Mixed Continuous Cultures of Polyvinyl Alcohol-Utilizing Symbionts Pseudomonas putida VM15A and Pseudomonas sp. Strain VM15C

Stable mixed continuous cultures of Pseudomonas sp. strain VM15C and Pseudomonas putida VM15A, the former of which produced a polyvinyl alcohol (PVA)-degrading enzyme and the latter of which produced an essential growth factor for PVA utilization by strain VM15C, were established with PVA as the sole source of carbon and energy with chemostat cultivation. A high extent of PVA degradation was achieved at dilution rates of less than 0.030/h. The predominant strain in the cultures was the primary metabolizer of PVA, strain VM15C. The growth supporter, strain VM15A, existed as a minor population, although its population was maintained at an almost constant level throughout a dilution region in which the VM15C population decreased markedly as the dilution rate was raised. A crude growth factor which was prepared from a culture supernatant of strain VM15A and increased the specific growth rate of strain VM15C with PVA in an axenic batch culture was also effective for enhancing the VM15C population and PVA degradation in the mixed continuous culture at a high dilution rate (0.064/h). This indicated that the growth-limiting substrate for strain VM15C in the mixed continuous culture is the growth factor produced by strain VM15A.     

Pyrroloquinoline Quinone-Dependent Cytochrome Reduction in Polyvinyl Alcohol-Degrading Pseudomonas sp. Strain VM15C

A polyvinyl alcohol (PVA) oxidase-deficient mutant of Pseudomonas sp. strain VM15C, strain ND1, was shown to possess PVA dehydrogenase, in which pyrroloquinoline quinone (PQQ) functions as a coenzyme. The mutant grew on PVA and required PQQ for utilization of PVA as an essential growth factor. Incubation of the membrane fraction of the mutant with PVA caused cytochrome reduction of the fraction. Furthermore, it was found that in spite of the presence of PVA oxidase, the membrane fraction of strain VM15C grown on glucose without PQQ required PQQ for cytochrome reduction during incubation with PVA. The results provide evidence that PVA dehydrogenase couples with the electron transport chain of PVA-degrading bacteria but that PVA oxidase does not.     

Symbiotic Utilization of Polyvinyl Alcohol by Mixed Cultures

Polyvinyl alcohol (PVA)-utilizing cultures were obtained from various sources. They were mixed cultures even after cyclical transfer to liquid and plate media with PVA as a sole source of carbon. Component bacteria were isolated from the several mixed cultures, and it was shown that PVA was utilized symbiotically by two bacterial members which could not utilize PVA in each respective pure culture. From a mixed culture, strains VM15, VM15A (Pseudomonas putida) and VM15C (Pseudomonas sp.) were isolated as members essential for PVA utilization. VM15C was the predominant strain in the mixed-culture population and produced PVA-degrading enzyme. The culture supernatant of VM15A enabled VM15C to grow on PVA. VM15A was presumed to supply VM15C with a unique growth stimulant which was distinct from usual growth factors.     

Localization of Polyvinyl Alcohol Oxidase Produced by a Bacterial Symbiont, Pseudomonas sp. Strain VM15C

An axenic culture of a polyvinyl alcohol (PVA)-degrading symbiont, Pseudomonas sp. strain VM15C, was established on PVA with a crude preparation of the growth factor (factor A) produced by the symbiotic partner Pseudomonas putida VM15A. An increase of factor A in the culture medium enhanced the cell-associated PVA oxidase activity as well as the growth rate, but decreased production of extracellular PVA oxidase. PVA oxidase in cells grown on PVA was present in the periplasmic space at a higher ratio than in cells grown on peptone. PVA degradation occurred rapidly with washed cells. PVA was also degraded by immobilized cells entrapped in agar gels.     

Purification of membrane-bound polyvinyl alcohol oxidase in Pseudomonas sp. VM15C

Abstract Polyvinyl alcohol (PVA) was utilized by a symbiotic mixed culture which was composed of Pseudomonas putida VM15A and Pseudomonas sp. VM14C. The PVA oxidase was found in the culture fluid, membrane, and cytosol fractions of VM15C. The membrane-bound PVA oxidase was purified by several steps of chromatography. The enzyme (p I = 9.6) exhibited the maximum activity at pH 8.0 to 8.4 and 45°C, and utilized secondary alcohol as well as PVA. The enzyme showed the PVA dehydrogenating activity linking with phenazine ethosulfate, indicating the possibility that PVA oxidation is coupled with an electron transport chain on the bacterial membrane.     

Production of Polyvinyl Alcohol Oxidase by a Symbiotic Mixed Culture

Production of polyvinly alcohol (PVA) oxidase by Pseudomonas sp. strain VM15C, a PVA degrader of a symbiotic PVA-utilizing mixed culture, was examined in various cultures. Despite the absence of PVA in the culture in nutrient broth, VM15C showed approximately the same productivity of PVA oxidase activity as that in the culture with PVA as the sole carbon source, whereas the productivity in the culture with glucose was lower than that of either the nutrient broth or the PVA culture. PVA oxidase activity produced in the nutrient broth culture was predominantly present in the cells, and most of the activity appeared to be in the cytoplasm. In contrast, in the culture with PVA as the sole carbon source, the activity was present in the culture fluid in a larger ratio than in the nutrient broth culture. Thus, production of PVA oxidase activity by this strain was constitutive and repressible, although localization of the produced activity was changed by growth conditions.     

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.     

Existence of a novel enzyme, pyrroloquinoline quinone-dependent polyvinyl alcohol dehydrogenase, in a bacterial symbiont, Pseudomonas sp. strain VM15C.

A novel enzyme, pyrroloquinoline quinone (PQQ)-dependent polyvinyl alcohol (PVA) dehydrogenase, was found in and partially purified from the membrane fraction of a PVA-degrading symbiont, Pseudomonas sp. strain VM15C. The enzyme required PQQ for PVA dehydrogenation with phenazine methosulfate, phenazine ethosulfate, and 2,6-dichlorophenolindophenol as electron acceptors and did not show PVA oxidase activity leading to H2O2 formation. The enzyme was active toward low-molecular-weight secondary alcohols rather than primary alcohols. A membrane-bound PVA oxidase was also present in cells of VM15C. Although the purified oxidase showed a substrate specificity similar to that of PQQ-dependent PVA dehydrogenase and about threefold-higher PVA-dehydrogenating activity with phenazine methosulfate or phenazine ethosulfate than PVA oxidase activity with H2O2 formation, it was shown that the enzyme does not contain PQQ as the coenzyme, and PQQ did not affect its activity. Incubation of the membrane fraction of cells with PVA caused a reduction in the cytochrome(s) of the fraction.     

Properties and roles of bacterial symbionts of polyvinyl alcohol-utilizing mixed cultures

From several polyvinyl alcohol (PVA)-utilizing mixed cultures, two component bacterial strains essential for PVA utilization were isolated, and their properties and roles in PVA utilization were studied. Each pair of essential component strains consisted of a type I strain, which produced a PVA-degrading enzyme and constituted the predominant population of the mixed culture in PVA, and a type II strain, which produced a certain growth stimulant for the former strain. All of the type I strains were taxonomically identical and assigned as Pseudomonas sp. In contrast, type II strains were taxonomically different from each other, belonging to Pseudomonas spp. and Alcaligenes sp. PVA utilization occurred in each mixed culture of a type I strain with Pseudomonas putida VM15A as a substitute for the type II strain of the original pair and also in each mixed culture of a type II strain with Pseudomonas sp. VM15C. The growth rates of these substituted, mixed cultures differed from each other.     

Properties and roles of bacterial symbionts of polyvinyl alcohol-utilizing mixed cultures.

From several polyvinyl alcohol (PVA)-utilizing mixed cultures, two component bacterial strains essential for PVA utilization were isolated, and their properties and roles in PVA utilization were studied. Each pair of essential component strains consisted of a type I strain, which produced a PVA-degrading enzyme and constituted the predominant population of the mixed culture in PVA, and a type II strain, which produced a certain growth stimulant for the former strain. All of the type I strains were taxonomically identical and assigned as Pseudomonas sp. In contrast, type II strains were taxonomically different from each other, belonging to Pseudomonas spp. and Alcaligenes sp. PVA utilization occurred in each mixed culture of a type I strain with Pseudomonas putida VM15A as a substitute for the type II strain of the original pair and also in each mixed culture of a type II strain with Pseudomonas sp. VM15C. The growth rates of these substituted, mixed cultures differed from each other.     

Cloning and Characterization of the Gene Encoding Pyrroloquinoline Quinone-dependent Poly (vinyl alcohol) Dehydrogenase of Pseudomonas sp. Strain VM15C

A gene library of poly (vinyl alcohol) (PVA)-degrading Pseudomonas sp. strain VM15C was constructed in Escherichia coli with the vector pUC18. Screening of this library with a chromogenic PVA dehydrogenase assay resulted in the isolation of a clone that carries the gene (pdh) for the PVA dehydrogenase, and the entire nucleotide sequence of its structural gene was determined. The gene encodes a protein of 639 amino acid residues (68,045 Da) and in the deduced amino acid sequence, some putative functional sites, a signal sequence, a heme c-binding site, and a PQQ-binding site, were detected. The amino acid sequence showed low similarity to other types of quinoprotein dehydrogenases. PVA dehydrogenase expressed in E. coli clones required PQQ. Ca²⁺, and Mg²⁺ stimulated the activity. PVA-dependent heme c reduction occurred with exogenous PQQ in cell extracts of the E. coli clone. The PVA dehydrogenase in the E. coli clone was localized in the cytoplasm.     

Isolation and characterization of a novel poly(vinyl alcohol)-degrading bacterium, Sphingopyxis sp. PVA3

We have isolated a poly(vinyl alcohol) (PVA)-degrading bacterium from an activated sludge sample obtained from the drainage of a dyeing factory. Enrichment cultures were performed in media containing PVA as the sole or major carbon source. After several rounds of cultivation on liquid and solid media, we were able to isolate a single colony with PVA-degrading ability (strain PVA3). The bacterium could degrade PVA in the absence of symbionts or cofactors such as pyrroloquinoline quinone (PQQ). Over 90% of PVA, at an initial concentration of 0.1%, was degraded within a 6-day cultivation. Degradation was confirmed by both iodometric methods and gel permeation chromatography. Examination of the PVA attached to the cells revealed a large increase in carbonyl groups, suggesting the oxidation of hydroxyl groups of the polymer on the surfaces of cells. Addition of PQQ to the culture medium did not enhance the growth and the PVA-degrading rates of strain PVA3. Furthermore, we found that cells grown on PVA generated hydrogen peroxide upon the addition of PVA. The results strongly suggest that the initial oxidation of PVA is mediated via a PVA oxidase, and not a PQQ-dependent dehydrogenase. A biochemical and phylogenetic characterization of the bacterium was performed. The sequence of the 16S ribosomal RNA gene of the bacterium indicated a phylogenetic position of the strain within the genus Sphingopyxis, and the strain was therefore designated Sphingopyxis sp. PVA3.     

Factors affecting the production of pyrroloquinoline quinone by the methylotrophic bacterium W3A1

Two variants of the methylotrophic bacterium W3A1, designated W3A1-S (slimy) and W3A1-NS (nonslimy), were compared with respect to their ability to grow in batch culture on the C1 substrates methylamine, methanol, and trimethylamine. Substrate utilization, cell density, pH, cellular and soluble polysaccharide production, and concentrations of the enzymes methylamine dehydrogenase, trimethylamine dehydrogenase, and methanol dehydrogenase produced were measured as a function of growth. The ability of the two bacterial variants to excrete the redox cofactor pyrroloquinoline quinone into the growth medium was also investigated. The two variants were similar with respect to all properties measured, except that W3A1-S produced significantly more capsular polysaccharides than variant W3A1-NS. Pyrroloquinoline quinone was excreted when either variant was grown on any of the C1 substrates investigated but was maximally produced when the methylamine concentration was 0.45% (wt/vol). This cofactor is excreted only as bacterial growth enters the stationary phase, a time when the levels of trimethylamine dehydrogenase and the quinoproteins methanol dehydrogenase and methylamine dehydrogenase begin to decline. It is not known whether the pyrroloquinoline quinone found in the medium is made de novo for excretion, derived from the quinoprotein pool, or both. Pyrroloquinoline quinone excretion has been observed with other methylotrophs, but this is the first instance where the excretion was observed with substrates other than methanol.     

Factors affecting the production of pyrroloquinoline quinone by the methylotrophic bacterium W3A1.

Two variants of the methylotrophic bacterium W3A1, designated W3A1-S (slimy) and W3A1-NS (nonslimy), were compared with respect to their ability to grow in batch culture on the C1 substrates methylamine, methanol, and trimethylamine. Substrate utilization, cell density, pH, cellular and soluble polysaccharide production, and concentrations of the enzymes methylamine dehydrogenase, trimethylamine dehydrogenase, and methanol dehydrogenase produced were measured as a function of growth. The ability of the two bacterial variants to excrete the redox cofactor pyrroloquinoline quinone into the growth medium was also investigated. The two variants were similar with respect to all properties measured, except that W3A1-S produced significantly more capsular polysaccharides than variant W3A1-NS. Pyrroloquinoline quinone was excreted when either variant was grown on any of the C1 substrates investigated but was maximally produced when the methylamine concentration was 0.45% (wt/vol). This cofactor is excreted only as bacterial growth enters the stationary phase, a time when the levels of trimethylamine dehydrogenase and the quinoproteins methanol dehydrogenase and methylamine dehydrogenase begin to decline. It is not known whether the pyrroloquinoline quinone found in the medium is made de novo for excretion, derived from the quinoprotein pool, or both. Pyrroloquinoline quinone excretion has been observed with other methylotrophs, but this is the first instance where the excretion was observed with substrates other than methanol.     

cis-benzeneglycol production using a mutant Pseudomonas strain

The biotransformation of commodity aromatic chemicals into dihydroxy derivatives was studied. A strain isolated from the invironment, Pseudomonas JI104, used benzene, toluene, and other hydrocarbons as sole carbon and energy sources. We selected mutants unable to grow with benzene, and among these, screened for strains with deficient cis-benzenglycol dehydrogenase able to stably produce cis-benzeneglycol when another carbon source was co-metabolized.We exained the possibility of cis-benzeneglycol production by growing the mutant strain in the presence of benzene vapor. Ethanol was the carbon and energy source most adapted to the cis-benzeneglycol production phase, and lactate or propanol could also be used. Glucose inhibited the production of the metabolite.The growth rates were barely affected by the presence of benzene at a reduced partial pressure (less than 20% of saturation), showing that continuous culture is possible. In a batch process, 0.54g·1⁻¹ of a cell suspension produced 5.1 mmol·1⁻¹cis-benzeneglycol in 27 h, using ethanol as the energy source.     

Ethanol effect on batch and fed-batch Arthrospira platensis growth

The ability of Arthrospira platensis to use ethanol as a carbon and energy source was investigated by batch process and fed-batch process. A. platensis was cultivated under the effect of a single addition (batch process) and a daily pulse feeding (fed-batch process) of pure ethanol, at different concentrations, to evaluate cell concentration (X) and specific growth rate (μ). A marked increase was observed in the cell concentration of A. platensis in runs with ethanol addition when compared to control cultures without ethanol addition. The fed-batch process using an ethanol concentration of 38 mg L^?1 days^?1 reached the maximum cell concentration of 2,393 ± 241 mg L^?1, about 1.5-fold that obtained in the control culture. In all experiments, the maximum specific growth rate was observed in the early exponential phase of cell growth. In the fed-batch process, μ decreased more slowly than in the batch process and control culture, resulting in the highest final cell concentration. Ethanol can be used as a feasible carbon and energy source for A. platensis growth via a fed-batch process.     

Biodegradation kinetics of 4-fluorocinnamic acid by a consortium of Arthrobacter and Ralstonia strains

Arthrobacter sp. strain G1 is able to grow on 4-fluorocinnamic acid (4-FCA) as sole carbon source. The organism converts 4-FCA into 4-fluorobenzoic acid (4-FBA) and utilizes the two-carbon side-chain for growth with some formation of 4-fluoroacetophenone as a dead-end side product. We also have isolated Ralstonia sp. strain H1, an organism that degrades 4-FBA. A consortium of strains G1 and H1 degraded 4-FCA with Monod kinetics during growth in batch and continuous cultures. Specific growth rates of strain G1 and specific degradation rates of 4-FCA were observed to follow substrate inhibition kinetics, which could be modeled using the kinetic models of Haldane–Andrew and Luong–Levenspiel. The mixed culture showed complete mineralization of 4-FCA with quantitative release of fluoride, both in batch and continuous cultures. Steady-state chemostat cultures that were exposed to shock loadings of substrate responded with rapid degradation and returned to steady-state in 10–15 h, indicating that the mixed culture provided a robust system for continuous 4-FCA degradation.     

Metabolism of Dibenzofuran by Pseudomonas sp. Strain HH69 and the Mixed Culture HH27

A Pseudomonas sp. strain, HH69, and a mixed culture, designated HH27, were isolated by selective enrichment from soil samples. The pure strain and the mixed culture grew aerobically on dibenzofuran as the sole source of carbon and energy. Degradation proceeded via salicylic acid which was branched into the gentisic acid and the catechol pathway. Both salicylic acid and gentisic acid accumulated in the culture medium of strain HH69. The acids were slowly metabolized after growth ceased. The enzymes responsible for their metabolism showed relatively low activities. Besides the above-mentioned acids, 2-hydroxyacetophenone, benzopyran-4-one (chromone), several 2-substituted chroman-4-ones, and traces of the four isomeric monohydroxydiben-zofurans were identified in the culture medium. 2,2′,3-Trihydroxybiphenyl was isolated from the medium of a dibenzofuran-converting mutant derived from parent strain HH69, which can no longer grow on dibenzofuran. This gives evidence for a novel type of dioxygenases responsible for the attack on the biarylether structure of the dibenzofuran molecule. A meta-fission mechanism for cleavage of the dihydroxylated aromatic nucleus of 2,2′,3-trihydroxybiphenyl is suggested as the next enzymatic step in the degradative pathway.     

Energetics of Growth of a Defined Mixed Culture of Desulfovibrio vulgaris and Methanosarcina barkeri: Interspecies Hydrogen Transfer in Batch and Continuous Cultures

Interspecies hydrogen transfer was studied in Desulfovibrio vulgaris-Methanosarcina barkeri mixed cultures. Experiments were performed under batch and continuous growth culture conditions. Lactate or pyruvate was used as an energy source. In batch culture and after 30 days of simultaneous incubation, these organisms were found to yield 1.5 mol of methane and 1.5 mol of carbon dioxide per mol of lactate fermented. When M. barkeri served as the hydrogen acceptor, growth yields of D. vulgaris were higher compared with those obtained on pyruvate without any electron acceptor other than protons. In continuous culture, all of the carbon derived from the oxidation of lactate was recovered as methane and carbon dioxide, provided the dilution rate was minimal. Increasing the dilution rate induced a gradual accumulation of acetate, causing acetate metabolism to cease at above μ = 0.05 h⁻¹. Under these conditions all of the methane produced originated from carbon dioxide. The growth yields of D. vulgaris were measured when sulfate or M. barkeri was the electron acceptor. Two key observations resulted from the present study. First, although sulfate was substituted by M. barkeri, metabolism of D. vulgaris was only slightly modified. The coculture-fermented lactate produced equimolar quantities of carbon dioxide and methane. Second, acetogenesis and methane formation from acetate were completely separable.     

Degradation of cocaine by a mixed culture of Pseudomonas fluorescens MBER and Comamonas acidovorans MBLF.

A mixed culture that could utilize cocaine as the sole source of carbon and energy for growth was isolated by selective enrichment. The individual microorganisms within this mixed culture were identified as Pseudomonas fluorescens (termed MBER) and Comamonas acidovorans (termed MBLF). Each microorganism was shown to be unable to grow to any appreciable extent on 10 mM cocaine in the absence of the other. C. acidovorans MBLF was found to possess an inducible cocaine esterase which catalyzed the hydrolysis of cocaine to ecgonine methyl ester and benzoate. C. acidovorans was capable of growth on benzoate at concentrations below 5 mM but was unable to metabolize ecgonine methyl ester. P. fluorescens MBER was capable of growth on either benzoate as the sole source of carbon or ecgonine methyl ester as the sole source of carbon and nitrogen. P. fluorescens MBER was found to initiate the degradation of ecgonine methyl ester via ecgonine, pseudoecgonine, and pseudoecgonyl-coenzyme A. Subcellular studies resulted in the identification of an ecgonine methyl esterase, an ecgonine epimerase, and a pseudoecgonyl-coenzyme A synthetase which were induced by growth on ecgonine methyl ester or ecgonine. Further metabolism of the ecgonine moiety is postulated to involve nitrogen debridging, with the production of carbonyl-containing intermediates.