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.

Growth associated polyhydroxybutyrate production by the novel Zobellellae tiwanensis strain DD5 from banana peels under submerged fermentation.

In view of environmental pollution by fossil fuel-based plastics, it has become imperative to find out an alternative biodegradable plastic for sustainability. In this context, polyhydroxy butyrate (PHB) production was carried out by the Zobellella sp. DD5 using inexpensive banana peels as the carbon source. Under optimized condition, 1.13 g/L (47.3%) of PHB was produced by the bacteria in growth associated mechanism. The CO group of PHB was detected from the high intense absorption band (1719 cm-1) of FTIR spectroscopic analysis. NMR and GC-MS results are also identical with the chemical shift signal CH, CH2 and CH3 group of PHB. The PHB is crystalline in nature and degree of crystallinity (Xc) - 34.38%, melting temperature (Tm) - 169 °C, thermal decomposition temperature (Td) - 248 °C as detected by XRD and DTA respectively. Rough surface morphology of PHB film was validated by AFM and SEM imaging that improves biodegradability of the PHB. The Young's modulus, tensile strength and elongation at break depicted hard and brittle nature of PHB. Fluorescence-activated cell sorting (FACS) confirmed cytocompatibility of PHB at 500 µg/mL in human embryonic kidney (HEK-293) cell line. The non-cytotoxic PHB can be used for various biomedical and agricultural applications in future.

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.

Zobellella denitrificans strain MW1, a newly isolated bacterium suitable for poly(3-hydroxybutyrate) production from glycerol.

AIMS: To search for new bacteria for efficient production of polyhydroxyalkanoates (PHAs) from glycerol. METHODS AND RESULTS: Samples were taken from different environments in Germany and Egypt, and bacteria capable of growing in mineral salts medium with glycerol as sole carbon source were enriched. From a wastewater sediment sample in Egypt, a Gram-negative bacterium (strain MW1) was isolated that exhibited good growth and that accumulated considerable amounts of polyhydroxybutyrate (PHB) from glycerol and also from other carbon sources. The 16S rRNA gene sequence of this isolate exhibited 98.5% and 96.2% similarity to Zobellella denitrificans strain ZD1 and to Zobellella taiwanensis strain ZT1 respectively. The isolate was therefore affiliated as strain MW1 of Z. denitrificans. Strain MW1 grows optimally on glycerol at 41 degrees C and pH 7.3 and accumulated PHB up to 80.4% (w/w) of cell dry weight. PHB accumulation was growth-associated. Although it was not an absolute requirement, 20 g l(-1) sodium chloride enhanced both growth (5 g cell dry weight per litre) and PHB content (87%, w/w). Zobellella denitrificans strain MW1 is also capable to accumulate the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer if sodium propionate was used as cosubstrate in addition to glycerol. CONCLUSIONS: A new PHB-accumulating strain was isolated and identified. This strain is able to utilize glycerol for growth and PHB accumulation to high content especially in the presence of NaCl that will enable the utilization of waste glycerol from biodiesel industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on accumulation of PHA in a member of the new genus Zobellella. Furthermore, utilization of glycerol as the sole carbon source for fast growth and PHB biosynthesis, growth in the presence of NaCl and high PHB contents of the cells will make this newly isolated bacterium a potent candidate for industrial production of PHB from crude glycerol occurring as byproduct during biodiesel production.

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%.

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.