Polyhydroxybutyrate Accumulation in Bacillus megaterium and Optimization of Process Parameters Using Response Surface Methodology

The accumulation of polyhydroxybutyrate of Bacillus megaterium is growth associated and significantly dependent on carbon sources. In the present investigation B. megaterium strain isolated from soil was studied for PHB production in fructose minimal media. The PHB production was found to be growth associated. The polymer production by the strain was found to vary from 24 to 48 % content (w/w) of the dry cell weight. Box Bohn design was used to study the interactive effect of four variables on cell growth and PHB production. The optimized medium conditions with the constrain to maximize cell growth and PHB content were glucose 4.32 g/L, Mannitol 4.52 g/L and Na succinate 3.45 g/L and PHB yield 1.38 g/L amounting to 49 % of dry cell weight which is more than 1.8 folds the basal medium. The polymer production by the strain was found to vary from 12.18 to 57.2 % content (w/w) of the dry cell weight.     

Aerobic thermophilic bacteria enhance biogas production

The enhancing effect of aerobic thermophilic (AT) bacteria on the production of biogas from anaerobically digested sewage sludge (methanogenic sludge) was investigated. Sewage sludge (5%, w/w) was incubated at 65°C with shaking for a few months to prepare the AT seed sludge. AT sludge was prepared by incubation of the AT seed sludge (5%, v/v) and sewage sludge (5%, w/w) at 65°C with shaking. The addition of this AT sludge (1.2% ± 0.5% of total volatile solids) to methanogenic sludge enhanced the production of biogas. The optimum volume of the addition and the pretreatment temperature of the AT sludge for optimum biogas production were 5% (v/v) and 65°C. Batch-fed anaerobic digestion was covered with the addition of various AT sludges. The AT sludge prepared with the AT seed sludge improved the biogas production by 2.2 times relative to that from the sewage sludge addition. The addition of sludge without AT seed sludge weakly enhanced biogas production. An aerobic thermophilic bacterium (strain AT1) was isolated from the AT seed sludge. Strain AT1 grew well in a synthetic medium. The production of biogas from the anaerobic digestion of sewage sludge was improved by the addition of 5% (v/v) AT1 bacterial culture compared with that from the sewage sludge addition. The addition of AT1 culture reduced the volatile solids by 21%, which was higher than the 12.6% achieved with the sewage sludge addition. The AT1 bacterial culture enhanced the biogas production more than the AT seed sludge. The phylogenetic analysis of the 16S rRNA gene revealed that strain AT1 is closely related to Geobacillus thermodenitrificans (100% sequence similarity). The improvement in the production of biogas with the AT sludge could be caused by thermophilic bacterial activity in the AT sludge.     

Partial characterization of the exopolysaccharide from Oscillatoria trichoides Szafer and the role of released polysaccharide in sequestration of Cr6+

The structural investigation and the chromium adsorptive potential of an exopolysaccharide (EPS) released during the growth of an indigenous cyanobacterium, Oscillatoria trichoides Szafer, were investigated in a laboratory‐scale study. The results showed that, of the total EPS produced, 410.53 milligrams/gram (mg g^?1) were released polysaccharides (RPS) and 11.36 mg g^?1 were capsular polysaccharides (CPS). The sorption of hexavalent chromium (Cr⁶⁺) by the RPS achieved a maximum amount of metal removal (q_max) value of 76.92 mg g^?1 of polysaccharide dry weight. The highest coefficient of determination (0.9742) for the Langmuir adsorption model indicates best fitness of the model in explaining the sorption as a unilayer process. Equilibrium studies indicated that 30 to 40 milligrams per liter initial chromium concentration and a pH of 2 were optimal for biosorption of chromium by the RPS. Scanning electron microscopy with energy‐dispersive X‐ray spectroscopy analysis of Cr⁶⁺‐treated RPS showed the presence of 3.76% bound chromium. Compositional analysis of the EPS showed the presence of carbohydrates, proteins, pyruvic acid, and hexosamines. High‐performance liquid chromatography analysis demonstrated the presence of hexoses, as neutral sugars and glucuronic acid as an acidic sugar. The presence of carboxylic groups was also detected by infrared spectroscopy. The presence of these chemical constituents may serve as binding sites for the metal ions; therefore, the RPS of this species appears to be a promising biosorbent for Cr⁶⁺.     

Utilization of Broken Rice for the Production of Poly(3-hydroxybutyrate)

The feasibility of utilizing non edible rice (broken rice) for production of fine materials such as poly(3-hydroxybutyrate) (PHB) was considered as one of the alternative ways of keeping the environment clean for sustainable development. Thus, production of PHB from broken rice by simultaneous saccharification and fermentation (SSF) was investigated. During the SSF process, the rice (15% w/v) material was hydrolyzed to glucose, which was utilized by Cupriavidus necator for growth and production of PHB. The PHB content reached 38% at 58 h fermentation. The PHB had weight average molar mass (Mw) and polydipersity index of 3.82 × 10⁵ (g/mol) and 4.15, respectively. Differential calorimetric scan of the PHB showed a melting temperature (Tm) of 176 °C. Given that the PHB was a homopolymer (which consisted of (R)-3-hydroxybutyric acid monomers), it was thought that broken rice could be a raw material for production of both PHB and (R)-3-hydroxybutyric acid. This SSF process would not only help in the utilization of broken rice or non edible rice, but would also serve as a model for utilization of other raw materials that contain starch for production of PHB.     

Archaeal Poly (3-hydroxybutyrate) Polymer Production from Glycerol: Optimization by Taguchi Methodology

In this study the possibility of poly (3-hydroxybutyrate) production from glycerol was investigated and optimized by Halorcula sp. IRU1, a novel archaea isolated from Urmia lake, Iran in batch experiments. Using Taguchi methodology, three important independent parameters (glycerol, yeast extract and KH₂PO₄) were evaluated for their individual and interactive effects on poly (3-hydroxybutyrate) production. It was shown that the glycerol concentration was the most significant factor affecting the yield of poly (3-hydroxybutyrate). The optimum factor levels were a glycerol concentration of 8% (v/v), yeast extract 0.8% (w/v) and KH₂PO₄ 0.002% (w/v). The predicted value obtained for poly (3-hydroxybutyrate) production under these conditions was about 81.87%. We can conclude that Haloarcula sp. IRU1 has a high potential for synthesis of poly (3-hydroxybutyrate) from glycerol.     

Effects of Poly(Ethylene Glycol) on the Production of Poly(β-Hydroxybutyrate) by Azotobacter vinelandii UWD

Azotobacter vinelandii UWD, ATCC 53799, an engineered strain derived from Azotobacter vinelandii UW was used in the poly(ethylene glycol) (PEG)-modulated synthesis of poly(-hydroxybutyrate) (PHB). To the best of our knowledge, this is the first report on modulating the production of PHB by amending the fermentation broth with PEG using A. vinelandii UWD. It was determined that A. vinelandii UWD is prone to back-mutation to the parent strain; hence fermentation experiments require the use of the antibiotic rifampicin. Diethylene glycol (DEG) and PEGs with molecular weights of 400, 2000, and 3400 Da and pentaerythritol ethoxylate (PEE) were used in the modulated fermentation experiments in a concentration of 2% (w/v). The molecular weight of the resulting polymers was reduced by up to 78%. No impact on the productivity of the strain was observed. Spectroscopic evidence showed that PEG-modulated synthesis resulted in the covalent attachment of the ethylene glycol moiety only when a small molecule, DEG, was used. PEGs had the same effects on the polymer formation in terms of molecular weight reduction as DEG, but no spectroscopic evidence was found for the formation of a covalent linkage between PHB and higher molecular weight PEGs.     

Recovery of yttrium from fluorescent powder of cathode ray tube,CRT: Zn removal by sulphide precipitation

This work is focused on the recovery of yttrium and zinc from fluorescent powder of cathode ray tube (CRT). Metals are extracted by sulphuric acid in the presence of hydrogen peroxide. Leaching tests are carried out according to a 2² full factorial plan and the highest extraction yields for yttrium and zinc equal to 100% are observed under the following conditions: 3 M of sulphuric acid, 10% v/v of H₂O₂ concentrated solution at 30% v/v, 10% w/w pulp density, 70 °C and 3 h of reaction.Two series of precipitation tests for zinc are carried out: a 2² full factorial design and a completely randomized factorial design. In these series the factors investigated are pH of solution during the precipitation and the amount of sodium sulphide added to precipitate zinc sulphide. The data of these tests are used to describe two empirical mathematical models for zinc and yttrium precipitation yields by regression analysis. The highest precipitation yields for zinc are obtained under the following conditions: pH equal to 2–2.5% and 10–12% v/v of Na₂S concentrated solution at 10% w/v. In these conditions the coprecipitation of yttrium is of 15–20%.Finally further yttrium precipitation experiments by oxalic acid on the residual solutions, after removing of zinc, show that yttrium could be recovered and calcined to obtain the final product as yttrium oxide. The achieved results allow to propose a CRT recycling process based on leaching of fluorescent powder from cathode ray tube and recovery of yttrium oxide after removing of zinc by precipitation. The final recovery of yttrium is 75–80%.     

Application of recombinant gene technology for production of polyhydroxyalkanoic acids: Biosynthesis of poly(4-hydroxybutyric acid) homopolyester

Screening of a large number of bacteria revealed several strains, which utilize 1,4-butanediol and/or 4-hydroxybutyric acid (4HB) as a carbon source for growth and for synthesis of polyhydroxyalkanoic acids (PHA) containing 4HB as one constituent among others (mostly 3-hydroxybutyric acid). However, none of the wild-type strains investigated in this study was able to produce a homopolyester consisting solely of 4HB. Only several poly(3-hydroxybutyric acid)-leaky mutants ofAlcaligenes eutrophus strain JMP222 synthesized poly(4HB) homopolyester, which amounted to approximately 10% (w/w) of the cellular dry matter. If the PHA synthase structural gene ofA. eutrophus strain H16 was expressed in these mutants, the amount of poly(4HB) was increased to approximately 30% (w/w). The occurrence of poly(4HB) was demonstrated by gas chromatographic as well as¹H and¹³C nuclear magnetic resonance spectroscopic analysis.Paper presented at the Bio/Environmentally Degradable Polymer Society—Second National Meeting, August 19–21, 1993, Chicago, Illinois.     

Bacterial Poly(Hydroxyalkanoate) Polymer Production from the Biodiesel Co-product Stream

A co-product stream from soy-based biodiesel production (CSBP) containing glycerol, fatty acid soaps, and residual fatty acid methyl esters (FAME) was utilized as a fermentation feedstock for the bacterial synthesis of poly(3-hydroxybutyrate) (PHB) and medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) polymers. Pseudomonas oleovorans NRRL B-14682 and P. corrugata 388 grew and synthesized PHB and mcl-PHA, respectively, when cultivated in up to 5% (w/v) CSBP. In shake flask culture, P. oleovorans grew to 1.3 ± 0.1 g/L (PHA cellular productivity = 13–27% of the bacterial cell dry weight; CDW) regardless of the initial CSBP concentration, whereas P. corrugata reached maximum cell yields of 2.1 g/L at 1% CSBP, which tapered off to 1.7 g/L as the CSBP media concentration was increased to 5% (maximum PHA cellular productivity = 42% of the CDW at 3% CSBP). While P. oleovorans synthesized PHB from CSBP, P. corrugata produced mcl-PHA consisting primarily of 3-hydroxyoctanoic acid (C_8:0; 39 ± 2 mol%), 3-hydroxydecanoic acid (C_10:0; 26 ± 2 mol%) and 3-hydroxytetradecadienoic acid (C_14:2; 15 ± 1 mol%). The molar mass (M_n) of the PHB polymer decreased by 53% as the initial CSBP culture concentration was increased from 1% to 5% (w/v). In contrast, the M_n of the mcl-PHA polymer produced by P. corrugata remained constant over the range of CSBP concentrations used.     

Extracellular Biopolymer Production by Aureobasidium pullulans MTCC 2195 Using Jackfruit Seed Powder

In this present paper, statistical screening and optimization of jackfruit seed powder based medium components were investigated for pullulan production from Aureobasidium pullulans. Seven medium variables jackfruit seed powder, K₂HPO₄, yeast extract, (NH₄)₂SO₄, NaCl, MgSO₄·7H₂O and ZnSO₄·7H₂O were screened by employing Plackett–Burman (PB) method. PB method showed jackfruit seed powder, ZnSO₄·7H₂O, K₂HPO₄ and yeast extract were significant. Central composite design of response surface method applied to optimize the significant variables identified from the PB experiment. Statistical analysis of the experimental results showed optimal values were found to be jackfruit seed powder 2 % (w/v), K₂HPO₄ 0.55 % (w/v), yeast extract 0.30 % (w/v) and ZnSO₄·7H₂O 0.006 % (w/v) with maximum pullulan concentration of 18.76 (g/L). Maximum pullulan concentration of 17.95 (g/L) was observed in the validation experiment. This experimental result explained the model was fitted 96 % as compare with the result predicted by response surface method.     

Degradation of Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Some Soil Aspergillus spp.

Systematic screening of 45 soil fungi for degradation polyhydroxyalkanoic acids (PHAs) has led to the selection of 6 potent Aspergillus isolates belonging to A. flavus, A. oryzae, A. parasiticus, and A. racemosus. Degradation of PHAs as determined by tube assay method revealed that these Aspergillus spp. were more efficient in degrading poly(3-hydroxybutyrate) [P(3HB)] compared to copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (P3HB-co-16% 3HV). Moreover, the extent of degradation in mineral base medium was much better than those in complex organic medium. For all the Aspergillus spp. tested, maximum degradation was recorded at a temperature of 37°C with significant inhibition of growth. The optimum pH range for degradation was 6.5–7.0 with degradation being maximum at pH 6.8. The extent of polymer degradation increased with increase in substrate concentration, the optimum concentration for most of the cultures being 0.4% and 0.2% (w/v) for P(3HB) and P(3HB-co-16%3HV) respectively. Supplementation of the degradation medium with additional carbon sources exerted significant inhibitory effect on both P(3HB) and P(3HB-co-16%3HV) degradation.     

Utilization of molasses spentwash for production of bioplastics by waste activated sludge

Present study describes the treatment of molasses spentwash and its use as a potential low cost substrate for production of biopolymer polyhydroxybutyrate (PHB) by waste activated sludge. Fluorescence microscopy revealed the presence of PHB granules in sludge biomass which was further confirmed by fourier transform-infra-red spectroscopy (FT-IR) and ¹³C nuclear magnetic resonance (NMR). The processing of molasses spentwash was carried out for attaining different ratios of carbon and nitrogen (C:N). Highest chemical oxygen demand (COD) removal and PHB accumulation of 60% and 31% respectively was achieved with raw molasses spentwash containing inorganic nitrogen (C:N ratio = 28) followed by COD removal of 52% and PHB accumulation of 28% for filtered molasses containing inorganic nitrogen (C:N ratio = 29). PHB production yield (Y_p/s) was highest (0.184 g g^?1 COD consumed) for deproteinized spentwash supplemented with nitrogen. In contrast, the substrate consumption and product formation were higher in case of raw spentwash. Though COD removal was lowest from deproteinized spentwash, evaluation of kinetic parameters suggested higher rates of conversion of available carbon to biomass and PHB. Thus the process provided dual benefit of conversion of two wastes viz. waste activated sludge and molasses spentwash into value-added product-PHB.     

Large-scale production of poly(3-hydroxyvaleric acid) by fermentation ofChromobacterium violaceum,processing, and characterization of the homopolyester

A fed-batch process was developed, which allowed biotechnological production of the homopolyester poly(3-hydroxyvaleric acid) [poly(3HV)], in a mineral salts medium containing valeric acid as carbon source and complex nutrients as supplements byChromobacterium violaceum at a 10- and 300-L fermentation scale. This process yielded up to 40 g dry cell matter per L fermentation broth, and the cells contained up to 70% (w/w) poly(3HV). Poly(3HV), which was extracted from the cells with chloroform and was precipitated from this solvent with ethanol, was processed to test bars by injection molding or by press processing and to fibers by melt spinning. The unprocessed and processed poly(3HV) material was characterized with respect to the molecular weight and with respect to thermal, rheological, and mechanical properties. It was shown that it is possible to process biodegradable poly(3HV) thermoplastically and to obtain a polymer suitable for applications with low strength requirements.     

Microbial Poly(L-Lactide)-Degrading Enzyme Induced by Amino Acids, Peptides, and Poly(L-Amino Acids)

Poly(L-lactide)(PLA)-degrading activities of a fungus, Tritirachium album, and two strains of actinomycetes,Lentzea waywayandensis and Amycolatopsis orientalis, were inducible by some proteins (poly-L-amino acid), peptides and amino acids. Extracellular PLA-degrading activity of the culture filtrates was detected when these strains grew in liquid basal medium containing 0.1% (w/v) of (poly-L-amino acids), peptides or amino acids as the enzyme inducer. In addition to PLA-degrading activity, succinyl-(L-alanyl-L-alanyl-L-alanine)-p-nitroanilide (Suc-(Ala)₃-pNA)-degrading activity was observed, implying that the enzymes produced were protease-type. The enzyme activities produced varied between different strains and different inducers. Silk fibroin was the best inducer for A. orientalis and that elastin was the best inducer for L. waywayandensis and T. album.     

Solid state dehalogenation of PCBs in contaminated soil using NaBH4

In this work we present the results of an experimental study on the abatement of polychlorinated biphenyl (PCBs) in contaminated soil using a high energy milling technique, that promotes a reaction only by impact between milling bodies. A sample of soil from a controlled landfill was treated with powdered NaBH(4) using two different hydride/soil ratios (5 and 2.5% w/w). The efficiency of the dehalogenation/hydrogenation reaction was studied as a function of the milling time (3.5 up to 30 h). After each run, the total PCBs content and the production of inorganic chloride were measured. The complete abatement was obtained with a starting PCBs concentration of about 2600 mg/kg. The residual PCBs concentration resulted to be <0.2 mg/kg. The final products of the treatment were biphenyl and NaCl. Other toxic or hazardous organic by-products were not generated. Boron was found as boric acid.     

Environmentally-Friendly Synthesis of Ag Nanoparticles by Fusarium sporotrichioides for the Production of PVA/Bentonite/Ag Composite Nanofibers

Membranes and filters made of nanofibers can have many medicines and water treatment applications. The use of silver nanoparticles (AgNPs) with antibacterial activity in these structures improve their efficiency. However, due to the toxicity of the compounds used in the chemical synthesis of AgNPs, in this study, AgNPs were obtained through a biological process using Fusarium sporotrichioides. AgNPs preparation conditions were optimized, including F. sporotrichioides medium and AgNO₃ concentration. Next, a PVA nanofiber membrane with bentonite and AgNPs (Bio-AgNPs or Chem-AgNPs) was prepared using electrospinning. The optimal conditions for the production of Bio-AgNPs were the culture of F. sporotrichioides in the MGYP culture medium and 12 M of AgNO₃. The Bio-AgNPs particle size and zeta potential were 58 nm and ??16.8 mV, respectively, with antibacterial activity. The PVA/NB/AgNPs nanofibers operation conditions included 7.5% w/w PVA, 3% w/w bentonite, and AgNPs 5% w/w at a voltage of 11 kV, feed rate of 0.5 mL/h, and 15 cm distance between the needle and the collector. The average diameter of the PVA/NB/Bio-AgNPs nanofibers was 230 nm. Also, the presence of silver in the nanofibers was confirmed through EDX and XRD methods. The antibacterial assay of the nanofibers showed that the inhibition zone of PVA/NB/Bio-AgNPs against E. coli and S. aureus was 0.62 and 0.36 mm, which is better than PVA/NB/Chem-AgNPs and comparable with chloramphenicol. The produced membrane is suitable for water treatment, food packaging, and wound dressing because of its good thermal, mechanical, and antibacterial properties.

     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from various carbon sources by batch-fed cultures ofAlcaligenes eutrophus

Copolyesters of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced at 30°C from various carbon sources byAlcaligenes eutrophus under batch-fed growth conditions. The production of P(3HB-co-3HV) from butyric and pentanoic acids was effective under nitrogenlimited conditions, and the conversion of carbon sources into copolyester was as high as 56 wt% at a C/N molar ratio of 40. In contrast, under excess-nitrogen conditions (C/N<10), cell growth was good, while P(3HB-co-3HV) production was partially inhibited. The production of P(3HB-co-3HV) from fructose and propionic acid was almost completely inhibited under excess-nitrogen conditions.     

Production,Characterization and Bioemulsifying Activity of an Exopolysaccharide Produced by <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. Isolated from Freshwater

This study aimed to evaluate the emulsion stability of solutions containing exopolysaccharide and culture medium of a Sphingomonas sp. strain with various hydrophobic compounds. The exopolysaccharide characterized belongs to a sphingan group, however, not being a gellan gum as produced by certain Sphingomonas strains. In general, the emulsifying indexes found in this study were above 70% for gasoline, hexane, kerosene and used frying oil. Nonetheless, the best results were achieved in kerosene solutions, which showed an index of 80% after 24 h, remaining stable for more than 168 h in combinations with various EPS concentrations. Interestingly, diesel oil best results were singly achieved in solution pH of 11, showing an index of around 65%. Furthermore, hexane obtained an index of 100% after 24 h when culture medium was used. Thus, these findings highlight the use of EPS as a potential bioemulsifier agent to enhance hydrocarbon degradation and emulsification effects in environmental biotechnology.     

Effect of alkali catalyst on biodiesel production in South Korea from mixtures of fresh soybean oil and waste cooking oil

Biodiesel from waste cooking oil (WCO) and soybean oil (SO) mixture was produced by changing the alkali catalyst (NaOH) content and the WCO to SO ratio in the feedstock. All the prepared biodiesel samples satisfied the standard requirement in terms of free glycerol, density, and acid value. The minimum catalyst content and the highest WCO composition to get biodiesel from the WCO/SO mixture feedstock without ruining the biodiesel properties were 1.0 and 60 wt %, respectively. This conclusion implies that the waste cooking oil mixture, which contains 40 wt % fresh soybean oil, could be treated like the fresh soybean oil to produce biodiesel, and that this behavior would be helpful to reduce the biodiesel production cost when waste cooking oil used as feedstock. The unsaturated methyl esters such as linoleic, and oleic acid were dominant (almost 80 % w/w) in the fresh soybean oil. However the saturated methyl ester was increased due to the double bond breaking during the frying process. These results may deteriorate the biodiesel quality by changing the methyl ester composition.     

Fabrication and Characterization of Biodegradable Composite Films Made of Using Poly(caprolactone) Reinforced with Chitosan

Chitosan was dissolved in 2?% aqueous acetic acid solution and the films were prepared by solution casting. Values of tensile strength (TS), tensile modulus (TM), elongation at break (Eb?%) and water vapor permeability (WVP) of the chitosan films were found to be 30?MPa, 450?MPa, 8?% and 4.7?g?mm/m²?day?kPa, respectively. Poly(caprolactone) (PCL) films were prepared from its granules by compression molding and the values of TS, TM, Eb and WVP were 14?MPa, 220?MPa, 70?% and 1.54?g?mm/m²?day?kPa, respectively. PCL was reinforced with chitosan films, and composite films were prepared by compression molding. Amount of chitosan in the composite films varied from 10 to 50?% (w/w). It was found that with the incorporation of chitosan films in PCL, both the values of TS and TM of composite films increased significantly. The highest mechanical properties were found at 50?% (w/w) of chitosan content. The Oxygen transmission rate (OTR) of composite film was found to decrease significantly than PCL films. Thermal properties of the composite were also improved as compared to PCL. The water uptake test of the composite also showed promising results with a good stability of composite films. The interface of the composite was investigated by scanning electron microscopy and showed good interfacial adhesion between PCL and chitosan films.