t-DCE (> 90%). The microcosm fed ammonia was the least effective. None of the microcosms effectively degraded 1,1,1-trichloroethane. At the Moffett Field groundwater test site, in situ removal of c-DCE and TCE coincided with biostimulation through phenol and oxygen injection and utilization, with c-DCE removed more rapidly than TCE. Greater TCE and c-DCE removal was observed when the phenol concentration was increased. Over 90% removal of c-DCE and TCE was observed in the 2-m biostimulated zone. This compares with 40 to 50% removal of c-DCE and 15 to 25% removal of TCE achieved by methane-grown microorganisms previously evaluated in an adjacent in situ test zone. The in situ removal with phenol-grown microorganisms agrees qualitatively with the microcosm studies, with the rates and extents of removal ranked as follows: c-DCE > TCE > t-DCE. These studies demonstrate the potential for in situ TCE bioremediation using microorganisms grown on phenol.

Influence of the Endogenous Storage Lipid Poly-beta-Hydroxybutyrate on the Reducing Power Availability during Cometabolism of Trichloroethylene and Naphthalene by Resting Methanotrophic Mixed Cultures.

The role of the storage lipid poly-beta-hydroxybutyrate (PHB) in trichloroethylene transformation by methanotrophic mixed cultures was investigated. Naphthalene oxidation rates were used to assay for soluble methane monooxygenase activity. The PHB content of methanotrophic cells grown in reactors varied diurnally as well as from day to day. A positive correlation between the amount of PHB in the cells and the naphthalene oxidation rate as well as between PHB and the trichloroethylene transformation rate and capacity was found. Addition of beta-hydroxybutyrate increased the naphthalene oxidation rates significantly. PHB content in cells could be manipulated by incubation at different methane-to-nitrogen ratios. A positive correlation between the naphthalene oxidation rate and the PHB content after these incubations could be seen. Both the PHB content and the naphthalene oxidation rates decreased with time in resting methanotrophic cells exposed to oxygen. However, this decrease in the naphthalene oxidation rate cannot be explained by the decrease in the PHB content alone. Probably a deactivation of the methane monooxygenase itself is also involved.

Inhibition of Butyrate Oxidation by Formate during Methanogenesis.

A mixed methanogenic culture fed with glucose was perturbed with butyrate and formate to investigate the role of formate in the acetogenesis of butyrate. A free energy analysis suggests that formate rather than H(2) was the interspecies electron carrier for butyrate conversion into acetate for the culture studied.

Characterization of a methane-utilizing bacterium from a bacterial consortium that rapidly degrades trichloroethylene and chloroform.

A mixed culture of bacteria grown in a bioreactor with methane as a carbon and energy source rapidly oxidized trichloroethylene and chloroform. The most abundant organism was a crescent-shaped bacterium that bound the fluorescent oligonucleotide signature probes that specifically hybridize to serine pathway methylotrophs. The 5S rRNA from this bacterium was found to be 93.5% homologous to the Methylosinus trichosporium OB3b 5S RNA sequence. A type II methanotrophic bacterium, isolated in pure culture from the bioreactor, synthesized soluble methane monooxygenase during growth in a copper-limited medium and was also capable of rapid trichloroethylene oxidation. The bacterium contained the gene that encodes the soluble methane monooxygenase B component on an AseI restriction fragment identical in size to a restriction fragment present in AseI digests of DNA from bacteria in the mixed culture. The sequence of the 16S rRNA from the pure culture was found to be 92 and 94% homologous to the 16S rRNAs of M. trichosporium OB3b and M. sporium, respectively. Both the pure and mixed cultures oxidized naphthalene to naphthol, indicating the presence of soluble methane monooxygenase. The mixed culture also synthesized soluble methane monooxygenase, as evidenced by the presence of proteins that cross-reacted with antibodies prepared against purified soluble methane monooxygenase components from M. trichosporium OB3b on Western blots (immunoblots). It was concluded that a type II methanotrophic bacterium phylogenetically related to Methylosinus species synthesizes soluble methane monooxygenase and is responsible for trichloroethylene oxidation in the bioreactor.