Microbial community structure in three deep-sea carbonate crusts.

Carbonate crusts in marine environments can act as sinks for carbon dioxide. Therefore, understanding carbonate crust formation could be important for understanding global warming. In the present study, the microbial communities of three carbonate crust samples from deep-sea mud volcanoes in the eastern Mediterranean were characterized by sequencing 16S ribosomal RNA (rRNA) genes amplified from DNA directly retrieved from the samples. In combination with the mineralogical composition of the crusts and lipid analyses, sequence data were used to assess the possible role of prokaryotes in crust formation. Collectively, the obtained data showed the presence of highly diverse communities, which were distinct in each of the carbonate crusts studied. Bacterial 16S rRNA gene sequences were found in all crusts and the majority was classified as alpha-, gamma-, and delta- Proteobacteria. Interestingly, sequences of Proteobacteria related to Halomonas and Halovibrio sp., which can play an active role in carbonate mineral formation, were present in all crusts. Archaeal 16S rRNA gene sequences were retrieved from two of the crusts studied. Several of those were closely related to archaeal sequences of organisms that have previously been linked to the anaerobic oxidation of methane (AOM). However, the majority of archaeal sequences were not related to sequences of organisms known to be involved in AOM. In combination with the strongly negative delta 13C values of archaeal lipids, these results open the possibility that organisms with a role in AOM may be more diverse within the Archaea than previously suggested. Different communities found in the crusts could carry out similar processes that might play a role in carbonate crust formation.

Analysis of diversity among 3-chlorobenzoate-degrading strains of Rhodopseudomonas palustris.

The phenotypic and genetic characteristics of 14 strains of the purple nonsulfur bacterium Rhodopseudomonas palustris were studied to assess diversity within this species. While all strains had certain phenotypic characteristics in common, including the ability to metabolize benzoate and degrade 2- and 3-chlorobenzoate, there were also significant differences among the strains such as the rate of growth in media containing benzoate as a carbon source. Genetic characterization of the strains revealed there were three divergent lineages in the species. Based on 16S rRNA gene sequences, the 14 strains could be grouped into three distinct clusters (A, B, and C), and this clustering was congruent with that based on gene sequences of form II ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). Although BOX-PCR genomic DNA fingerprints of all 14 strains exhibited differences, analysis of the fingerprint images and UPGMA/product-moment analysis of similarities showed there were three groupings that were entirely consistent with clusters based on other characteristics of the strains. Thus, regardless of the method of analysis used, strains in groups A and B consistently clustered together and were separate from those of group C. These results suggest that strains in groups A-B and C represent phylogenetically related clones that have diverged from one another. This indicates that at least three lineages of Rhodopseudomonas palustris exist among the strains included in this study, and that each may be particularly well adapted to a distinct ecological niche.

Lactobacillus diolivorans sp. nov., a 1,2-propanediol-degrading bacterium isolated from aerobically stable maize silage.

Inoculation of maize silage with Lactobacillus buchneri (5 x 10(5) c.f.u. g(-1) of maize silage) prior to ensiling results in the formation of aerobically stable silage. After 9 months, lactic acid bacterium counts are approximately 10(10) c.f.u. g(-1) in these treated silages. An important subpopulation (5.9 x 10(7) c.f.u. g(-1)) is able to degrade 1,2-propanediol, a fermentation product of L. buchneri, under anoxic conditions to 1-propanol and propionic acid. From this group of 1,2-propanediol-fermenting, facultatively anaerobic, heterofermentative lactobacilli, two rod-shaped isolates were purified and characterized. Comparative 16S rDNA sequence analysis revealed that the newly isolated bacteria have identical 16S rDNA sequences and belong phylogenetically to the L. buchneri group. DNA-DNA hybridizations, whole-cell protein fingerprinting and examination of phenotypic properties indicated that these two isolates represent a novel species, for which the name Lactobacillus diolivorans sp. nov. is proposed. The type strain is LMG 19667T (= DSM 14421T).

Coexistence of a sulphate-reducing Desulfovibrio species and the dehalorespiring Desulfitobacterium frappieri TCE1 in defined chemostat cultures grown with various combinations of sulfate and tetrachloroethene.

A two-member co-culture consisting of the dehalorespiring Desulfitobacterium frappieri TCE1 and the sulphate-reducing Desulfovibrio sp. strain SULF1 was obtained via anaerobic enrichment from soil contaminated with tetrachloroethene (PCE). In this co-culture, PCE dechlorination to cis-dichloroethene was due to the activity of the dehalorespiring bacterium only. Chemostat experiments with lactate as the primary electron donor for both strains along with varying sulphate and PCE concentrations showed that the sulphate-reducing strain outnumbered the dehalogenating strain at relatively high ratios of sulphate/PCE. Stable co-cultures with both organisms present at similar cell densities were observed when both electron acceptors were supplied in the reservoir medium in nearly equimolar amounts. In the presence of low sulphate/PCE ratios, the Desulfitobacterium sp. became the numerically dominant strain within the chemostat co-culture. Surprisingly, in the absence of sulphate, strain SULF1 did not disappear completely from the co-culture despite the fact that there was no electron acceptor provided with the medium to be used by this sulphate reducer. Therefore, we propose a syntrophic association between the sulphate-reducing and the dehalorespiring bacteria via interspecies hydrogen transfer. The sulphate reducer was able to sustain growth in the chemostat co-culture by fermenting lactate and using the dehalogenating bacterium as a 'biological electron acceptor'. This is the first report describing growth of a sulphate-reducing bacterium in a defined two-member continuous culture by syntrophically coupling the electron and hydrogen transfer to a dehalorespiring bacterium.

Specific growth rate plays a critical role in hydrogen peroxide resistance of the marine oligotrophic ultramicrobacterium sphingomonas alaskensis strain RB2256.

The marine oligotrophic ultramicrobacterium Sphingomonas alaskensis RB2256 has a physiology that is distinctly different from that of typical copiotrophic marine bacteria, such as Vibrio angustum S14. This includes a high level of inherent stress resistance and the absence of starvation-induced stress resistance to hydrogen peroxide. In addition to periods of starvation in the ocean, slow, nutrient-limited growth is likely to be encountered by oligotrophic bacteria for substantial periods of time. In this study we examined the effects of growth rate on the resistance of S. alaskensis RB2256 to hydrogen peroxide under carbon or nitrogen limitation conditions in nutrient-limited chemostats. Glucose-limited cultures of S. alaskensis RB2256 at a specific growth rate of 0.02 to 0.13 h(-1) exhibited 10,000-fold-greater viability following 60 min of exposure to 25 mM hydrogen peroxide than cells growing at a rate of 0.14 h(-1) or higher. Growth rate control of stress resistance was found to be specific to carbon and energy limitation in this organism. In contrast, V. angustum S14 did not exhibit growth rate-dependent stress resistance. The dramatic switch in stress resistance that was observed under carbon and energy limitation conditions has not been described previously in bacteria and thus may be a characteristic of the oligotrophic ultramicrobacterium. Catalase activity varied marginally and did not correlate with the growth rate, indicating that hydrogen peroxide breakdown was not the primary mechanism of resistance. More than 1,000 spots were resolved on silver-stained protein gels for cultures growing at rates of 0.026, 0.076, and 0.18 h(-1). Twelve protein spots had intensities that varied by more than twofold between growth rates and hence are likely to be important for growth rate-dependent stress resistance. These studies demonstrated the crucial role that nutrient limitation plays in the physiology of S. alaskensis RB2256, especially under oxidative stress conditions.

Sphingomonas alaskensis sp. nov., a dominant bacterium from a marine oligotrophic environment.

Seven Gram-negative strains, isolated in 1990 from a 10(6)-fold dilution series of seawater from Resurrection Bay, a deep fjord of the Gulf of Alaska, were identified in a polyphasic taxonomic study. Analysis of 16S rDNA sequences and DNA-homology studies confirmed the phylogenetic position of all strains in the genus Sphingomonas and further indicated that all of the strains constitute a single homogeneous genomic species, distinct from all validly described Sphingomonas species. The ability to differentiate the species, both phenotypically and chemotaxonomically, from its nearest neighbours justifies the proposal of a new species name, Sphingomonas alaskensis sp. nov., for this taxon. Strain LMG 18877T (= RB2256T = DSM 13593T) was selected as the type strain.

Anaerobic conversion of lactic acid to acetic acid and 1, 2-propanediol by Lactobacillus buchneri.

The degradation of lactic acid under anoxic conditions was studied in several strains of Lactobacillus buchneri and in close relatives such as Lactobacillus parabuchneri, Lactobacillus kefir, and Lactobacillus hilgardii. Of these lactobacilli, L. buchneri and L. parabuchneri were able to degrade lactic acid under anoxic conditions, without requiring an external electron acceptor. Each mole of lactic acid was converted into approximately 0.5 mol of acetic acid, 0.5 mol of 1,2-propanediol, and traces of ethanol. Based on stoichiometry studies and the high levels of NAD-linked 1, 2-propanediol-dependent oxidoreductase (530 to 790 nmol min(-1) mg of protein(-1)), a novel pathway for anaerobic lactic acid degradation is proposed. The anaerobic degradation of lactic acid by L. buchneri does not support cell growth and is pH dependent. Acidic conditions are needed to induce the lactic-acid-degrading capacity of the cells and to maintain the lactic-acid-degrading activity. At a pH above 5.8 hardly any lactic acid degradation was observed. The exact function of anaerobic lactic acid degradation by L. buchneri is not certain, but some results indicate that it plays a role in maintaining cell viability.

The presence of Acetobacter sp. in ensiled forage crops and ensiled industrial byproducts.

The presence of acetic acid bacteria (AAB) in whole crop maize silage, whole crop wheat silage, pressed sugar beet pulp silage, grass silage and brewer's grains silage was investigated. AAB could be isolated from whole crop maize silage, whole crop wheat silage and pressed sugar beet pulp silage, but could not be detected in grass silage (> 100 silo's tested) or brewer's grains silage (5 silo's tested). Thirty AAB isolates were characterized to genus level. All isolates, i.e. 20 from whole crop maize silage, 5 from whole crop wheat silage and 5 from pressed sugar beet pulp silage, belonged to the genus Acetobacter. Two isolates from maize silage were further characterized. Partial 16S rRNA analyses revealed that one isolate was closely related to Acetobacter aceti (98% sequence homology), the other to Acetobacter pomorum (98% sequence homology). These results combined with the substrate utilization profiles indicate that these isolates probably represent thus far undescribed species of Acetobacter.

Biomarker evidence for widespread anaerobic methane oxidation in Mediterranean sediments by a consortium of methanogenic archaea and bacteria. The Medinaut Shipboard Scientific Party.

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible Nautile. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in (13)C (delta(13)C values are as low as -95 per thousand). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of (13)C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.

Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium frappieri TCE1.

Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 microm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35 degrees C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of Desulfitobacterium frappieri, because it exhibited 99.7% relatedness to the D. frappieri type strain, strain PCP-1. Growth with H(2), formate, L-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H(2)) are oxidized to acetate and CO(2). When L-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce cis-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g., Desulfitobacterium sp. strain PCE1 and Desulfitobacterium hafniense) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 micromol of chloride released. min(-1). mg of protein(-1)). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.

Degradation of 3-chlorobenzoate under low-oxygen conditions in pure and mixed cultures of the anoxygenic photoheterotroph Rhodopseudomonas palustris DCP3 and an aerobic Alcaligenes species.

The presence or absence of molecular oxygen has been shown to play a crucial role in the degradability of haloaromatic compounds. In the present study, it was shown that anaerobic phototrophic 3-chlorobenzoate (3CBA) metabolism by Rhodopseudomonas palustris DCP3 is oxygen tolerant up to a concentration of 3 microM O2. Simultaneous oxidation of an additional carbon source permitted light-dependent anaerobic 3CBA degradation at oxygen input levels which, in the absence of such an additional compound, would result in inhibition of light-dependent dehalogenation. Experiments under the same experimental conditions with strain DCP3 in coculture with an aerobic 3CBA-utilizing heterotroph, Alcaligenes sp. strain L6, revealed that light-dependent dehalogenation of 3CBA did not occur. Under both oxygen limitation (O2 < 0.1 microM) and low oxygen concentrations (3 microM O2), all the 3CBA was metabolized by the aerobic heterotroph. These data suggest that biodegradation of (halo)aromatics by photoheterotrophic bacteria such as R. palustris DCP3 may be restricted to anoxic photic environments.

Oxygen Consumption by Desulfovibrio Strains with and without Polyglucose.

The kinetics of oxygen reduction by Desulfovibrio salexigens Mast1 and the role of polyglucose in this activity were examined and compared with those of strains of D. desulfuricans and D. gigas. Oxidation rates were highest at air saturation (up to 40 nmol of O(2) min mg of protein) and declined with decreasing oxygen concentrations. Studies with cell extracts (CE) indicated that NADH oxidase was entirely responsible for the oxygen reduction in strain Mast1. In D. desulfuricans CSN, at least three independent systems appeared to reduce oxygen. Two were active at all oxygen concentrations (NADH oxidase and NADPH oxidase), and one was maximally active at less than 10 muM oxygen. In contrast to D. gigas and D. salexigens strains, the D. desulfuricans strains also contained NADH peroxidase and NADPH peroxidase activities and did not accumulate polyglucose under nonlimiting growth conditions. At air saturation, initial activities of the oxidases and peroxidases of cells harvested at the end of the log phase were on the order of 20 to 140 nmol of O(2) min mg of protein. In all strains, these enzymes were relatively stable but were susceptible to inactivation as soon as substrates were added to the assay mixture. Under those conditions, all oxidation activity disappeared after ca. 1 h of incubation. The same finding was observed with whole cells of D. desulfuricans CSN and D. desulfuricans ATCC 27774, but inactivation was less pronounced with cells of D. salexigens Mast1. It appeared that the presence of polyglucose in the whole cells retarded the process of inactivation of NADH oxidase, but this property was lost in crude CE. In spite of the effect of polyglucose on the oxidative potential, oxygen-dependent growth of D. salexigens Mast1 could be demonstrated neither in batch nor in continuous culture.

Stable-Isotope Analysis of a Combined Nitrification-Denitrification Sustained by Thermophilic Methanotrophs under Low-Oxygen Conditions.

To simulate growth conditions experienced by microbiota at O(inf2)-limited interfaces of organic matter in compost, an experimental system capable of maintaining dual limitations of oxygen and carbon for extended periods, i.e., a pO(inf2)-auxostat, has been used. (sup15)N tracer studies on thermophilic (53(deg)C) decomposition processes occurring in manure-straw aggregates showed the emission of dinitrogen gas from the reactor as a result of simultaneous nitrification and denitrification at low pO(inf2) values (0.1 to 2.0%, vol/vol). The N loss was confirmed by nitrogen budget studies of the system. Depending on the imposed pO(inf2), 0.6 to 1.4 mmol of N/day (i.e., 20 to 40% of input N) was emitted as N(inf2). When the pO(inf2) was raised, the rates of both nitrification and denitrification increased instantaneously, indicating that ammonia oxidation was limited by oxygen. In auxostats permanently running at pO(inf2) >= 2% (vol/vol), the free ammonium pool was almost completely oxidized and was converted to nitrite plus nitrate and N(inf2) gas. Labelling of the auxostat with [(sup13)C]carbonate was conducted to reveal whether nitrification was of autotrophic or heterotrophic origin. Incorporation of (sup13)CO(inf2) into population-specific cellular compounds was evaluated by profiling the saponifiable phospholipid fatty acids (FAs) by using capillary gas chromatography and subsequently analyzing the (sup13)C/(sup12)C ratios of the individual FAs, after their combustion to CO(inf2), by isotope ratio mass spectrometry. Apart from the observed label incorporation into FAs originating from a microflora belonging to the genus Methylococcus (type X group), supporting nitrification of a methylotrophic nature, this analysis also corroborated the absence of truly autotrophic nitrifying populations. Nevertheless, the extent to which ammonia oxidation continued to exist in this thermophilic community suggested that a major energy gain could be associated with it.

Complete degradation of tetrachloroethene in coupled anoxic and oxic chemostats.

Anaerobic tetrachloroethene (C2Cl4)-dechlorinating bacteria were enriched in slurries from chloroethene-contaminated soil. With methanol as electron donor, C2Cl4 and trichloroethene (C2HCl3) were reductively dechlorinated to cis-1,2-dichloroethene (cis-C2H2Cl2), whereas, with L-lactate or formate, complete dechlorination of C2Cl4 via C2HCl3, cis-C2H2Cl2 and chloroethene (C2H3Cl) to ethene was obtained. In oxic soil slurries with methane as a substrate, complete co-metabolic degradation of cis-C2H2Cl2 was obtained, whereas C2HCl3 was partially degraded. With toluene or phenol both of the above were readily co-metabolized. Complete degradation of C2Cl4 was obtained in sequentially coupled anoxic and oxic chemostats, which were inoculated with the slurry enrichments. Apparent steady states were obtained at various dilution rates (0.02-0.4 h-1) and influent C2Cl4-concentrations (100-1000 microM). In anoxic chemostats with a mixture of formate and glucose as the carbon and electron source, C2Cl4 was transformed at high rates (above 140 micromol 1-1 h-1, corresponding to 145 nmol Cl- min-1 mg protein-1), into cis-C2H2Cl2 and C2H3Cl. Reductive dechlorination was not affected by addition of 5 mM sulphate, but strongly inhibited after addition of 5 mM nitrate. Our results (high specific dechlorination rates and loss of dechlorination capacity in the absence of C2Cl4) suggest that C2Cl4-dechlorination in the anoxic chemostat was catalysed by specialized dechlorinating bacteria. The partially dechlorinated intermediates, cis-C2H2Cl2 and C2H3Cl, were further degraded by aerobic phenol-metaboizing bacteria. The maximum capacity for chloroethene (the sum of tri-, di- and monochloro derivatives removed) degradation in the oxic chemostat was 95 micromol 1-1 h-1 (20 nmol min-1 mg protein-1), and that of the combined anoxic --> oxic reactor system was 43.4 micromol 1-1 h-1. This is significantly higher than reported thus far.

scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis.

A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counter-parts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequence showed a high degree of identity to the beta-succinyl-CoA synthetase of Escherichia coli (46%), the mitochondrial beta-succinyl-CoA synthetase from pig (48%) and the hydrogenosomal beta-succinyl-CoA synthetase from Trichomonas vaginalis (49%). The G + C content of the succinyl-CoA synthetase coding sequence (43.8%) was considerably higher than that of the 5' (14.8%) and 3' (13.3%) non-translated flanking sequences, as has been observed for other genes from N. frontalis. The codon usage pattern was biased, with only 34 codons used and a strong preference for a pyrimidine (T) in the third positions of the codons. The coding sequence of the beta-succinyl-CoA synthetase cDNA was cloned in an E. coli expression vector encoding a 6(His) tag. The recombinant protein was purified by affinity binding and used to produce polyclonal antibodies. The anti-succinyl-CoA synthetase serum recognized a 45 kDa protein from a N. frontalis fraction enriched for hydrogenosomes and similar polypeptides in two related anaerobic fungi, Piromyces rhizinflata (45 kDa) and Caecomyces communis (47 kDa). Immunocytochemical experiments suggest that succinyl-CoA synthetase is located in the hydrogenosomal matrix. Staining for SCS activity in native electrophoretic gels revealed a band with an apparent molecular weight of approximately 330 kDa. The C-terminus of the succinyl-CoA synthetase sequence was devoid of the typical targeting signals identified so far in microbody proteins, indicating that N. frontalis uses a different signal for sorting SCSB into hydrogenosomes. Based on comparisons with other proteins we propose a putative N-terminal targeting signal for succinyl-CoA synthetase of N. frontalis that shows some of the features of mitochondrial targeting sequences.

Isolation of Alcaligenes sp. strain L6 at low oxygen concentrations and degradation of 3-chlorobenzoate via a pathway not involving (chloro)catechols.

Isolations of 3-chlorobenzoate (3CBA)-degrading aerobic bacteria under reduced O2 partial pressures yielded organisms which metabolized 3CBA via the gentisate or the protocatechuate pathway rather than via the catechol route. The 3CBA metabolism of one of these isolates, L6, which was identified as an Alcaligenes species, was studied in more detail. Resting-cell suspensions of L6 pregrown on 3CBA oxidized all known aromatic intermediates of both the gentisate and the protocatechuate pathways. Neither growth on nor respiration of catechol could be detected. Chloride production from 3CBA by L6 was strictly oxygen dependent. Cell-free extracts of 3CBA-grown L6 cells exhibited no catechol dioxygenase activity but possessed protocatechuate 3,4-dioxygenase, gentisate dioxygenase, and maleylpyruvate isomerase activities instead. In continuous culture with 3CBA as the sole growth substrate, strain L6 demonstrated an increased oxygen affinity with decreasing steady-state oxygen concentrations.

Responses to Stress and Nutrient Availability by the Marine Ultramicrobacterium Sphingomonas sp. Strain RB2256.

Sphingomonas sp. strain RB2256 was isolated from Resurrection Bay in Alaska and possibly represents the dominant bacterial species in some oligotrophic marine environments. Strain RB2256 has a high-affinity nutrient uptake system when growing under nutrient-limiting conditions, and growing cells are very small (<0.08 (mu)m(sup3)). These characteristics indicate that RB2256 is highly evolved for withstanding nutrient limitations and grazing pressure by heterotrophic nanoflagellates. In this study, strain RB2256 was subjected to nutrient starvation and other stresses (high temperature, ethanol, and hydrogen peroxide). It was found that growing cells were remarkably resistant, being able to survive at a temperature of 56(deg)C, in 25 mM hydrogen peroxide, or in 20% ethanol. In addition, growing cells were generally as resistant as starved cells. The fact that vegetative cells of this strain are inherently resistant to such high levels of stress-inducing agents indicates that they possess stress resistance mechanisms which are different from those of other nondifferentiating bacteria. Only minor changes in cell volume (0.03 to 0.07 (mu)m(sup3)) and maximum specific growth rate (0.13 to 0.16 h(sup-1)) were obtained for cells growing in media with different organic carbon concentrations (0.8 to 800 mg of C per liter). Furthermore, when glucose-limited, chemostat-grown cultures or multiple-nutrient-starved batch cultures were suddenly subjected to excess glucose, maximum growth rates were reached immediately. This immediate response to nutrient upshift suggests that the protein-synthesizing machinery is constitutively regulated. In total, these results are strong evidence that strain RB2256 possesses novel physiological and molecular strategies that allow it to predominant in natural seawater.

Desulfitobacterium sp. strain PCE1, an anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene or ortho-chlorinated phenols.

A strictly anaerobic bacterium, strain PCE1, was isolated from a tetrachloroethene-dechlorinating enrichment culture. Cells of the bacterium were motile curved rods, with approximately four lateral flagella. They possessed a gram-positive type of cell wall and contained cytochrome c. Optimum growth occurred at pH 7.2-7.8 and 34-38 degrees C. The organism grew with L-lactate, pyruvate, butyrate, formate, succinate, or ethanol as electron donors, using either tetrachloroethene, 2-chlorophenol, 2,4,6-trichlorophenol, 3-chloro-4-hydroxy-phenylacetate, sulfite, thiosulfate, or fumarate as electron acceptors. Strain PCE1 also grew fermentatively with pyruvate as the sole substrate. L-Lactate and pyruvate were oxidized to acetate. Tetrachloroethene was reductively dechlorinated to trichloroethene and small amounts ( 5%) of cis-1,2-dichloroethene and trans-1,2-dichloroethene. Chlorinated phenolic compounds were dechlorinated specifically at the ortho-position. On the basis of 16S rRNA sequence analysis, the organism was identified as a species within the genus Desulfitobacterium, which until now only contained the chlorophenol-dechlorinating bacterium, Desulfitobacterium dehalogenans.

Complete degradation of tetrachloroethene by combining anaerobic dechlorinating and aerobic methanotrophic enrichment cultures.

Degradation of tetrachloroethene (perchloroethylene, PCE) was investigated by combining the metabolic abilities of anaerobic bacteria, capable of reductive dechlorination of PCE, with those of aerobic methanotrophic bacteria, capable of co-metabolic degradation of the less-chlorinated ethenes formed by reductive dechlorination of PCE. Anaerobic communities reductively dechlorinating PCE, trichloroethene (TCE) and dichloroethenes were enriched from various sources. The maximum rates of dechlorination observed for various chloroethenes in these batch enrichments were: PCE to TCE (341 mumol l-1 day-1), TCE to cis-dichloroethene (159 mumol l-1 day-1), cis-dichloroethene to chloroethene (99 mumol l-1 day-1) and trans-dichloroethene to chloroethene (22 mumol l-1 day-1). A mixture of these enrichments was inoculated into an anoxic fixed-bed upflow column. In this column PCE was converted mainly into cis-1,2-dichloroethene, small amounts of TCE and chloroethene, and chloride. Enrichments of aerobic methanotrophic bacteria were grown in an oxic fixed-bed downflow column. Less-chlorinated ethenes, formed in the anoxic column, were further metabolized in this oxic methanotrophic column. On the basis of analysis of chloride production and the disappearance of chlorinated ethenes it was demonstrated that complete degradation of PCE was possible by combining these two columns. Operation of the two-column system under various process conditions indicated that the sensitivity of the methanotrophic bacteria to chlorinated intermediates represented the bottle-neck in the sequential anoxic/oxic degradation process of PCE.

Substrate uptake and utilization by a marine ultramicrobacterium.

A facultatively oligotrophic ultramicrobacterium (strain RB2256) isolated from an Alaskan fjord by extinction dilution in seawater, was grown in batch culture and under single- and dual-substrate-limitation of alanine and glucose in a chemostat. The nature of the uptake systems, and the uptake kinetics and utilization patterns of alanine and glucose were investigated. Glucose uptake was inducible, the system exhibited a narrow substrate specificity, and part of the uptake system was osmotic-shock-sensitive. Half-saturation constants for glucose were between 7 and 74 microM during glucose limitation. The initial step in glucose metabolism was the synthesis of sugar polymers, even during glucose-limited growth. The alanine uptake system was constitutively expressed and was binding-protein-dependent. In addition to L-alanine, nine other amino acids inhibited accumulation of [14C]L-alanine, indicating broad substrate specificity of the alanine transporter. Half-saturation constants between 1.3 and 1.8 microM were determined for alanine uptake during alanine limitation. Simultaneous utilization of glucose and alanine occurred during substrate-limited growth in the chemostat, and during growth in batch culture at relatively high (mM) substrate concentrations. However, the half-saturation constant for alanine transport during dual-substrate-limitation, i.e. in the presence of glucose, increased almost fivefold. We conclude that mixed substrate utilization is an inherent property of this organism.