Integration of poly-3-(hydroxybutyrate-co-hydroxyvalerate) production by Haloferax mediterranei through utilization of stillage from rice-based ethanol manufacture in India and its techno-economic analysis.

Haloferax mediterranei has potential for economical industrial-scale production of polyhydroxyalkanoate (PHA) as it can utilize cheap carbon sources, has capacity for nonsterile cultivation and allows simple product recovery. Molasses-based Indian distilleries are converting themselves to cereal-based distilleries. Waste stillage (14 l) of rice-based ethanol industry was used for the production of PHA by H. mediterranei in the simple plug-flow reactor configuration of the activated sludge process. Cells utilized stillage and accumulated 63 ± 3 % PHA of dry cell weight and produced 13.12 ± 0.05 g PHA/l. The product yield coefficient was 0.27 while 0.14 g/l h volumetric productivity was reached. Simultaneous lowering of 5-day biochemical oxygen demand and chemical oxygen demand values of stillage by 82 % was attained. The biopolymer was characterized as poly-3-(hydroxybutyrate-co-17.9 mol%-hydroxyvalerate) (PHBV). Directional properties of decanoic acid jointly with temperature-dependent water solubility in decanoic acid were employed for two-step desalination of the spent stillage medium in a cylindrical baffled-tank with an immersed heater and a stirrer holding axial and radial impellers. 99.3 % of the medium salts were recovered and re-used for PHA production. The cost of PHBV was estimated as US$2.05/kg when the annual production was simulated as 1890 tons. Desalination contributed maximally to the overall cost. Technology and cost-analysis demonstrate that PHA production integrated with ethanol manufacture is feasible in India. This study could be the basis for construction of a pilot plant.

Production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) by Haloferax mediterranei using rice-based ethanol stillage with simultaneous recovery and re-use of medium salts.

Haloferax mediterranei holds promise for competitive industrial-scale production of polyhydroxyalkanoate (PHA) because cheap carbon sources can be used thus lowering production costs. Although high salt concentration in production medium permits a non-sterile, low-cost process, salt disposal after process completion is a problem as current environmental standards do not allow total dissolved solids (TDS) above 2000 mg/l in discharge water. As the first objective of this work, the waste product of rice-based ethanol industry, stillage, was used for the production of PHA by H. mediterranei in shake flasks. Utilization of raw stillage led to 71 ± 2% (of dry cell weight) PHA accumulation and 16.42 ± 0.02 g/l PHA production. The product yield coefficient was 0.35 while 0.17 g/l h volumetric productivity was attained. Simultaneous reduction of BOD5 and COD values of stillage by 83% was accomplished. The PHA was isolated by osmotic lysis of cells, purification by sodium dodecyl sulfate and organic solvents. The biopolymer was identified as poly-3-(hydroxybutyrate-co-15.4 mol%-hydroxyvalerate) (PHBV). This first report on utilization of rice-based ethanol stillage for PHBV production by H. mediterranei is currently the most cost effective. As the second objective, directional properties of decanoic acid together with temperature dependence of water solubility in decanoic acid were applied for two-stage desalination of the spent stillage medium. We report for the first time, recovery and re-use of 96% of the medium salts for PHA production thus removing the major bottleneck in the potential application of H. mediterranei for industrial production of PHBV. Final discharge water had TDS content of 670 mg/l.

Enhanced biotransformation of fluoranthene by intertidally derived Cunninghamella elegans under biofilm-based and niche-mimicking conditions.

The aims of the investigation were to ascertain if surface attachment of Cunninghamella elegans and niche intertidal conditions provided in a bioreactor influenced biotransformation of fluoranthene by C. elegans. A newly designed polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) holding eight equidistantly spaced rectangular strips mounted radially on a circular disc allowed comparison of fluoranthene biotransformation between CCFs with a hydrophobic surface (PMMA-CCF) and a hydrophilic glass surface (GS-CCF) and a 500-ml Erlenmeyer flask (EF). Fluoranthene biotransformation was higher by 22-fold, biofilm growth was higher by 3-fold, and cytochrome P450 gene expression was higher by 2.1-fold when C. elegans was cultivated with 2% inoculum as biofilm culture in PMMA-CCF compared to planktonic culture in EF. Biotransformation was enhanced by 7-fold with 10% inoculum. The temporal pattern of biofilm progression based on three-channel fluorescence detection by confocal laser scanning microscopy demonstrated well-developed, stable biofilm with greater colocalization of fluoranthene within extracellular polymeric substances and filaments of the biofilm grown on PMMA in contrast to a glass surface. A bioreactor with discs rotating at 2 revolutions per day affording 6-hourly emersion and immersion mimicked the niche intertidal habitat of C. elegans and supported biofilm formation and transformation of fluoranthene. The amount of transformed metabolite was 3.5-fold, biofilm growth was 3-fold, and cytochrome P450 gene expression was 1.9-fold higher in the process mimicking the intertidal conditions than in a submerged process without disc rotation. In the CCF and reactor, where biofilm formation was comparatively greater, higher concentration of exopolysaccharides allowed increased mobilization of fluoranthene within the biofilm with consequential higher gene expression leading to enhanced volumetric productivity.

Production enhancement and characterization of the polyhydroxyalkanoate produced by Natrinema ajinwuensis (as synonym) ≡ Natrinema altunense strain RM-G10.

Application of halophiles can decrease the cost of polyhydroxyalkanoate (PHA) production or bioplastic which are an alternative to the petroleum-derived plastic. Extremely halophilic archaeon, Natrinema ajinwuensis RM-G10 accumulated 61.02±0.68% PHA of its cell dry mass at 72h in repeated batch cultures yielding 0.210±0.001gL-1h-1 volumetric productivity after selection of the best cultivation conditions. Transmission electron microscopy showed the presence of PHA granules inside the archaeal cells. Characterization by gas chromatographic analysis, gas chromatographic- mass spectrophotometric analysis, thermogravimetric analysis, differential scanning calorimetric analysis, X-ray diffraction analysis, Fourier transform infra red spectroscopy and nuclear magnetic resonance spectroscopy revealed the polymer to be poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with 13.93mol% 3-hydroxyvalerate content and having 35.45% crystallinity, -12.3°C glass transition temperature, 143°C and 157.5°C melting temperatures and 284°C degradation temperature. This is the first report on production enhancement (on a small scale) and characterization of the polyhydroxyalkanoate produced by Natrinema ajinwuensis (as synonym) ≡ Natrinema altunense strain RM-G10 and the Natrinema genus in general.

Alteration of Zeta potential and membrane permeability in bacteria: a study with cationic agents.

In the present study, we have tried to establish the correlation between changes in Zeta potential with that of cell surface permeability using bacteria (Escherichia coli and Staphylococcus aureus). An effort has been made to establish Zeta potential as a possible marker for the assessment of membrane damage, with a scope for predicting alteration of cell viability. Cationic agents like, cetyl trimethyl ammonium bromide and polymyxin B were used for inducing alteration of Zeta potential, and the changes occurring in the membrane permeability were studied. In addition, assessment of poly-dispersity index (PDI), cell viability along with confocal microscopic analysis were performed. Based on our results, it can be suggested that alteration of Zeta potential may be correlated to the enhancement of membrane permeability and PDI, and it was observed that beyond a critical point, it leads to cell death (both Gram-positive and Gram-negative bacteria). The present findings can not only be used for studying membrane active molecules but also for understanding the surface potential versus permeability relationship.

Utilization of vinasse for production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) by Haloferax mediterranei.

Vinasse, a highly polluting waste of the ethanol industry was utilized for the production of polyhydroxyalkanoate (PHA) by the extremely halophilic archaeon, Haloferax mediterranei in shake-flasks. Following pre-treatment through adsorption on activated carbon, 25%-50% (v/v) pre-treated vinasse was utilized leading to 70% maximum accumulation of PHA. Maximum PHA concentration of 19.7 g/l, product yield coefficient (based on total carbohydrates) of 0.87 and 0.21 g/l h volumetric productivity were achieved. Concomitant lowering of BOD5 of pre-treated vinasse by at least 78% and COD by at least 80% was attained at the end of this process. The PHA was recovered by osmotic lysis of the cells and purification by sodium hypochlorite and organic solvents. Through UV-vis spectroscopy, gas chromatography, differential scanning calorimetry and nuclear magnetic resonance spectroscopy, the PHA was identified as poly-3-(hydroxybutyrate-co-hydroxyvalerate). The 3-hydroxyvalerate content was 12.36 mol % (utilizing 25% pre-treated vinasse) and 14.09 mol % (utilizing 50% pre-treated vinasse). High salt concentration in the medium allowed this process without sterile conditions and thus reduction in costs of sterilization can be envisaged. Activated charcoal pre-treatment of vinasse is economical than competing processes such as ultrafiltration of whey, extrusion and enzymatic treatment of rice and corn starch. Without impacting sugar prices, this process can easily be integrated into a distillery that has fermentation equipment and trained personnel. High PHA content, productivity, zero-cost carbon source, low-cost isolation of a high-purity product and potential integration into ethanol manufacturing unit with concomitant wastewater treatment should merit further development of this process to higher scales.

Utilization of vinasse for the production of polyhydroxybutyrate by Haloarcula marismortui.

Vinasse, a recalcitrant waste of the ethanol industry was employed for the production of polyhydroxyalkanoate (PHA) by the extremely halophilic archaeon, Haloarcula marismortui in shake flasks. The PHA was recovered by osmotic lysis of the cells and subsequent purification by sodium hypochlorite and organic solvents. Through UV-vis spectroscopy, differential scanning calorimetry, Fourier transform infrared, and nuclear magnetic resonance spectroscopy, the PHA was found to have characteristics very similar to that of the standard polyhydroxybutyrate (PHB) from Sigma. Inhibitory effect of polyphenols contained in vinasse was assessed by a quick and reliable cup-plate agar-diffusion method. Raw vinasse (10%) was utilized leading to accumulation of 23% PHA (of cell dry weight) and following an efficacious pre-treatment process through adsorption on activated carbon, 100% pre-treated vinasse could be utilized leading to 30% accumulation of PHB by H. marismortui. Maximum specific growth rate, specific production rate, and volumetric productivity attained using 10% raw vinasse were comparable to that obtained using a previously reported nutrient deficient medium (NDM), while the values with 100% pre-treated vinasse were higher than that determined using NDM medium. This is the first report of polyhydroxybutyrate production by a halophilic microorganism utilizing vinasse.

Inhibition of membrane-associated methyltransferases by a cholesterol-based metal chelator.

We have designed, synthesized, and characterized a metal chelating compound that is based on the structure of cholesterol and contains the high affinity metal chelating group, lysine nitrilotriacetic acid (Lys-NTA). Using the enzyme isoprenylcysteine carboxylmethyltransferase (Icmt) from yeast as a model integral membrane metalloenzyme, we find that this agent potently inhibits Icmt activity with an IC(50) value between 35 and 75 microM, which is at least 40 times more potent than the best known Icmt metal chelating inhibitor, Zincon. We propose that the rigid hydrophobic cholesterol moiety promotes partitioning into the membrane, enabling the metal-binding NTA group(s) to inactivate the enzyme by metal chelation. Because this compound is based on a naturally occurring membrane lipid and appears to chelate metals buried deeply within water insoluble environments, this agent may also be useful as a general tool for identifying previously unappreciated metal dependencies of other classes of membrane proteins.