Production of polyhydroxyalkanoates using sewage and cheese whey.

Recently, polyhydroxyalkanoates (PHAs) have been produced using raw sewage in our laboratory; however, the production concentrations are low. Therefore, this study aimed to enhance PHA production by applying different strategies. PHA production was higher in sewage-containing medium than in mineral salt medium and was enhanced 22-fold after glucose supplementation. A relatively high degree of glucose consumption (83.6 ± 1.59 %) was also achieved. Bacteria incubated with cheese whey diluted with sewage showed higher PHA production than bacteria incubated with cheese whey diluted with distilled water did. The expression of the PHA synthase gene (phaC) was evaluated via real-time polymerase chain reaction using low- and high-carbon-containing sewage. Relatively higher phaC expression levels were observed in high-carbon-containing sewage but at lower nitrogen concentrations. The characteristics of the produced PHA were comparable to those of standard PHA. Therefore, this study revealed that the bacterium Bacillus sp. CYR1 can produce PHA from low- or high-carbon-containing wastewater.

Quantification of the Monomer Compositions of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and Poly(3-hydroxyvalerate) by Alkaline Hydrolysis and Using High-Performance Liquid Chromatography.

With the growing interest in bioplastics, there is an urgent need to develop rapid analysis methods linked to production technology development. This study focused on the production of a commercially non-available homopolymer, poly(3-hydroxyvalerate) (P(3HV)), and a commercially available copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), through fermentation using two different bacterial strains. The bacteria Chromobacterium violaceum and Bacillus sp. CYR1 were used to produce P(3HV) and P(3HB-co-3HV), respectively. The bacterium Bacillus sp. CYR1 produced 415 mg/L of P(3HB-co-3HV) when incubated with acetic acid and valeric acid as the carbon sources, whereas the bacterium C. violaceum produced 0.198 g of P(3HV)/g dry biomass when incubated with sodium valerate as the carbon source. Additionally, we developed a fast, simple, and inexpensive method to quantify P(3HV) and P(3HB-co-3HV) using high-performance liquid chromatography (HPLC). As the alkaline decomposition of P(3HB-co-3HV) releases 2-butenoic acid (2BE) and 2-pentenoic acid (2PE), we were able to determine the concentration using HPLC. Moreover, calibration curves were prepared using standard 2BE and 2PE, along with sample 2BE and 2PE produced by the alkaline decomposition of poly(3-hydroxybutyrate) and P(3HV), respectively. Finally, the HPLC results obtained by our new method were compared using gas chromatography (GC) analysis.

Review of the Developments of Bacterial Medium-Chain-Length Polyhydroxyalkanoates (mcl-PHAs).

Synthetic plastics derived from fossil fuels-such as polyethylene, polypropylene, polyvinyl chloride, and polystyrene-are non-degradable. A large amount of plastic waste enters landfills and pollutes the environment. Hence, there is an urgent need to produce biodegradable plastics such as polyhydroxyalkanoates (PHAs). PHAs have garnered increasing interest as replaceable materials to conventional plastics due to their broad applicability in various purposes such as food packaging, agriculture, tissue-engineering scaffolds, and drug delivery. Based on the chain length of 3-hydroxyalkanoate repeat units, there are three types PHAs, i.e., short-chain-length (scl-PHAs, 4 to 5 carbon atoms), medium-chain-length (mcl-PHAs, 6 to 14 carbon atoms), and long-chain-length (lcl-PHAs, more than 14 carbon atoms). Previous reviews discussed the recent developments in scl-PHAs, but there are limited reviews specifically focused on the developments of mcl-PHAs. Hence, this review focused on the mcl-PHA production, using various carbon (organic/inorganic) sources and at different operation modes (continuous, batch, fed-batch, and high-cell density). This review also focused on recent developments on extraction methods of mcl-PHAs (solvent, non-solvent, enzymatic, ultrasound); physical/thermal properties (Mw, Mn, PDI, Tm, Tg, and crystallinity); applications in various fields; and their production at pilot and industrial scales in Asia, Europe, North America, and South America.

Bioelectricity generation using long-term operated biocathode: RFLP based microbial diversity analysis.

In the present work, power generation and substrate removal efficiencies of long-term operated microbial fuel cells, containing abiotic cathodes and biocathodes, were evaluated for 220 days. Among the two microbial fuel cell (MFC) types, the one containing biocathode showed higher power density (54 mW/m2), current density (122 mA/m2) coulombic efficiency (33%), and substrate removal efficiency (94%) than the abiotic cathode containing MFC. Voltammetric analysis also witnessed higher and sustainable electron discharge for the MFC with biocathode, when compared with the abiotic cathode MFC. Over the tested period, both MFC have shown a cell voltage drop, after 150 and 165, days, for the MFC with biocathode and abiotic cathodes, respectively. Polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP) analysis identified 281 clones. Bacteria belonging to Acinetobacter, Acidovorax, Pseudomonas and Burkholderia were observed in the abiotic cathode MFC. Bacteria belonging to Geobacter, Cupriavidus and Acidobacteria were observed in the biocathode MFC. Almost similar types of archaea (Methanosarcinales, Methanolinea, Nitrososphaera and Methanomicrobiales) were observed in both MFCs.

3,3'-Thiodipropionic acid (TDP), a possible precursor for the synthesis of polythioesters: identification of TDP transport proteins in Variovorax paradoxus TBEA6.

3,3'-Thiodipropionic acid (TDP) is an antioxidant, which can be used as precursor carbon source to synthesize polythioesters. The bacterium Variovorax paradoxus TBEA6 strain can use TDP as a single source of carbon and energy. In the present study, experiments were carried out to identify proteins involved in the transport of TDP into the cells of strain TBEA6. Hence, eight putative tctC genes, which encode for the TctC proteins, were amplified from genomic DNA of TBEA6 strain using polymerase chain reaction and expressed in E. coli BL21 cells. Cells were grown in auto-induction medium, and protein purification was done using His Spin Trap affinity columns. Purity and molecular weight of each protein were confirmed by SDS-PAGE analysis. Protein-ligand interactions were monitored in thermoshift assays using the real-time PCR system. Two TctC proteins (locus tags VPARA-44430 and VPARA-01760) out of eight proteins showed a significant shift in their melting temperatures when they interact with the ligand (TDP or gluconate). The responsible genes were deleted in the genome of TBEA6 using suicide plasmid pJQ200mp18Tc, and single deletion mutants of the two candidate genes were subsequently generated. Finally, growth of the wild-type strain (TBEA6) and the two mutant strains (ΔVPARA-44430 and ΔVPARA-01760) were monitored and compared using TDP or gluconate as carbon sources. Wild type strains were successfully grown with TDP or gluconate. From the two mutant strains, one (ΔVPARA-44430) was unable to grow with TDP indicating that the tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.Key Points• Putative tctC genes from V. paradoxus TBEA6 were heterologously expressed in E. coli.• Protein-ligand interactions monitored in thermoshift assays using the real-time PCR.• tctC gene with locus tag VPARA-44430 is involved in the uptake of TDP.

Medium-Chain Fatty Acids (MCFA) Production Through Anaerobic Fermentation Using Clostridium kluyveri: Effect of Ethanol and Acetate.

Medium-chain fatty acids (MCFA) are saturated monocarboxylic acids and can be used as antimicrobials, corrosion inhibitors, precursors in biodiesel, and bioplastic production. In the present study, MCFA production was evaluated with acetate and ethanol using the bacteria Clostridium kluyveri. Effects of substrate, electron donor, and methane inhibitor on MCFA production were evaluated. Bacteria successfully converted the ethanol and acetate to butyrate (C4), caproate (C6), and caprylate (C8) by chain elongation process. The highest concentrations of butyrate (4.6 g/l), caproate (3.2 g/l), and caprylate (0.5 g/l) were obtained under methane inhibition conditions than other conditions. The productions of butyrate and caproate were 1.6 and 1.48 times higher under methane inhibition conditions, respectively. Results denoted that the bacteria C. kluyveri can be used for conversion of acetate and ethanol into useful products like butyrate and caproate.

Bio-Augmentation of Cupriavidus sp. CY-1 into 2,4-D Contaminated Soil: Microbial Community Analysis by Culture Dependent and Independent Techniques.

In the present study, a 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterial strain CY-1 was isolated from the forest soil. Based on physiological, biochemical and 16S rRNA gene sequence analysis it was identified as Cupriavidus sp. CY-1. Further 2,4-D degradation experiments at different concentrations (200 to 800 mg l(-1)) were carried out using CY-1. Effect of NaCl and KNO3 on 2,4-D degradation was also evaluated. Degradation of 2,4-D and the metabolites produced during degradation process were analyzed using high pressure liquid chromatography (HPLC) and GC-MS respectively. The amount of chloride ions produced during the 2,4-D degradation were analyzed by Ion chromatography (IC) and it is stoichiometric with 2,4-D dechlorination. Furthermore two different types of soils collected from two different sources were used for 2,4-D degradation studies. The isolated strain CY-1 was bio-augmented into 2,4-D contaminated soils to analyze its degradation ability. Culture independent methods like denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP), and culture dependent methods like colony forming units (CFU) and most probable number (MPN) were used to analyze the survivability of strain CY-1 in contaminated soil. Results of T-RFLP were coincident with the DGGE analysis. From the DGGE, T-RFLP, MPN and HPLC results it was concluded that strain CY-1 effectively degraded 2,4-D without disturbing the ecosystem of soil indigenous microorganisms.

Acidogenic spent wash valorization through polyhydroxyalkanoate (PHA) synthesis coupled with fermentative biohydrogen production.

The production of polyhydroxyalkanoates (PHAs) by Bacillus tequilensis biocatalyst using spent wash effluents as substrate was evaluated to increase the versatility of the existing PHA production process and reduce production cost. In this study, spent wash was used as a substrate for biohydrogen (H2) production and the resulting acidogenic effluents were subsequently employed as substrate for PHA production. Maximum H2 production of 39.8L and maximum PHA accumulation of 40% dry cell weight was attained. Good substrate removal associated with decrement in acidification (53% to 15%) indicates that the VFA generated were effectively utilized for PHA production. The PHA composition showed presence of copolymer [P (3HB-co-3HV)] with varying contents of hydroxybutyrate and hydroxyvalerate. The results obtained suggest that the use of spent wash effluents as substrate can considerably reduce the production cost of PHA with simultaneous waste valorization. PHA synthesis with B. tequilensis and spent wash effluents is reported for the first time.

Microaerophilic microenvironment at biocathode enhances electrogenesis with simultaneous synthesis of polyhydroxyalkanoates (PHA) in bioelectrochemical system (BES).

Microaerophilic microenvironment at biocathode was evaluated for electrogenesis along with the polyhydroxyalkanoates (PHA) accumulation in bio-electrochemical system (BES). The electrogenic activity (512 mV; 15.2 mW/m(2)) was extended for longer periods (144 h) which might be attributed to the lowering of losses due to the controlled microbial metabolism. Growth limiting stress at cathode due to lower oxygen levels and its effective utilization by the protons and electrons coming from anode, might have diverted the microbial metabolism towards PHA synthesis instead of oxidation. PHA accumulation (19% of dry cell weight (DCW)) was observed with higher hydroxy butyrate (HB) (89%) concentration at 48 th h in the cathodic biocatalyst and was re-utilized by the end of experiment. Bio-electro kinetics studied through voltammetry and Tafel analysis further supported the observed electrogenesis in microaerophilic reduction microenvironment, in terms of redox catalytic currents, Tafel slopes, exchange current densities and polarization resistance.

Influence of aerobic and anoxic microenvironments on polyhydroxyalkanoates (PHA) production from food waste and acidogenic effluents using aerobic consortia.

The functional role of aerobic and anoxic microenvironments on polyhydroxyalkanoates (PHA) production using food waste (UFW) and effluents from acidogenic biohydrogen production process (FFW) were studied employing aerobic mixed culture as biocatalyst. Anoxic microenvironment documented higher PHA production, while aerobic microenvironment showed higher substrate degradation. FFW showed higher PHA accumulation (39.6%) than UFW (35.6%) due to ready availability of precursors (fatty acids). Higher fraction of poly-3-hydroxy butyrate (PHB) was observed compared to poly-3-hydroxy valerate (PHV) in the accumulated PHA in the form of co-polymer [P3(HB-co-HV)]. Dehydrogenase, phosphatase and protease enzymatic activities were monitored during process operation. Integration with fermentative biohydrogen production yielded additional substrate degradation under both aerobic (78%) and anoxic (72%) microenvironments apart from PHA production. Microbial community analysis documented the presence of aerobic and facultative organisms capable of producing PHA. Integration strategy showed feasibility of producing hydrogen along with PHA by consuming fatty acids generated during acidogenic process in association with increased treatment efficiency.

Endocrine disruptive estrogens role in electron transfer: bio-electrochemical remediation with microbial mediated electrogenesis.

Bioremediation of selected endocrine disrupting compounds (EDCs)/estrogens viz. estriol (E3) and ethynylestradiol (EE2) was evaluated in bio-electrochemical treatment (BET) system with simultaneous power generation. Estrogens supplementation along with wastewater documented enhanced electrogenic activity indicating their function in electron transfer between biocatalyst and anode as electron shuttler. EE2 addition showed more positive impact on the electrogenic activity compared to E3 supplementation. Higher estrogen concentration showed inhibitory effect on the BET performance. Poising potential during start up phase showed a marginal influence on the power output. The electrons generated during substrate degradation might have been utilized for the EDCs break down. Fuel cell behavior and anodic oxidation potential supported the observed electrogenic activity with the function of estrogens removal. Voltammetric profiles, dehydrogenase and phosphatase enzyme activities were also found to be in agreement with the power generation, electron discharge and estrogens removal.

Aerobic remediation of petroleum sludge through soil supplementation: microbial community analysis.

The effect of soil concentration on the aerobic degradation of real-field petroleum sludge was studied in slurry phase reactor. Total petroleum hydrocarbons (TPH) and polycyclic aromatic hydrocarbons (PAHs) showed effective removal but found to depend on the soil concentration. Aromatic fraction (48.12%) documented effective degradation compared to aliphatics (47.31%), NSO (28.69%) and asphaltenes (26.66%). PAHs profile showed efficient degradation of twelve individual aromatic compounds where lower ring compounds showed relatively higher degradation efficiency compared to the higher ring compounds. The redox behaviour and dehydrogenase activity showed a linear increment with the degradation pattern. Microbial community composition and changes during bioremediation were studied using denaturing gradient gel electrophoresis (DGGE). Among the 12 organisms identified, Proteobacteria was found to be dominant representing 50% of the total population (25% of γ-proteobacteria; 16.6% of ß-proteobacteria; 8.3% of α-proteobacteria), while 33.3% were of uncultured bacteria and 16.6% were of firmicutes.

Influence of carbohydrates and proteins concentration on fermentative hydrogen production using canteen based waste under acidophilic microenvironment.

Functional role of biomolecules viz., carbohydrates and proteins on acidogenic biohydrogen (H(2)) production was studied through the treatment of canteen based composite food waste. The performance was evaluated in an anaerobic sequencing batch reactor (AnSBR) at pH 6 with five variable organic loading conditions (OLR1, 0.854; OLR2, 1.69; OLR3, 3.38; OLR4, 6.54 and OLR5, 9.85kgCOD/m(3)-day). Experimental data depicted the feasibility of H(2) production from the stabilization of food waste and was found to depend on the substrate load. Among the five loading conditions studied, OLR4 documented maximum H(2) production (69.95mmol), while higher substrate degradation (3.99kgCOD/m(3)-day) was observed with OLR5. Specific hydrogen yield (SHY) vary with the removal of different biomolecules and was found to decrease with increase in the OLR. Maximum SHY was observed with hexose removal at OLR1 (139.24mol/kg Hexose(R) at 24h), followed by pentoses (OLR1, 108.26mol/kg Pentose(R) at 48h), proteins (OLR1, 109.71mol/kg Protein(R) at 48h) and total carbohydrates (OLR1, 58.31mol/kg CHO(R) at 24h). Proteins present in wastewater helped to maintain the buffering capacity but also enhanced the H(2) production by supplying readily available organic nitrogen to the consortia. Along with carbohydrates and proteins, total solids also registered good removal.

Fermentative effluents from hydrogen producing bioreactor as substrate for poly(beta-OH) butyrate production with simultaneous treatment: an integrated approach.

The feasibility of bioplastics production as poly(beta-OH)butyrate (PHB) was studied with individual volatile fatty acids (VFA) and acid-rich effluents from a biohydrogen producing reactor (HBR) as primary substrates employing aerobic consortia as biocatalyst under anoxic microenvironment. Butyrate as substrate showed higher PHB productivity (33%) followed by acetate (32%), acids mixture (16%) and propionate (11%) among synthetic VFA studied. Acid-rich effluents from HBR yielded higher PHB productivity (25%) especially at lower substrate loading conditions. Decrement observed in PHB production (from 25% to 6%) with increase in substrate load might be due to the presence of high concentration of residual carbon along with acid metabolites. Neutral redox operation showed effective PHB production compared to acidic and basic conditions due to associated higher metabolic activity of the biocatalyst. The integrated approach helped to treat additional COD from acid-rich HBR effluents apart from by-product recovery.

Phosphatase and dehydrogenase activities in anodic chamber of single chamber microbial fuel cell (MFC) at variable substrate loading conditions.

Performance of microbial fuel cell (MFC) was evaluated with the function of phosphatase and dehydrogenase activities at increasing organic loading rate (OLR) (0.195kg chemical oxygen demand (COD)/m(3)-day; 0.458kg COD/m(3)-day; 0.911kg COD/m(3)-day; 1.589kg COD/m(3)-day). Variation in enzyme activities along with power generation and substrate degradation was observed during MFC operation with the function of organic loading rate (OLR). Phosphatase activity showed a decreasing trend with time from 24 to 36th hour depending on OLR which is a good sign of substrate utilization. Dehydrogenase activity was observed to be high at the 12th hour irrespective of the OLR. However, the activity was increased with increasing OLR. Higher dehydrogenase activity was observed at 1.589kg COD/m(3)-day representing the possibility of higher redox reactions. Higher power output was recorded at the 12th hour with 53.58mW/m(2) (0.195kg COD/m(3)-day) and 24th hour with 60.29mW/m(2) (0.458kg COD/m(3)-day) and 76.17mW/m(2) (0.911kg COD/m(3)-day). At higher OLR studied (1.589kg COD/m(3)-day), maximum power generation (49.86mW/m(2)) was observed at 12th hour indicating decreased performance. Electron discharge and recovery properties observed during MFC operation were supporting higher performance at 0.911kg COD/m(3)-day. Increase in OLR showed improvement in substrate degradation [OLR1, 56.32% (0.11kg COD/m(3)-day); OLR2, 56.42% (0.26kg COD/m(3)-day); OLR3, 59.53% (0.54kg COD/m(3)-day); OLR4, 64.40% (1.78kg COD/m(3)-day)].