Chemostat Enrichment Research Articles

Nutrient-Limited Enrichments of Nitrifiers From Soil Yield Consortia of Nitrosocosmicus-Affiliated AOA and Nitrospira-Affiliated NOB.

The discovery of ammonia-oxidizing archaea (AOA) and complete ammonia-oxidizing (comammox) bacteria widespread in terrestrial ecosystems indicates an important role of these organisms in terrestrial nitrification. Recent evidence indicated a higher ammonia affinity of comammox bacteria than of terrestrial AOA and ammonia-oxidizing bacteria (AOB), suggesting that comammox bacteria could potentially represent the most low-nutrient adapted nitrifiers in terrestrial systems. We hypothesized that a nutrient-limited enrichment strategy could exploit the differences in cellular kinetic properties and yield enrichments dominated by high affinity and high yield comammox bacteria. Using soil with a mixed community of AOA, AOB, and comammox Nitrospira, we compared performance of nutrient-limited chemostat enrichment with or without batch culture pre-enrichment in two different growth media without inhibitors or antibiotics. Monitoring of microbial community composition via 16S rRNA and amoA gene sequencing showed that batch enrichments were dominated by AOB, accompanied by low numbers of AOA and comammox Nitrospira. In contrast, nutrient-limited enrichment directly from soil, and nutrient-limited sub-cultivation of batch enrichments consistently yielded high enrichments of Nitrosocosmicus-affiliated AOA associated with multiple canonical nitrite-oxidizing Nitrospira strains, whereas AOB numbers dropped below 0.1% and comammox Nitrospira were lost completely. Our results reveal competitiveness of Nitrosocosmicus sp. under nutrient limitation, and a likely more complex or demanding ecological niche of soil comammox Nitrospira than simulated in our nutrient-limited chemostat experiments.

Growth yield and selection of nosZ clade II types in a continuous enrichment culture of N2O respiring bacteria

Nitrous oxide (N2 O) reducing microorganisms may be key in the mitigation of N2 O emissions from managed ecosystems. However, there is still no clear understanding of the physiological and bioenergetic implications of microorganisms possessing either of the two N2 O reductase genes (nosZ), clade I and the more recently described clade II type nosZ. It has been suggested that organisms with nosZ clade II have higher growth yields and a lower affinity constant (Ks ) for N2 O. We compared N2 O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N2 O as a sole electron acceptor and growth limiting factor and acetate as electron donor. From the sequencing of the 16S rRNA gene, FISH and quantitative PCR of nosZ and nir genes, we concluded that betaproteobacterial denitrifying organisms dominated the enrichments with members within the family Rhodocyclaceae being highly abundant. When comparing cultures with different nosZI/nosZII ratios, we did not find support for (i) a more energy conserving N2 O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N2 O, defined by μmax /Ks , in organisms with nosZ clade II.

Characterization of the newly isolated ω-oxidizing yeast Candida sorbophila DS02 and its potential applications in long-chain dicarboxylic acid production.

α, ω-Dicarboxylic acids (DCAs) are multipurpose chemicals widely used in polymers, perfumes, plasticizers, lubricants, and adhesives. The biotransformation of DCAs from alkanes and fatty acids by microorganisms has attracted recent interest, since synthesis via chemical oxidation causes problems in terms of the environment and safety. We isolated an ω-oxidizing yeast from a wastewater disposal facility of a petrochemical factory by chemostat enrichment culture. The haploid strain identified as Candida sorbophila DS02 grew on glucose and dodecane, exhibiting greater cell shrinkage on the latter. In flask cultures with mixed alkanes (C10-16) and fatty acid methyl esters (C10-16), DS02 used mixed alkanes simultaneously unlike Candida tropicalis and Yarrowia lipolytica and showed high substrate resistance. In flask cultures with acrylic acid-a known inhibitor of β-oxidation-DS02 produced 0.28g/l dodecanedioic acid (DDDA) from dodecane, similar to wild-type C. tropicalis ATCC 20336. In fed-batch fermentation, DS02 produced 9.87g/l DDDA, which was 5.7-fold higher than wild-type C. tropicalis. These results suggest that C. sorbophila strain DS02 has potential applications for the large-scale production of DCA.

Role of nitrite in the competition between denitrification and DNRA in a chemostat enrichment culture

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) are two microbial processes that compete for oxidized nitrogen compounds in the environment. The objective of this work was to determine the role of nitrite versus nitrate as terminal electron acceptor on the competition between DNRA and denitrification. Initially, a mixed culture chemostat was operated under nitrate limitation and performed DNRA. Stepwise, the influent nitrate was replaced with nitrite until nitrite was the sole electron acceptor and N-source present. Despite changing the electron acceptor from nitrate to nitrite, the dominant process remained DNRA and the same dominant organism closely related to Geobacter lovleyi was identified. Contrary to previous studies conducted with a complex substrate in marine microbial communities, the conclusion of this work is that nitrate versus nitrite as electron acceptor does not generally control the competition between DNRA and denitrification. Our results show that the effect of this ratio must be interpreted in combination with other environmental factors, such as the type and complexity of the electron donor, pH, or sulfide concentrations.

DNRA and Denitrification Coexist over a Broad Range of Acetate/N-NO3- Ratios, in a Chemostat Enrichment Culture.

Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) compete for nitrate in natural and engineered environments. A known important factor in this microbial competition is the ratio of available electron donor and elector acceptor, here expressed as Ac/N ratio (acetate/nitrate-nitrogen). We studied the impact of the Ac/N ratio on the nitrate reduction pathways in chemostat enrichment cultures, grown on acetate mineral medium. Stepwise, conditions were changed from nitrate limitation to nitrate excess in the system by applying a variable Ac/N ratio in the feed. We observed a clear correlation between Ac/N ratio and DNRA activity and the DNRA population in our reactor. The DNRA bacteria dominated under nitrate limiting conditions in the reactor and were outcompeted by denitrifiers under limitation of acetate. Interestingly, in a broad range of Ac/N ratios a dual limitation of acetate and nitrate occurred with co-occurrence of DNRA bacteria and denitrifiers. To explain these observations, the system was described using a kinetic model. The model illustrates that the Ac/N effect and concomitant broad dual limitation range related to the difference in stoichiometry between both processes, as well as the differences in electron donor and acceptor affinities. Population analysis showed that the presumed DRNA-performing bacteria were the same under nitrate limitation and under dual limiting conditions, whereas the presumed denitrifying population changed under single and dual limitation conditions.

Unique Kinetic Properties of Phenol-Degrading Variovorax Strains Responsible for Efficient Trichloroethylene Degradation in a Chemostat Enrichment Culture

A chemostat enrichment of soil bacteria growing on phenol as the sole carbon source has been shown to exhibit quite high trichloroethylene (TCE)-degrading activities. To identify the bacterial populations responsible for the high TCE-degrading activity, a multidisciplinary survey of the chemostat enrichment was conducted by employing molecular-ecological and culture-dependent approaches. Three chemostat enrichment cultures were newly developed under different phenol-loading conditions (0.25, 0.75, and 1.25 g liter(-1) day(-1)) in this study, and the TCE-degrading activities of the enrichments were measured. Among them, the enrichment at 0.75 g liter(-1) day(-1) (enrichment 0.75) expressed the highest activity. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments detected a Variovorax ribotype as the strongest band in enrichment 0.75; however, it was not a major ribotype in the other samples. Bacteria were isolated from enrichment 0.75 by direct plating, and their 16S rRNA genes and genes encoding the largest subunit of phenol hydroxylase (LmPHs) were analyzed. Among the bacteria isolated, several strains were affiliated with the genus Variovorax and were shown to have high-affinity-type LmPHs. The LmPH of the Variovorax strains was also detected as the major genotype in enrichment 0.75. Kinetic analyses of phenol and TCE degradation revealed, however, that these strains exhibited quite low affinity for phenol compared to other phenol-degrading bacteria, while they showed quite high specific TCE-degrading activities and relatively high affinity for TCE. Owing to these unique kinetic traits, the Variovorax strains can obviate competitive inhibition of TCE degradation by the primary substrate of the catabolic enzyme (i.e., phenol), contributing to the high TCE-degrading activity of the chemostat enrichments. On the basis of physiological information, mechanisms accounting for the way the Variovorax population overgrew the chemostat enrichment are discussed.

Chemostat approach for the directed evolution of biodesulfurization gain-of-function mutants.

Chemostat enrichment is a classical microbiological method that is well suited for use in directed-evolution strategies. We used a two-phase sulfur-limited chemostat to select for gain-of-function mutants with mutations in the biodesulfurization (Dsz) system of Rhodococcus erythropolis IGTS8, enriching for growth in the presence of organosulfur compounds that could not support growth of the wild-type strain. Mutations arose that allowed growth with octyl sulfide and 5-methylbenzothiophene as sole sulfur sources. An isolate from the evolved chemostat population was genetically characterized and found to contain mutations in two genes, dszA and dszC. A transversion (G to T) in dszC codon 261 resulted in a V261F mutation that was determined to be responsible for the 5-methylbenzothiophene gain-of-function phenotype. By using a modified RACHITT (random chimeragenesis on transient templates) method, mutant DszC proteins containing all possible amino acids at that position were generated, and this mutant set was assayed for the ability to metabolize 5-methylbenzothiophene, alkyl thiophenes, and dibenzothiophene. No mutant with further improvements in these catalytic activities was identified, but several clones lost all activity, confirming the importance of codon 261 for enzyme activity.

Chemostat enrichments of human feces with resistant starch are selective for adherent butyrate-producing clostridia at high dilution rates.

Resistant starch (RS) enrichments were made using chemostats inoculated with human feces from two individuals at two dilution rates (D = 0.03 h(-1) and D = 0.30 h(-1)) to select for slow- and fast-growing amylolytic communities. The fermentations were studied by analysis of short-chain fatty acids, amylase and alpha-glucosidase activities, and viable counts of the predominant culturable populations and the use of 16S rRNA-targeted oligonucleotide probes. Considerable butyrate was produced at D = 0. 30 h(-1), which corresponded with reduced branched-chain fatty acid formation. At both dilution rates, high levels of extracellular amylase activity were produced, while alpha-glucosidase was predominantly cell associated. Bacteroides and bifidobacteria predominated at the low dilution rate, whereas saccharolytic clostridia became more important at D = 0.30 h(-1). Microscopic examination showed that within 48 h of inoculation, one particular bacterial morphotype predominated in RS enrichments at D = 0.30 h(-1). This organism attached apically to RS granules and formed rosette-like structures which, with glycocalyx formation, agglomerated to form biofilm networks in the planktonic phase. Attempts to isolate this bacterium in pure culture were repeatedly unsuccessful, although a single colony was eventually obtained. On the basis of its 16S rDNA sequence, this RS-degrading, butyrate-producing organism was identified as being a previously unidentified group I Clostridium sp. A 16S rRNA-targeted probe was designed using this sequence and used to assess the abundance of the population in the enrichments. At 240 h, its contributions to total rRNA in the chemostats were 5 and 23% at D = 0.03 and 0.30 h(-1), respectively. This study indicates that bacterial populations with significant metabolic potential can be overlooked using culture-based methodologies. This may provide a paradigm for explaining the discrepancy between the low numbers of butyrate-producing bacteria that are isolated from fecal samples and the actual production of butyrate.

Characterization of 3-chlorobenzoate degrading aerobic bacteria isolated under various environmental conditions

The rates of bacterial growth in nature are often restricted by low concentrations of oxygen or carbon substrates. In the present study the metabolic properties of 24 isolates that had been isolated using various concentrations of 3-chlorobenzoate, benzoate and oxygen as well as using continuous culture at high and low growth rates were determined to investigate the effects of these parameters on the metabolism of monoaromatic compounds. Bacteria were enriched from different sampling sites and subsequently isolated. In batch culture this was done both under low oxygen (2% O 2) and air-saturated concentrations. Chemostat enrichments were performed under either oxygen or 3-chlorobenzoate limiting conditions. Bacteria metabolizing aromatics with gentisate or protocatechuate as intermediates (gp bacteria) as well as bacteria metabolizing aromatic compounds via catechols (cat bacteria) were isolated from batch cultures when either benzoate or 3CBA were used as C sources, regardless of the enrichment conditions applied. In contrast, enrichments performed in chemostats at low dilution rates resulted in gp-type organisms only, whereas at high dilution rates cat-type organisms were enriched, irrespective of the oxygen and 3-chlorobenzoate concentration used during enrichment. It is noteworthy that the gp-type of bacteria possessed relatively low μ max values on 3CBA and benzoate along with relatively high substrate and oxygen affinities for these compounds. This is in contrast with cat-type of bacteria, which seemed to be characterized by high maximum specific growth rates on the aromatic substrates and relatively high apparent half saturation constants. In contrast, bacteria degrading chlorobenzoate via gentisate or protocatechuate may possibly be better adapted to conditions leading to growth at reduced rates such as low oxygen and low substrate concentrations.

Characterization of 3-chlorobenzoate degrading aerobic bacteria isolated under various environmental conditions.

The rates of bacterial growth in nature are often restricted by low concentrations of oxygen or carbon substrates. In the present study the metabolic properties of 24 isolates that had been isolated using various concentrations of 3-chlorobenzoate, benzoate and oxygen as well as using continuous culture at high and low growth rates were determined to investigate the effects of these parameters on the metabolism of monoaromatic compounds. Bacteria were enriched from different sampling sites and subsequently isolated. In batch culture this was done both under low oxygen (2% O(2)) and air-saturated concentrations. Chemostat enrichments were performed under either oxygen or 3-chlorobenzoate limiting conditions. Bacteria metabolizing aromatics with gentisate or protocatechuate as intermediates (gp bacteria) as well as bacteria metabolizing aromatic compounds via catechols (cat bacteria) were isolated from batch cultures when either benzoate or 3CBA were used as C sources, regardless of the enrichment conditions applied. In contrast, enrichments performed in chemostats at low dilution rates resulted in gp-type organisms only, whereas at high dilution rates cat-type organisms were enriched, irrespective of the oxygen and 3-chlorobenzoate concentration used during enrichment. It is noteworthy that the gp-type of bacteria possessed relatively low µ(max) values on 3CBA and benzoate along with relatively high substrate and oxygen affinities for these compounds. This is in contrast with cat-type of bacteria, which seemed to be characterized by high maximum specific growth rates on the aromatic substrates and relatively high apparent half saturation constants. In contrast, bacteria degrading chlorobenzoate via gentisate or protocatechuate may possibly be better adapted to conditions leading to growth at reduced rates such as low oxygen and low substrate concentrations.

Chlorinated biphenyl mineralization by individual populations and consortia of freshwater bacteria

Comparative studies were performed to investigate the contribution of microbial consortia, individual microbial populations, and specific plasmids to chlorinated biphenyl biodegradation among microbial communities from a polychlorinated biphenyl-contaminated freshwater environment. A bacterial consortium, designated LPS10, was shown to mineralize 4-chlorobiphenyl (4CB) and dehalogenate 4,4'-dichlorobiphenyl. The LPS10 consortium involved three isolates: Pseudomonas testosteroni (LPS10A), which mediated the breakdown of 4CB and 4,4'-dichlorobiphenyl to 4-chlorobenzoic acid; an isolate tentatively identified as an Arthrobacter sp. (LPS10B), which mediated 4-chlorobenzoic acid degradation; and Pseudomonas putida bv. A (LPS10C), whose role in the consortium has not been determined. None of these isolates contained detectable plasmids or sequences homologous to the 4CB-degradative plasmid pSS50. A freshwater isolate, designated LBS1C1, was found to harbor a 41-megadalton plasmid that was related to the 35-megadalton plasmid pSS50, and this isolate was shown to mineralize 4CB. In chemostat enrichments with biphenyl and 4CB as primary carbon sources, the LPS10 consortium was found to outcomplete bacterial populations harboring plasmids homologous to pSS50. These results demonstrate that an understanding of the biodegradative capacity of individual bacterial populations as well as interacting populations of bacteria must be considered in order to gain a better understanding of polychlorinated biphenyl biodegradation in the environment.

Mixed chemostat cultures of obligately aerobic and fermentative or methanogenic bacteria grown under oxygen-limiting conditions

Defined mixed cultures of an obligately aerobic Pseudomonas testosteroni and anaerobic Veillonella alcalescens strain were grown under oxygen and lactate limitation in chemostats with different oxygen supply rates. The aerobic and the anaerobic bacteria were shown to coexist and to complete for common substrates over a wide range of oxygen supply rates. Under similar conditions but with formate as the major substrate chemostat enrichments gave rise to undefined mixed cultures of aerobic, fermentative and methanogenic bacteria. The relevance of these observations to natural mineralization processes is discussed.

Mixed chemostat cultures of obligately aerobic and fermentative or methanogenic bacteria grown under oxygen-limiting conditions

Defined mixed cultures of an obligately aerobic Pseudomonas testosteroni and anaerobic Veillonella alcalescens strain were grown under oxygen and lactate limitation in chemostats with different oxygen supply rates. The aerobic and the anaerobic bacteria were shown to coexist and to complete for common substrates over a wide range of oxygen supply rates. Under similar conditions but with formate as the major substrate chemostat enrichments gave rise to undefined mixed cultures of aerobic, fermentative and methanogenic bacteria. The relevance of these observations to natural mineralization processes is discussed.

Chemostat enrichment of sulphate-reducing bacteria from the large gut

Electron donor‐limited chemostat enrichments were used to isolate sulphate‐reducing bacteria (SRB) from human faeces. When acetate, lactate, propionate, butyrate or amino acids were used as electron donors, SRB identical to those found using traditional isolational methods (agar shake dilution series) were obtained. However, chemostat enrichments facilitated the isolation of SRB able to metabolize mixtures of alcohols (C1‐C5) and mixtures of fatty acids (C2‐C6) which had not been detected by the direct isolation techniques.

Thiobacillus strain Q, a chemolithoheterotrophic sulphur bacterium

The physiological properties of an organism isolated from a selective chemostat enrichment using acetate and thiosulphate as the limiting substrates, provisionally called Thiobacillus Q, were investigated. Although the organism made up 85% of the community in the enrichment culture, its expected chemolithotrophic nature was not apparent in batch experiments. The growth yield was not enhanced by the addition of thiosulphate to an acetate containing mineral medium, even though up to 50% of the thiosulphate was oxidized. Under acetate limitation in the chemostat, there was a linear increase in yield with thiosulphate addition up to a concentration of 7 mM. Higher thiosulphate concentrations resulted in loss of thiosulphate oxidizing capacity and a decrease in the biomass to the level obtained with acetate alone. This loss may be due to the presence of inhibitory (50–100 μM) levels of sulphite which is probably produced as an intermediate of the biological thiosulphate oxidation. Experiments with sulphide showed that Thiobacillus Q could also use it as an additional energy source. The complete lack of autotrophic growth, both in batch and chemostat experiments, together with the absence of even very low amounts of the key enzymes of the Calvin cycle demonstrated that this organism is a typical chemolithoheterotroph. Although this organism has provisionally been placed in the genus Thiobacillus, standard taxonomic procedures showed a close relationship with Pseudomonas alcaligenes. This study stresses the importance of quantitative chemostat studies in establishing the role of inorganic oxidations in energy metabolism and in the understanding of the role of heterotrophic sulphur oxidation in natural environments.

Major differences between glutamate-fermenting species isolated from chemostat enrichments at different dilution rates

Major differences between glutamate-fermenting species isolated from chemostat enrichments at different dilution rates

Major differences between glutamate-fermenting species isolated from chemostat enrichments at different dilution rates

From chemostat enrichments conducted at dilution rates of 0.025, 0.12 and 0.25 h−1 glutamate- and aspartate-fermenting bacteria were isolated. The dominant aspartate-fermenting strains in all these enrichments belonged to the genus Campylobacter, whereas 3 dissimilar types of glutamate-fermenting bacteria predominated at the different dilution rates. One of these strains was identified as Clostridium cochlearium. The remaining two were designated as strain DKglu16 (glutamate → acetate + propionate + ammonium + carbon dioxide) and DKglu21 (glutamate → acetate + formate + ammonium + carbon dioxide). Grown in continuous culture under glutamate limitation, strain DKglu16 (μmax= 0.13 h−1; Ks= 1.9 μM) outcompeted C. cochlearium(μmax= 0.36 h−1; Ks= 7 μM) at low dilution rates, but was outgrown at higher rates of dilution (0.044 h−1). In glutamate-limited continuous culture the competitiveness of strain DKglu16 increased considerably when lactate was added to the feed in addition to glutamate.

Chemostat enrichment and isolation of Hyphomicrobium EG. A dimethyl-sulphide oxidizing methylotroph and reevaluation of Thiobacillus MS1.

A stable mixed bacterial culture was obtained by chemostat enrichment using dimethyl-sulphoxide as a carbon and energy source. This culture could not only rapidly oxidize dimethyl-sulphoxide but also dimethyl-sulphide. Enzyme determinations indicated that an important part of it consisted of methylotrophs, which assimilated carbon via the serine pathway. Indeed plate counts revealed the majority of the community to be a Hyphomicrobium species. This organism, designated Hyphomicrobium EG, is an obligate methylotroph which can only grow aerobically on several different C1-compounds. Its performance on dimethyl-sulphoxide was compared with that of the community and of another recently isolated strain, Hyphomicrobium S. The mixed culture, Hyphomicrobium EG and Hyphomicrobium S had a mu max of 0.08, 0.08 and 0.014 h-1 respectively. The KS for dimethyl-sulphoxide was the same for all three cultures (3-6 microM), whereas that for dimethyl-sulphide of Hyphomicrobium EG after growth on dimethyl-sulphoxide was 3-fold higher than that of the other two cultures (48 and 16 microM respectively). After growth on dimethyl-sulphide it improved to 3 microM. Dimethyl-sulphide respiration was maximal at a concentration of 100 microM; higher concentrations were inhibitory. One of the accompanying organisms, a pink methylotroph, was able to derive energy from the oxidation of thiosulphate. Available cultures of Thiobacillus MS1 that were reported to be able to utilize dimethyl-sulphide could no longer metabolize this compound.

Changes in the Nitrate-reducing Community of an Anaerobic Saltmarsh Sediment in Response to Seasonal Selection by Temperature

Measurement of the nitrate-reducing potential by bacteria in saltmarsh sediment, using a thermal gradient block incubator, revealed seasonal physiological changes in the community. A mesophilic part of the nitrate-reducing community was always present, although it achieved maximum development at the end of the summer and minimum development at the end of the winter. In contrast, a distinct psychrotrophic part of the community achieved maximum development at the end of the winter but disappeared during summer. Chemostat enrichment of nitrate-reducing bacteria at 10 °C isolated predominantly Pseudomonas spp., but Vibrio spp. predominated in enrichments at 25 °C. The observed seasonal changes in situ might reflect differential seasonal selection of these two groups of bacteria.

Comparative study of enrichment methods for the isolation of autotrophic nitrifying bacteria from soil, estuarine and marine sediments

A comparative study has been undertaken to determine the efficiency of methods for the enrichment and isolation of autotrophic nitrifying bacteria from soils and estuarine and marine sediments. Chemostat enrichments proved to be the most efficient means of isolating autotrophic NH+4 oxidisers whereas NO−2 oxidising bacteria were never successfully enriched by this method. In contrast, gel enrichment and traditional batch culture enrichments of nitrifying bacteria were comparatively time consuming procedures and the degree of enrichment obtained for NH+4 oxidising bacteria never approached that obtained with continuous culture enrichments. Gel enrichments, however, because they have continuous physicochemical gradients provide qualitative advantages in that morphologically distinct types of nitrifying bacteria can be isolated from the same gel.