Anaerobic degradation of cresols by denitrifying bacteria

The initial reactions in anaerobic metablism of methylphenols (cresols) and dimethylphenols were studied with denitrifying bacteria. A newly isolated strain, possibly a Paracoccus sp., was able to grow on o-or p-cresol as sole organic substrate with a generation time of 11 h; o-or p-cresol was completely oxidized to CO₂ with nitrate being reduced to N₂. A denitrifying Pseudomonas-like strain oxidized m-or p-cresol as the sole organic growth substrate completely to CO₂ with a generation time of 14 h. Demonstration of intermediates and/or in vitro measurement of enzyme activities suggest the following enzymatic steps:(1) p-Cresol was metabolized by both strains via benzoyl-CoA as central intermediate as follows: p-cresol 4-OH-benzaldehyde 4-OH-benzoate 4-OH-benzoly-CoA benzoyl-CoA. Oxidation of the methyl group to 4-OH-benzaldehyde was catalyzed by p-cresol methylhydroxylase. After oxidation of the aldehyde to 4-OH-benzoate, 4-OH-benzoyl-CoA is formed by 4-OH-benzoyl-CoA synthetase; subsequent reductive dehydroxylation of 4-OH-benzoyl-CoA to benzoyl-CoA is catalyzed by 4-OH-benzoyl-CoA reductase (dehydroxylating).(2) o-Cresol was metabolized in the Paracoccus-like strain via 3-CH₃-benzoyl-CoA as central intermediate as follows: o-cresol 4-OH-3-CH₃-benzoate 4-OH-3-CH₃-benzoyl-CoA 3-CH₃-benzoyl-CoA. The following enzymes were demonstrated: (a) An enzyme catalyzing an isototope exchange reaction between ¹⁴CO₂ and the carboxyl of 4-OH-3-CH₃-benzoate; this activity is thought to be a partial reaction catalyzed by an o-cresol carboxylase. (b) 4-OH-3-CH₃-benzoyl-CoA synthetase (AMP-forming) activating the carboxylation product 4-OH-3-CH₃-benzoate to its coenzyme A thioester. (c) 4-OH-3-CH₃-benzoyl-CoA reductase (dehydroxylating) catalyzing the reductive dehydroxylation of the 4-hydroxyl group with reduced benzyl viologen as electron donor to yield 3-CH₃-benzoyl-CoA. This thioester may also be formed by action of a coenzyme A ligase when 3-CH₃-benzoate is metabolized. 2,4-Dimethylphenol was metabolized via 4-OH-3-CH₃-benzoate and further to 3-CH₃-benzoyl-CoA.(3) The initial reactions of anaerobic metabolism of m-cresol in the Pseudomonas-like strain were not resolved. No indication for the oxidation of the methyl group nor for the carboxylation of m-cresol was found. In contrast, 2,4-and 3,4-dimethylphenol were oxidized to 4-OH-3-CH₃-and 4-OH-2-CH₃-benzoate, respectively, probably initiated by p-cresol methylhydroxylase; however, these compounds were not metabolized further.The hydroxyl and methyl groups are abbreviated as OH-and CH₃-, respectively     

Influence of electron acceptor,carbon, nitrogen,and phosphorus on polyhydroxyalkanoate (PHA) production by <Emphasis Type="Italic">Brachymonas</Emphasis> sp. P12

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorus-removing bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH₄Cl), and phosphorus (KH₂PO₄) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g⁻¹ biomass, but the PHB concentration of an anoxic culture decreased (−218 mg PHB g⁻¹ biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g⁻¹ biomass h⁻¹, was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.     

Isolation and characterization of a bacterium that mineralizes toluene in the absence of molecular oxygen

A bacterium tentatively identified as a Pseudomonas sp. was isolated from a laboratory aquifer column in which toluene was degraded under denitrifying conditions. The organism mineralized toluene in pure culture in the absence of molecular oxygen. In carbon balance studies using [ring-UL-¹⁴C]toluene, more than 50% of the radioactivity was recovered as ¹⁴CO₂. Nitrate and nitrous oxide served as electron acceptors for toluene mineralization. The organism was also able to degrade m-xylene, benzoate, benzaldehyde, p-cresol, p-hydroxybenzaldehyde, p-hydroxybenzoate and cyclohexanecarboxylic acid in the absence of molecular oxygen.     

New metabolic pathway for o-cresol degradation by Pseudomonas sp. CP4 as evidenced by 1H NMR spectroscopic studies

Degradation intermediates of o-, m- and p-cresols extracted from resting cells of Pseudomonas sp. CP4, a potent cresol- and phenol-degrading laboratory isolate, were analysed by using ¹H NMR spectroscopy at 270 MHz. Ortho-, meta- and para-cresols were found to be degraded to 2-methyl-4-oxalocrotonate. 3-Methylcatechol from o-cresol was degraded further to 2-ketohex-cis-4-enoate, 4-methylcatechol from m- and p-cresol was degraded to 2-ketohex-cis-4-enoate. Also 2-ketopent-4-enoate was found to be formed from p-cresol. Formation of 2-methyl-4-oxalocrotonate was envisaged as taking place from 5-hydroxy-2-methylmuconic semialdehyde, the ring-cleavage product of 4-methylresorcinol, a possible product by hydroxylation of o-cresol along with 3-methylcatechol. This is a deviation from the hitherto known pathways of o-cresol degradation. Based on these observations, pathways for the degradation of all three isomers of cresol are proposed.     

Biodegradation of p-cresol by immobilized cells of Bacillus sp. strain PHN 1

The Bacillus sp. strain PHN 1 capable of degrading p-cresol was immobilized in various matrices namely, polyurethane foam (PUF), polyacrylamide, alginate and agar. The degradation rates of 20 and 40 mM p-cresol by the freely suspended cells and immobilized cells in batches and semi-continuous with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 20 and 40 mM p-cresol than freely suspended cells and the cells immobilized in polyacrylamide, alginate and agar. The PUF- immobilized cells could be reused for more than 35 cycles, without losing any degradation capacity and showed more tolerance to pH and temperature changes than free cells. These results revealed that the immobilized cell systems are more efficient than freely suspended cells for degradation of p-cresol.     

Simultaneous degradation of 3-chlorobenzoate and phenolic compounds by a defined mixed culture ofPseudomonas spp.

A mixed culture of a chlorobenzoate-(3-CBA)-degradingPseudomonas aeruginosa, strain 3mT, and a phenol/cresols-degradingPseudomonas sp., strain CP4, simultaneously and efficiently degraded mixtures of 3-CBA and phenol/cresols. However, strains 3mT and CP4 usedortho- andmeta-ring cleavage pathways, respectively. Degradation of 3-CBA was complete when the 3-CBA was equal in amount to or less than that of phenol. CP4/3mT inoculum ratios (w/w) of 1:1 or 1:2 gave the most effective degradation of both the substrates in the mixture. The mixed culture degraded equimolar mixtures of 3-CBA/phenol up to 10mm. Equimolar mixtures of 3-CBA ando-, m- orp-cresol were also degraded by the mixed culture.The authors are with the Microbiology and Bioengineering Department, Central Food Technological Research Institute, Mysore-570013, India;     

An extracellular poly (3-hydroxybutyrate) depolymerase from Penicillium sp. DS9713a-01

Summary Penicillium sp. DS9713a-01 was obtained by ultraviolet (u.v.) light mutagenesis from the Penicillium sp. DS9713a which can degrade poly (3-hydroxybutyrate) (PHB). The enzymatic activity of DS9713a-01 was 97% higher than that of the wild-type strain. The DS9713a-01 mutant could completely degrade PHB films in 5 days; however, the wild-type strain achieved only 61% at the same time. The extracellular PHB depolymerase was purified from the culture medium containing PHB as the sole carbon source by filtration, ammonium sulfate precipitation and chromatography on Sepharose CL-6B. The molecular weight of the PHB depolymerase was about 15.1kDa determined by SDS-polyacrylamide gel electrophoresis. The optimum activity of the PHB depolymerase was observed at pH 8.6 and 50 °C. The enzyme was stable at temperatures below 37 °C and in the pH range from 8.0 to 9.2. The activity of PHB depolymerase could be activated or inhibited by some metal ions. The apparent K _m value was 0.164 mg ml⁻¹. Mass spectrometric analysis of the water-soluble products after enzymatic degradation revealed that the primary product was the monomer, 3-hydroxybutyric acid.     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

Enhancement of symbiotic nitrogen fixation by vitamin-secreting fluorescent Pseudomonas

Fluorescent Pseudomonas sp. strain 267 promotes growth of nodulated clover plants under gnotobiotic conditions. In the growth conditions (60 M FeCl₃), the production of siderophores of the pseudobactin-pyoverdin group was repressed. Plant growth enhancement results from secretion of B vitamins by Pseudomonas sp. strain 267. This was proven by stimulation of clover growth by naturally auxotrophic strains of Rhizobium leguminosarum bv. trifolii and marker strains E. coli thi^- and R. meliloti pan^- in the presence of the supernatant of Pseudomonas sp. strain 267. The addition of vitamins to the plant medium increased symbiotic nitrogen fixation by the clover plants.     

Specific ratio and survival of Pseudomonas CF600 as co-culture for phenol degradation in continuous cultivation

Studies were carried out to understand parallel survival of two strains when cultivated as co-culture on a single carbon source in continuous cultivation. Strains used were Pseudomonas sp. strain CF600 that is reported for degradation of phenol; and HKR1 a lab strain, which was isolated from a site contaminated with phenol. In continuous cultivation Pseudomonas sp. CF600 showed an accumulation of colored intermediate, 2-hydroxy muconic semialdehyde (HMS), when fed with phenol as a sole source of carbon under dissolved oxygen limiting condition (40% saturation level). Under the same cultivation condition when it was co-cultured with strain HKR1, complete degradation of phenol was observed with no accumulation of intermediate. Different dilution rates (0.03, 0.15, and 0.30) were set in the bioreactor during cultivation. It was also observed that both the strains follow a typical cell density ratio of 1:18 as strain HKR1: Pseudomonas sp. CF600 irrespective of the dilution rates used in the study to favor degradation of phenol. Pseudomonas sp. CF600 is reported to degrade phenol via a plasmid-encoded pathway (pVI150). The enzymes for this meta-cleavage pathway are clustered on 15 genes encoded by a single operon, the dmp operon. PCR using primers from the different catabolic loci of dmp operon, demonstrated that the strain HKR1 follows a different metabolic pathway for intermediate utilization.     

Substrate specificity of a long-chain alkylamine-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> sp isolated from activated sludge

A bacterium strain BERT, which utilizes primary long-chain alkylamines as nitrogen, carbon and energy source, was isolated from activated sludge. This rod-shaped motile, Gram-negative strain was identified as a Pseudomonas sp. The substrate spectrum of this Pseudomonas strain BERT includes primary alkylamines with alkyl chains ranging from C₃ to C₁₈, and dodecyl-1,3-diaminopropane. Amines with alkyl chains ranging from 8 to 14 carbons were the preferred substrates. Growth on dodecanal, dodecanoic acid and acetic acid and simultaneous adaptation studies indicated that this bacterium initiates degradation through a C_alkyl–N cleavage. The cleavage of alkylamines to the respective alkanals in Pseudomonas strain BERT is mediated by a PMS-dependent alkylamine dehydrogenase. This alkylamine dehydrogenase produces stoichiometric amounts of ammonium from octylamine. The PMS-dependent alkylamine was found to oxidize a broad range of long-chain alkylamines. PMS-dependent long-chain aldehyde dehydrogenase activity was also detected in cell-free extract of Pseudomonas strain BERT grown on octylamine. The proposed pathway for the oxidation of alkylamine in strain BERT proceeds from alkylamine to alkanal, and then to the fatty acid.     

Accumulation of eicosapentaenoic acid in <Emphasis Type="Italic">Nannochloropsis</Emphasis> sp. in response to elevated CO<Subscript>2</Subscript> concentrations

To increase eicosapentaenoic acid (EPA, 20:5, n-3) content in the marine alga Nannochloropsis sp., the effect of CO₂ concentration during cultivation has been investigated. In a batch culture under normal atmospheric conditions (0.037% CO₂), the EPA content per cell increased during the first 1.5 days and then decreased immediately even though the cells were in an exponential growth phase. Increasing the CO₂ concentration to 0.3% and 2% over day 1.5 retained the EPA content at the higher concentration for another 1 and 2 days, respectively, suggesting that the EPA accumulation is enhanced by elevated concentrations of CO₂. EPA accumulation in response to elevated CO₂ concentrations was also observed during a later growth phase when CO₂ was introduced after the decrease of EPA content. The addition of CO₂ caused a slight decrease in the pH of the medium though this was not the cause of the observed EPA accumulation as addition of acidic buffer did not affect the EPA content. The maximum EPA production was obtained when 2% CO₂ was supplied 12 h prior to the end of the exponential growth. The total EPA production during 4-day cultivation was about twice that obtained with ambient air. These results suggest that the available CO₂ concentration affects the EPA content in Nannochloropsis sp.     

Effect of anaerobic promoters on the microaerobic production of polyhydroxybutyrate (PHB) in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from Ralstonia eutropha for the micro- or anaerobic PHB production in recombinant Escherichia coli. Among the promoters, the one for alcohol dehydrogenase (P _adhE ) was found most effective. Recombinant E. coli JM 109 (pWCY09) harboring P _adhE and phbCAB achieved a 48% PHB accumulation in the cell dry weight after 48 h of static culture compared with only 30% PHB production under its native promoter. Sixty-seven percent PHB was produced in the dry weight (CDW) of an acetate pathway deleted (Δpta deletion) E. coli JW2294 harboring the vector pWCY09. In a batch process conducted in a 5.5-l NBS fermentor containing 3 l glucose LB medium, E. coli JW2294 (pWCY09) grew to 7.8 g/l CDW containing 64% PHB after 24 h of microaerobic incubation. In addition, molecular weight of PHB was observed to be much higher under microaerobic culture conditions. The high activity of P _adhE appeared to be the reason for improved micro- or anaerobic cell growth and PHB production while high molecular weight contributed to the static culture condition.     

A Polyhydroxybutyrate-Producing <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Isolated from Antarctic Environments with High Stress Resistance

Pseudomonas sp. 14-3, a strain that accumulates large quantities of polyhydroxybutyrate (PHB) when grown on octanoate, was isolated from Antarctic environments. This isolate was characterized on the basis of phenotypic features and partial sequencing of its 16S ribosomal RNA gene. Pseudomonas sp. 14-3 showed increased tolerance to both thermal and oxidative stress compared with three other Pseudomonas species. Stress tolerance of Pseudomonas sp. 14-3 was analyzed in polyhydroxyalkanoate accumulating and non-accumulating conditions, and increased levels of stress resistance were observed when PHB was produced. Pseudomonas sp. 14-3 was isolated from Antarctic regions, a habitat normally exposed to extreme conditions. An association between high PHB accumulation and high stress resistance in bacteria adapted to extreme environments is suggested.     

Rhodococcus sp. strain TM1 plays a synergistic role in the degradation of piperidine by Mycobacterium sp. strain THO100

Mycobacterium sp. strain THO100 and Rhodococcus sp. strain TM1 were isolated from a morpholine-containing enrichment culture of activated sewage sludge. Strain THO100, but not strain TM1, was able to degrade alicyclic amines such as morpholine, piperidine, and pyrrolidine. The mixed strains THO100 and TM1 showed a better growth on piperidine as the substrate than the pure strain THO100 because strain TM1 was able to reduce the level of glutaraldehyde (GA) produced during piperidine degradation. GA was toxic to strain THO100 (IC₅₀ = 28.3 μM) but less toxic to strain TM1 (IC₅₀ = 215 μM). Strain THO100 possessed constitutive semialdehyde dehydrogenases, namely Sad1 and Sad2, whose activities toward succinic semialdehyde (SSA) were strongly inhibited by GA. The two isozymes were identified as catalase–peroxidase (KatG = Sad1) and semialdehyde dehydrogenase (Sad2) based on mass spectrometric analyses of tryptic peptides and database searches of the partial DNA sequences of their genes. In contrast, strain TM1 containing another constitutive enzyme Gad1 could oxidize both SSA and GA. This study suggested that strain TM1 possessing Gad1 played a synergistic role in reducing the toxic and inhibitory effects of GA produced in the degradation of piperidine by strain THO100.     

Salinity-induced accumulation of poly-β-hydroxybutyrate in rhizobia indicating its role in cell protection

Summary Poly β-hydroxybutyrate (PHB) is an energy and carbon storage material accumulated in response to the limitation of an essential nutrient. The effect of different salt concentrations on growth and PHB accumulation of four different Sinorhizobium strains was examined. Irrespective of the strain, a defined trend in the accumulation of PHB inside the cells was observed. While minimum PHB content was accumulated at low or zero salinity, maximum was observed by the salt-tolerant strains at higher salt concentrations. This suggests a definite role for PHB in cell protection in saline conditions.     

Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in<Emphasis Type="Italic"> Bacillus megaterium</Emphasis>

Molecular analysis of a genomic region of Bacillus megaterium, a polyhydroxybutyrate (PHB)-producing microorganism, revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). Enzyme activity was measured throughout the different growth phases of B. megaterium and was found to correlate with PHB accumulation during the late-exponential growth phase. Ptb expression was repressed by glucose and activated by the branched amino acids isoleucine and valine. Overexpression of Act_Bm, a ⁵⁴ regulator from B. megaterium whose gene is located upstream from ptb, caused an increase in Ptb activity and PHB accumulation in B. megaterium.     

Periodic change in DO concentration for efficient poly-β-hydroxy-butyrate production using temperature-inducible recombinant<Emphasis Type="Italic">Escherichia coli</Emphasis> with proteome analysis

RecombinantEscherichia coli strain harboring the λp _R-p _L promotor and heterologus poly-β-hydroxybutyrate (PHB) biosynthesis genes was used to investigate the effect of culture conditions on the efficient PHB production. The expression ofphb genes was induced by a temperature upshift from 33°C to 38°C. The protein expression levels were measured by using two-dimensional electrophoresis, and the enzyme activities were also measured to understand the effect of culture temperature, carbon sources, and the dissolved oxygen (DO) concentration on the metabolic regulations. AcetylCoA is an important branch point for PHB production. The decrease in DO concentration lowers the citrate synthase activity, thus limit the flux toward the TCA cycle, and increase the flux for PHB production. Since NADPH is required for PHB production, the PHB production does not continue leading the overproduction of acetate and lactate. Based on these observations, a new operation was considered where DO concentration was changed periodically, and it was verified its usefulness for the efficient PHB production by experiments.     

Physiology of nitrogen fixation in Azospirillum lipoferum Br 17 (ATCC 29 709)

Changes of cellular activities during batch cultures with Azospirillum lipoferum strain Br 17 (ATCC 29 709) were observed within the growth cycle, at optimal pO₂ (0.002–0.003 atm). The relative growth rate for cells growing with N₂ as sole nitrogen source during log phase was =0.13 h^-1 and the doubling time was 5.3 h. Nitrogenase activity was not accompanied by hydrogen evolution at any growth stage, and a very active uptake hydrogenase was demonstrated. The hydrogenase activity increased towards the end of the growth period when glucose became limiting and N₂ fixation reached its maximal specific activity. Oxygen consumption and oxygen tolerance at the various growth stages, increased simultaneously with the uptake hydrogenase activity indicating a possible role of this enzyme in an oxygen protection mechanism of A. lipoferum nitrogenase. The efficiency of nitrogen fixation expressed as mg total nitrogen fixed in cells and supernatant per g glucose consumed, was 20 at the early log phase and increased to 48 at the late log phase. About 25% of the total fixed nitrogen was recovered in the culture supernatant.Abbreviations DOT Dissolved oxygen tension - PHB Poly--hydroxybutyric acid - O.D. Optical density (560 nm) - A.T.C.C. American type culture collection - NTA Nitrilotriacetic acid Graduate student of the Universidade Federal Rural do Rio de Janeiro, Brazil     

A novel n-alkane-degrading bacterium as a minor member of p-xylene-degrading sulfate-reducing consortium

A p-xylene-degrading, sulfate-reducing enrichment culture was characterized by analyzing the response of its members to changes in the available substrate. The culture was inoculated into media containing other substrates, resulting in the establishment of benzoate-, acetate-, and lactate-utilizing enrichment cultures. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the enriched cultures targeting 16S rRNA genes showed quite simple band patterns. The predominant band from the benzoate-utilizing enrichment culture was identical to that from the original enrichment culture utilizing p-xylene. A single, dominant DGGE band was observed in common from the acetate- and lactate-utilizing enrichment cultures. A novel sulfate-reducing bacterium, strain PL12, was isolated from the lactate-utilizing enrichment culture. The 16S rRNA gene sequence of strain PL12 was identical to that of the dominant DGGE band in the acetate- and lactate-utilizing enrichment cultures and distinct from the dominant sequences in the original p-xylene-degrading and benzoate-utilizing enrichment cultures. Phylogenetic analysis of the 16S rRNA gene sequences showed that the isolate belonged to the family Desulfobacteraceae in the class Deltaproteobacteria. The isolated strain PL12 could utilize n-hexane and n-decane as substrates, but could not utilize benzoate, p-xylene and other aromatic hydrocarbons. These results suggest that the p-xylene degradation observed in the original enrichment culture was performed by the dominant bacterium corresponding to DGGE band pXy-K-13 (Nakagawa et al. 2008). The novel strain PL12 might have been utilizing metabolites of p-xylene.