Lignin induced iron reduction by novel sp., Tolumonas lignolytic BRL6-1.

Lignin is the second most abundant carbon polymer on earth and despite having more fuel value than cellulose, it currently is considered a waste byproduct in many industrial lignocellulose applications. Valorization of lignin relies on effective and green methods of de-lignification, with a growing interest in the use of microbes. Here we investigate the physiology and molecular response of the novel facultative anaerobic bacterium, Tolumonas lignolytica BRL6-1, to lignin under anoxic conditions. Physiological and biochemical changes were compared between cells grown anaerobically in either lignin-amended or unamended conditions. In the presence of lignin, BRL6-1 accumulates higher biomass and has a shorter lag phase compared to unamended conditions, and 14% of the proteins determined to be significantly higher in abundance by log2 fold-change of 2 or greater were related to Fe(II) transport in late logarithmic phase. Ferrozine assays of the supernatant confirmed that Fe(III) was bound to lignin and reduced to Fe(II) only in the presence of BRL6-1, suggesting redox activity by the cells. LC-MS/MS analysis of the secretome showed an extra band at 20 kDa in lignin-amended conditions. Protein sequencing of this band identified a protein of unknown function with homology to enzymes in the radical SAM superfamily. Expression of this protein in lignin-amended conditions suggests its role in radical formation. From our findings, we suggest that BRL6-1 is using a protein in the radical SAM superfamily to interact with the Fe(III) bound to lignin and reducing it to Fe(II) for cellular use, increasing BRL6-1 yield under lignin-amended conditions. This interaction potentially generates organic free radicals and causes a radical cascade which could modify and depolymerize lignin. Further research should clarify the extent to which this mechanism is similar to previously described aerobic chelator-mediated Fenton chemistry or radical producing lignolytic enzymes, such as lignin peroxidases, but under anoxic conditions.

Analysis of Zobellella denitrificans ZD1 draft genome: Genes and gene clusters responsible for high polyhydroxybutyrate (PHB) production from glycerol under saline conditions and its CRISPR-Cas system.

Polyhydroxybutyrate (PHB) is biodegradable and renewable and thus considered as a promising alternative to petroleum-based plastics. However, PHB production is costly due to expensive carbon sources for culturing PHB-accumulating microorganisms under sterile conditions. We discovered a hyper PHB-accumulating denitrifying bacterium, Zobellella denitrificans ZD1 (referred as strain ZD1 hereafter) capable of using non-sterile crude glycerol (a waste from biodiesel production) and nitrate to produce high PHB yield under saline conditions. Nevertheless, the underlying genetic mechanisms of PHB production in strain ZD1 have not been elucidated. In this study, we discovered a complete pathway of glycerol conversion to PHB, a novel PHB synthesis gene cluster, a salt-tolerant gene cluster, denitrifying genes, and an assimilatory nitrate reduction gene cluster in the ZD1 genome. Interestingly, the novel PHB synthesis gene cluster was found to be conserved among marine Gammaproteobacteria. Higher levels of PHB accumulation were linked to higher expression levels of the PHB synthesis gene cluster in ZD1 grown with glycerol and nitrate under saline conditions. Additionally, a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas type-I-E antiviral system was found in the ZD1 genome along with a long spacer list, in which most of the spacers belong to either double-stranded DNA viruses or unknown phages. The results of the genome analysis revealed strain ZD1 used the novel PHB gene cluster to produce PHB from non-sterile crude glycerol under saline conditions.

Modeling of growth kinetics for an isolated marine bacterium, Oceanimonas sp. BPMS22 during the production of a trypsin inhibitor.

Protease inhibitors significantly control physiologically relevant protease activities. Protease inhibitors from marine microbial sources are unique due to their rough living environmental conditions. In the present study, a protein protease inhibitor (PI) was produced from marine Oceanimonas sp. BPMS22. Seven different media were screened for the growth of the bacterium and production of PI. Different carbon and nitrogen sources were screened and optimized for the specific protease inhibitor activity. Three different growth models were checked for the best fit of the bacterial growth. A modified Gompertz model was selected as the best model for the growth of Oceanimonas sp. BPMS22 with the maximum specific growth rate of 0.165 hr-1 and doubling time of 4.2 hr. The production of PI takes place during the non-growing phase of the bacterial growth. A kinetic model for the production of PI during non-growing phase was used for studying various process parameters. From the model, the maximum trypsin inhibitor formation rate of 0.3802 IU per mg of biomass per hour was observed at 49.91 hr.

Analysis of bacterial FAMEs using gas chromatography - vacuum ultraviolet spectroscopy for the identification and discrimination of bacteria.

The identification of microorganisms is very important in different fields and alternative methods are necessary for a rapid and simple identification. The use of fatty acids for bacterial identification is gaining attention as phenotypic characteristics are reflective of the genotype and are more easily analyzed. In this work, gas chromatography-vacuum ultraviolet spectroscopy (GC-VUV) was used to determine bacteria fatty acid methyl esters (FAMEs), to identify and discriminate different environmental bacteria based on their fatty acid profile. Microorganisms were grown in agar and their fatty acids extracted, saponified, and esterified before analysis. Unique FAME profiles were obtained for each microorganism mainly composed of branched, cyclopropane, hydroxy, saturated, and unsaturated fatty acid methyl esters. S. maltophilia showed a higher diversity of fatty acids while Bacillus species showed higher complexity in terms of branched-chain FAMEs, with several iso and anteiso forms. 12 different bacteria genera and 15 species were successfully differentiated based on their fatty acid profiles after performing PCA and cluster analysis. Some difficult to differentiate species, such as Bacillus sp., which are genetically very similar, were differentiated with the developed method.

Comparative Genomics of the Aeromonadaceae Core Oligosaccharide Biosynthetic Regions.

Lipopolysaccharides (LPSs) are an integral part of the Gram-negative outer membrane, playing important organizational and structural roles and taking part in the bacterial infection process. In Aeromonas hydrophila, piscicola, and salmonicida, three different genomic regions taking part in the LPS core oligosaccharide (Core-OS) assembly have been identified, although the characterization of these clusters in most aeromonad species is still lacking. Here, we analyse the conservation of these LPS biosynthesis gene clusters in the all the 170 currently public Aeromonas genomes, including 30 different species, and characterise the structure of a putative common inner Core-OS in the Aeromonadaceae family. We describe three new genomic organizations for the inner Core-OS genomic regions, which were more evolutionary conserved than the outer Core-OS regions, which presented remarkable variability. We report how the degree of conservation of the genes from the inner and outer Core-OS may be indicative of the taxonomic relationship between Aeromonas species.

Biodegradation of saline phenolic wastewater in a biological contact oxidation reactor with immobilized cells of Oceanimonas sp.

OBJECTIVE: To develop a method to treat saline phenolic wastewater in a biological contact oxidation reactor (BCOR) with immobilized cells of a marine microorganism, Oceanimonas sp., isolated from seawater. RESULTS: Cells were immobilized on fibre carriers in the BCOR. Saline wastewater with phenol at 1.5 g/l and NaCl at 6 % (w/v) was treated. In continuous assays, 99 % removal of phenol was achieved and a kinetic model for the phenol degradation is presented based on Monod's equation. CONCLUSION: The BOCR system using immobilized cells of Oceanimonas efficiently treats saline phenolic wastewaters without having decrease the salinity of the wastewater.

Oceanisphaera aquimarina sp. nov., Isolated from Oil-Contaminated Sediment of Ocean Coastal Area from South Korea.

Strain S33(T) was isolated from oil-contaminated sediment of Tae-an coastal region of South Korea. Cells are aerobic, motile, Gram staining-negative, and coccoid shaped. Strain S33(T) grew optimally at the temperature of 25 °C (range of 4-40 °C), pH 6.0 (range of pH 6.0-10.0), and in the presence of 1 % (w/v) NaCl (range of 0-10 %). Ubiquinone-8 was the predominant respiratory quinone. C16:0, summed feature 3 (comprising C16:1ω7c/C16:1ω6c) and C18:1ω7c were the major fatty acids. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. Strain S33(T) showed the ability to degrade benzene, toluene, and ethylbenzene after 3 days incubation. 16S rRNA gene sequence analysis showed that the strain S33(T) was most closely related to Oceanisphaera sediminis TW92(T) (97.3 %), Oceanisphaera profunda SM1222(T) (97.2 %), and Oceanisphaera ostreae T-w6(T) (97.1 %) and <97 % with other members of the genus Oceanisphaera. The genomic DNA G+C mol% content of strain S33(T) was 51.0 mol%. Based on distinct phenotypic, genotypic, and phylogenetic analysis, strain S33(T) was proposed to represent a novel species in the genus Oceanisphaera as Oceanisphaera aquimarina sp. nov. (= KEMB 1002-058(T) = JCM 30 794(T)).

A novel heterotrophic nitrifying and aerobic denitrifying bacterium, Zobellella taiwanensis DN-7, can remove high-strength ammonium.

A novel heterotrophic bacterium capable of heterotrophic nitrification and aerobic denitrification was isolated from ammonium contaminated landfill leachate and physiochemical and phylogenetically identified as Zobellella taiwanensis DN-7. DN-7 converted nitrate, nitrate, and ammonium to N2 as the primary end product. Single factor experiments suggested that the optimal conditions for ammonium removal were trisodium citrate as carbon source, C/N ratio 8, pH 8.0-10.0, salinity less than 3 %, temperature 30 °C, and rotation speed more than 150 rpm. Specifically, DN-7 could remove 1000.0 and 2000.0 mg/L NH4 (+)-N completely within 96 and 216 h, with maximum removal rates of 19.6 and 17.3 mg L(-1) h(-1), respectively. These results demonstrated that DN-7 is a promising candidate for application of high-strength ammonium wastewater treatments.

A new electrochemically active bacterium phylogenetically related to Tolumonas osonensis and power performance in MFCs.

A facultative anaerobic bacterium (designated as P2-A-1) was isolated from microbial fuel cells (MFCs) inoculated with sludge from a sewage treatment plant. Based on 16S rDNA sequence analysis, the strain was identified as Tolumonas osonensis OCF 7(T) according to its biochemical, physiological and morphological characteristics. Through parameters optimization, the P2-A-1 MFC reached the maximum power density of 424 mW/m(2) in the substrate of 2g/L sodium acetate. Further, a facile bacteria treatment approach by chemically "perforating" pores and channels on bacterial membrane was developed to significantly improve the power density. And 1mM of EDTA-treated cell yielded the highest power density of 509.1 mW/m(2) because the membrane permeability of cell was enhanced by verification of coenzyme Q and fatty acid composition tests. It offers a novel facultative anaerobic Gram-positive bacterium that can utilize a wide variety of substrates for power production, making it highly valuable for application in MFCs.

Oceanisphaera ostreae sp. nov., isolated from seawater of an oyster farm, and emended description of the genus Oceanisphaera Romanenko et al. 2003.

A Gram-stain-negative, motile, non-spore-forming and short rod- or rod-shaped bacterial strain, T-w6(T), was isolated from seawater of an oyster farm in the South Sea, Korea. Strain T-w6(T) grew optimally at 25 °C and in the presence of 2% (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain T-w6(T) joined the cluster comprising Oceanisphaera species with a bootstrap resampling value of 90.8%, and this cluster joined the clade comprising members of the genera Oceanimonas and Zobellella with a bootstrap resampling value of 100%. Strain T-w6(T) exhibited 16S rRNA gene sequence similarity of 95.9 and 96.6% to the type strains of Oceanisphaera litoralis and Oceanisphaera donghaensis, respectively. Strain T-w6(T) and the type strains of Oceanisphaera litoralis and Oceanisphaera donghaensis had Q-8 as the predominant ubiquinone and iso-C(15:0) 2-OH and/or C(16:1)ω7c, C(18:1)ω7c and C(16:0) as the major fatty acids. The major polar lipids were phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content of strain T-w6(T) was 56.6 mol%. Mean DNA-DNA relatedness of strain T-w6(T) with Oceanisphaera litoralis DSM 15406(T) and Oceanisphaera donghaensis KCTC 12522(T) was 13 and 10%, respectively. Phenotypic properties of strain T-w6(T) demonstrated that this strain could be distinguished from the other Oceanisphaera species. On the basis of the data presented, strain T-w6(T) is considered to represent a novel species of the genus Oceanisphaera, for which the name Oceanisphaera ostreae sp. nov. is proposed; the type strain is T-w6(T) (=KCTC 23422(T) =CCUG 60525(T)). An emended description of the genus Oceanisphaera is also presented.

Tolumonas osonensis sp. nov., isolated from anoxic freshwater sediment, and emended description of the genus Tolumonas.

A polyphasic taxonomic study was performed on a strain of an unknown Gram-negative, non-motile, saccharolytic, facultatively anaerobic bacterium, strain OCF 7(T), isolated from anoxic freshwater sediment. The strain grew optimally at 22 °C and pH 7.5, and was able to grow under strictly anaerobic conditions. Major fermentation products from glucose metabolism were formate, acetate, ethanol and lactate. Comparative 16S rRNA gene sequence analysis indicated that strain OCF 7(T) was phylogenetically related to the type strain of Tolumonas auensis (97.2 % similarity) within the family Aeromonadaceae of the Gammaproteobacteria. However, OCF 7(T) did not produce toluene from phenylacetate, phenylalanine, phenoxyacetate, phenylsuccinate or phenylbutyrate in the presence of glucose. Phenol was not produced from tyrosine or phenoxyacetate in the presence of glucose. Dominant fatty acids of this micro-organism included C(16 : 0), C(18 : 1)ω7c and C(16 : 1)ω7c (and/or iso-C(15 : 0) 2-OH). Major polar lipids were phosphatidylglycerol and phosphatidylethanolamine, and the respiratory quinone was menaquinone MK-8. The genomic DNA G+C content of strain OCF 7(T) was 52.1 mol%. Based on phylogenetic and phenotypic evidence, strain OCF 7(T) should be classified as a representative of a novel species of Tolumonas, for which the name Tolumonas osonensis sp. nov. is proposed; the type strain is OCF 7(T) ( = DSM 22975(T) = ATCC BAA-1908(T)). An emended description of the genus Tolumonas is also given.

Zobellella aerophila sp. nov., isolated from seashore sand, and emended description of the genus Zobellella.

A strictly aerobic, nitrate-reducing, motile, rod-shaped member of the class Gammaproteobacteria, designated strain JC2671(T), was isolated from a seashore sand sample from Dokdo, Korea. The isolate reduced nitrate to nitrite, but not to nitrogen, and required NaCl for growth. 16S rRNA gene sequence analysis indicated that the isolate belonged to the genus Zobellella within the order Alteromonadales with sequence similarities of 96.0-97.6 % to strains of Zobellella species with validly published names. However, in DNA-DNA hybridization studies, a low genomic relatedness (43 %) between strain JC2671(T) and the type strain of Zobellella denitrificans indicated that the isolate represented a novel genomic species. The polar lipid pattern (phosphatidylethanolamine and phosphatidylglycerol), predominant cellular fatty acids [C(16 : 0), summed feature 3 (C(16 : 1)ω6c and/or C(16 : 1)ω7c) and summed feature 8 (C(18 : 1)ω6c and/or C(18 : 1)ω7c)] and the DNA G+C content (59 mol%) of the novel strain were consistent with its assignment to the genus Zobellella. In contrast, a number of phenotypic characteristics, namely a requirement of NaCl for growth, the inability to grow under facultatively anaerobic conditions, the absence of nitrite reduction and differences in carbohydrate utilization and enzymic activities, clearly distinguished the novel isolate from other species of the genus Zobellella. Data from this polyphasic study indicate that strain JC2671(T) represents a novel species in the genus Zobellella, for which the name Zobellella aerophila sp. nov. is proposed. The type strain is JC2671(T) ( = KACC 15081(T)  = JCM 17110(T)). The description of the genus Zobellella has been emended accordingly.

Poly(3-hydroxybutyrate) production from glycerol by Zobellella denitrificans MW1 via high-cell-density fed-batch fermentation and simplified solvent extraction.

Industrial production of biodegradable polyesters such as polyhydroxyalkanoates is hampered by high production costs, among which the costs for substrates and for downstream processing represent the main obstacles. Inexpensive fermentable raw materials such as crude glycerol, an abundant by-product of the biodiesel industry, have emerged to be promising carbon sources for industrial fermentations. In this study, Zobellella denitrificans MW1, a recently isolated bacterium, was used for the production of poly(3-hydroxybutyrate) (PHB) from glycerol as the sole carbon source. Pilot-scale fermentations (42-liter scale) were conducted to scale up the high PHB accumulation capability of this strain. By fed-batch cultivation, at first a relatively high cell density (29.9 +/- 1.3 g/liter) was obtained during only a short fermentation period (24 h). However, the PHB content was relatively low (31.0% +/- 4.2% [wt/wt]). Afterwards, much higher concentrations of PHB (up to 54.3 +/- 7.9 g/liter) and higher cell densities (up to 81.2 +/- 2.5 g/liter) were obtained by further fed-batch optimization in the presence of 20 g/liter NaCl, with optimized feeding of glycerol and ammonia to support both cell growth and polymer accumulation over a period of 50 h. A high specific growth rate (0.422/h) and a short doubling time (1.64 h) were attained. The maximum PHB content obtained was 66.9% +/- 7.6% of cell dry weight, and the maximum polymer productivity and substrate yield coefficient were 1.09 +/- 0.16 g/liter/h and 0.25 +/- 0.04 g PHB/g glycerol, respectively. Furthermore, a simple organic solvent extraction process was employed for PHB recovery during downstream processing: self-flotation of cell debris after extraction of PHB with chloroform allowed a convenient separation of a clear PHB-solvent solution from the cells. Maximum PHB recovery (85.0% +/- 0.10% [wt/wt]) was reached after 72 h of extraction with chloroform at 30 degrees C, with a polymer purity of 98.3% +/- 1.3%.

Identification of novel denitrifying bacteria Stenotrophomonas sp. ZZ15 and Oceanimonas sp. YC13 and application for removal of nitrate from industrial wastewater.

Two novel denitrifying bacteria were successfully isolated from industrial wastewater and soil samples. Using morphological, biochemical/biophysical and 16S rRNA gene analyses, these two bacteria were identified as Stenotrophomonas sp. ZZ15 and Oceanimonas sp. YC13, respectively. Both of these two bacteria showed efficient NO(3) (-)-N removing abilities under a semi-anaerobic condition without obvious accumulation of NO(2) (-)-N, N(2)O-N and NH(4) (+)-N. NO(3) (-)-N removal from paper mill wastewater was also successful by treatments with either a denitrifier or an immobilization method. Therefore, this study provides valuable denitrifying bacteria in biotreatment of industrial wastewater and other environmental pollution caused by NO(3) (-)/NO(2) (-).

Zobellella denitrificans gen. nov., sp. nov. and Zobellella taiwanensis sp. nov., denitrifying bacteria capable of fermentative metabolism.

Two denitrifying strains of heterotrophic, facultatively anaerobic bacteria, designated ZD1(T) and ZT1(T), were isolated from sediment samples collected from mangrove ecosystems in Taiwan. The isolates were Gram-negative. Cells grown in broth cultures were straight rods that were motile by means of a single polar flagellum. The isolates grew optimally in 1-3 % NaCl, but NaCl was not an absolute requirement for growth; only strain ZT1(T) grew in 13-14 % NaCl. Both isolates grew between 10 and 45 degrees C, with optimum growth at 30-35 degrees C. They were capable of anaerobic growth by denitrifying metabolism using nitrate or nitrous oxide as terminal electron acceptors or, alternatively, by fermenting glucose, sucrose or mannitol as substrates. C(18 : 1)omega7c was the most abundant fatty acid (32.6-35.7 %). The other major fatty acids included C(16 : 1)omega7c (27.5-29.4 %) and C(16 : 0) (20.1-22.0 %). The two isolates had 16S rRNA gene sequence similarity of 96.8 % and shared 94.1-96.8 % sequence similarity with the most closely related species, Oceanimonas doudoroffii, Oceanimonas baumannii, Oceanimonas smirnovii and Oceanisphaera litoralis. They could be distinguished from these species in that they were capable of fermentative metabolism, had relatively high DNA G+C contents (62.0-64.0 mol%) and contained C(18 : 1)omega7c instead of C(16 : 1)omega7c as the most abundant fatty acid. Characterization data accumulated in this study revealed that the two denitrifying isolates could be classified as representatives of two novel species in a new genus, Zobellella gen. nov., with Zobellella denitrificans sp. nov. (type strain ZD1(T) = BCRC 17493(T) = JCM 13380(T)) as the type species and Zobellella taiwanensis sp. nov. (type strain ZT1(T) = BCRC 17494(T) = JCM 13381(T)) as a second species.

Combined solvent and water activity stresses on turgor regulation and membrane adaptation in Oceanimonas baumannii ATCC 700832.

Oceanimonas baumannii ATCC 700832 is a Gram negative marine bacterium capable of utilising phenol as asole carbon source. The ability of the bacterium to tolerate low water activity when utilising either succinate or phenol as a substrate in minimal medium was studied. The membrane lipid and protein composition showed two discreet adaptive phases as salinity increased. Firstly, when NaCl concentration was increased from 0.15% (w/v), the minimum at which growth was observed, to 1% NaCl (w/v), the ratio of zwitterionic to anionic phospholipids in the membrane increased significantly. At the same time the ratio of saturated to unsaturated fatty acids and the total membrane protein decreased significantly. The second phase was observed when salinity was increased from 1% to 7% NaCl (w/v) as the ratio of zwitterionic to anionic phospholipids decreased and membrane protein increased. However, the ratio of saturated to unsaturated fatty acids was unaffected. Salinity also affected the tolerance of cultures to elevated levels of phenol. Cultures grown in 0.15% NaCl (w/v) could tolerate 12 mM phenol, whereas in the presence of 1% NaCl (w/v) cultures continued to grow in up to 20 mM phenol and in 7% NaCl (w/v) cultures 8 mM phenol could be tolerated. Changes to the composition of the membrane phospholipids and fatty acids were also observed when phenol concentrations were at the maximum that could be tolerated. Under such conditions the ratio of zwitterionic to anionic phospholipids decreased twofold compared to cultures utilising 4 mM phenol as the substrate, in all salinities except in 7% NaCl (w/v) cultures, where there was no significant effect. The ratio of saturated to unsaturated fatty acids increased significantly in all salinities compared to cultures grown with 4 mM phenol.

Identification of indole-3-acetic acid producing freshwater wetland rhizosphere bacteria associated with Juncus effusus L.

Production of indole-3-acetic acid (IAA), a key physiological feature of culturable, O2-tolerant bacteria associated with the freshwater macrophyte Juncus effusus L., was examined over a period of 2 years. Up to 74% of rhizobacteria identified and tested produced IAA. The number of indoleacetic acid producers decreased in winter. IAA was produced even when L-tryptophan, a precursor of IAA, was not added to the medium. Most of the IAA-producing strains were dominated by strains that were not identifiable to species level on the basis of API testing. Based on 16S rRNA gene sequencing and fatty acid analysis, it was found that IAA-producing rhizosphere bacteria associated with the freshwater wetland plant Juncus effusus L. are representatives of several families, including the Enterobacteriaceae, Pseudomonadaceae, Aeromonadaceae, Burkholderiaceae, and Bacillaceae. This study identifies numerous potentially important bacterial physiological groups of freshwater wetlands. Additionally, the study provides a baseline for monitoring and assessing the mutualistic relationships of wetland plants with rhizosphere bacteria in freshwater wetlands.