Poly(hydroxyalkanoate) Biosynthesis from Crude Alaskan Pollock (Theragra chalcogramma) Oil

Six strains of Pseudomonas were tested for their abilities to synthesize poly(hydroxyalkanoate) (PHA) polymers from crude Pollock oil, a large volume byproduct of the Alaskan fishing industry. All six strains were found to produce PHA polymers from hydrolyzed Pollock oil with productivities (P; the percent of the cell mass that is polymer) ranging from 6 to 53% of the cell dry weight (CDW). Two strains, P. oleovorans NRRL B-778 (P = 27%) and P. oleovorans NRRL B-14682 (P = 6%), synthesized poly(3-hydroxybutyrate) (PHB) with number average molecular weights (M_n) of 206,000 g/mol and 195,000 g/mol, respectively. Four strains, P. oleovorans NRRL B-14683 (P = 52%), P. resinovorans NRRL B-2649 (P = 53%), P. corrugata 388 (P = 43%), and P. putida KT2442 (P = 39%), synthesized medium-chain-length PHA (mcl-PHA) polymers with M_n values ranging from 84,000 g/mol to 153,000 g/mol. All mcl-PHA polymers were primarily composed of 3-hydroxyoctanoic acid (C_8:0) and 3-hydroxydecanoic acid (C_10:0) amounting to at least 75% of the total monomers present. Unsaturated monomers were also present in the mcl-PHA polymers at concentrations between 13% and 16%, providing loci for polymer derivatization and/or crosslinking. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Isolation and Characterization of a <Emphasis Type="Italic">Burkholderia</Emphasis> sp. USM (JCM15050) Capable of Producing Polyhydroxyalkanoate (PHA) from Triglycerides,Fatty Acids and Glycerols

A consortium of microorganisms from oil polluted wastewater sample was cultivated to promote polyhydroxyalkanoate (PHA) accumulation before subjecting the mixed cultures to sucrose density gradient ultracentrifugation. This resulted in the fractionation of the bacterial cells according to their physical features such as size, morphology and/or densities. An isolate was identified as Burkholderia sp. USM (JCM15050), which was capable of converting palm oil products [crude palm kernel oil (CPKO), palm olein (PO), palm kernel acid oil (PKAO), palm stearin (PS), crude palm oil (CPO), palm acid oil (PAO) and palm fatty acid distillate (PFAD)], fatty acids and various glycerol by-products into poly(3-hydroxybutyrate) [P(3HB)]. Up to 70 and 60 wt% of P(3HB) could be obtained when 0.5%(v/v) CPKO and glycerol was fed, respectively. Among the various fatty acids tested, lauric acid followed by oleic acid and myristic acid gave the best cell growth and PHA accumulation. Compared to Cupriavidus necator H16, the present isolate showed better ability to grow on and produce PHA from various glycerol by-products generated by the palm oil industry. This study demonstrated for the first time an isolate that has the potential to utilize palm oil and glycerol derivatives for the biosynthesis of PHA.     

Accumulation of Polyhydroxyalkanoates by Rhizobacteria Underneath Nickel-Hyperaccumulators from Serpentine Ecosystem

Nickel-resistant bacteria isolated from underneath Ni-hyperaccumulators growing on serpentine soils were screened for production of polyhydroxyalkanoates. These rhizobacteria accumulated poly-3-hydroxybutyric acid [P(3HB)] accounting 3.9–67.7% of cell dry weight during growth in gluconate and/or glucose. Cupriavidus pauculus KPS 201 utilized only gluconate and accumulated about 67.7% P(3HB) while, Bacillus firmus AND 408 utilized both carbon sources for polymer synthesis. The isolates being resistant to Ni also accumulated substantial amount of P(3HB) when grown in presence of the heavy metal and this was revealed by transmission electron microscopic studies. Although B. firmus AND 408 produced only P(3HB) at higher concentrations of gluconate, C. pauculus KPS 201 synthesized copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) [P(3HB-co-3HV)]. In presence of 0.8% gluconate and 4 mM Ni, KPS 201 cells produced PHA amounting 81% CDW, which contained 76 and 24 mol% 3HB and 3HV monomers, respectively.     

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.     

Enzymatic Degradation and Aminolysis of Microbial Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) Single Crystals

Solution-grown single crystals of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] were hydrolyzed by polyhydroxybutyrate (PHB) depolymerase from Ralstonia pickettii T1. Enzymatic degradation proceeded from the edges of lamellar crystals, yielding serrated contour and small crystal fragments. Gel permeation chromatography analysis revealed that the molecular weights of the crystals decreased during enzymatic degradation, suggesting that the enzymatic hydrolysis of chain-folding regions at the crystal surfaces occurred in addition to the enzymatic degradation at crystal laterals or edges. After P(3HB-co-4HB) single crystals were aminolysed in 20% aqueous methylamine solution to remove the folded-chain regions and enzymatic degradation by lipase from Rhizopus oryzae to remove 4HB components at crystal surfaces of single crystal aminolyzed, it was found that a small amount (up to ca. 2 mol%) of 4HB component can be incorporated into the P(3HB) mother crystal lattice irrespective of the 4HB content.     

Synthesis of poly(hydroxyalkanoate) from hydrolyzed linseed oil

Pseudomonas putida was grown on a mixture of long-chain fatty acids obtained by hydrolysis of linseed oil. A poly(3-hydroxyalkanoate) containing 51.2% of unsaturated monomers was obtained. A considerable percentage (13.6%) was constituted by C₁₄ and C₁₆ monomers containing three double-bonds in the side chains. The polymer showed a high tendency to crosslink when it was kept in presence of air. In the crosslinked polymer no polyunsaturated monomers could be detected.     

Isolation and identification of potential fungal species for spent engine lubrication oil remediation in Peninsular Malaysia

Improper disposal and spills of spent engine oil into the environment can result in contamination, which eventually affects humans through the food chain. Mycoremediation is an effective and inexpensive alternative to clean up spent engine oil contamination. In recent work, the potential effectiveness of fungi for degrading spent engine oil was confirmed, with the species identified through molecular identification. Fungi that were able to grow in Bushnell Haas Broth supplied with spent engine oil were identified with the potential to utilize spent engine oil as a carbon source. Six species of fungi namely Penicillium simplicissimum, Aspergillus nidulans, Aspergillus niger, Trichoderma longibrachiatum, Aspergillus ustus, and Aspergillus flavus were successfully identified in this study. Over a course of seven days, P. simplicissimum (21.11 percent) was identified as the most effective fungi in degrading spent engine oil, followed by A. nidulans (17.75 percent), A. niger (15.85 percent), T. longibrachiatum (15.12 percent), A. ustus (15.02 percent), and A. flavus (11.80 percent). As these species of fungi were isolated from the natural environment in Peninsular Malaysia, the potential of using these fungi as mycoremediation of spent engine oil was therefore confirmed.     

Synthesis, Characterization, and Hydrolytic Degradation of Copolymers of 2-Methylene-1,3-dioxepane with Ethylene and with Styrene

2-Methylene-1,3-dioxepane (MDP) was copolymerized with ethylene (E) at a pressure of approximately 1000 psi and a temperature of approximately 70°C with AIBN as the free radical initiator. The copolymers obtained, poly(MDP-co-E), were characterized by elemental analysis, IR, ¹H-NMR and ¹³C-NMR spectroscopy, DSC, and GPC. The copolymers contained 2–15 mol% ester units. MDP was also copolymerized with styrene (S) at 120°C with di-t-butyl peroxide as the initiator to prepare the copolymer, poly(MDP-co-S). The number-average molecular weights of both types of copolymers were in the range of 6000 to 11,000, and the weight-average molecular weights were in the range of 9000 to 17,000. The melting temperatures of poly(MDP-co-E) decreased with increasing ester unit content in the copolymer. For the MDP-S copolymers, the glass transition temperatures decreased with increasing ester unit content. Both poly(MDP-co-E) and poly(MDP-co-S) were degraded by methanolysis, and their molecular weights decreased by the expected amounts based on the ester unit content.     

Synthesis of Short-chain-length/Medium-chain-length Polyhydroxyalkanoate (PHA) Copolymers in Peroxisome of the Transgenic Arabidopsis Thaliana Harboring the PHA Synthase Gene from Pseudomonas sp. 61-3

In this paper, the photosynthetic production of short-chain-length/medium-chain-length polyhydroxyalkanoate (PHA) copolymers is reported. The wild-type and highly active doubly mutated PHA synthase 1 (S325T/Q481K, abbreviated ST/QK) genes from Pseudomonas sp. 61-3 were introduced into Arabidopsis thaliana. Peroxisome targeting signal 1 (PTS1) was used to target PHA synthases into the peroxisome to synthesize PHA from the intermediates of the β-oxidation pathway. The transgenic Arabidopsis produced PHA copolymers consisting of 40–57 mol% 3-hydroxybutyrate, 21–49 mol% 3-hydroxyvalerate, 8–18 mol% 3-hydroxyhexanoate, and 2–8 mol% 3-hydroxyoctanoate. The maximum PHA contents were 220μ g/g cell dry weight (cdw) in leaves, and 36μ g/g cdw in stems, respectively. The expression of the ST/QK mutated PHA synthase in leaves gene did not lead to significant difference in PHA content and monomer composition of PHAs, compared to the wild-type PHA synthase gene, suggesting that the supply of monomers may be a rate-determining step of PHA biosynthesis in the peroxisome. However, in stems, there were significant differences dependent on whether the wild-type or ST/QK mutated PHA synthase was expressed. These results suggest that tissue-specific monomer availability is important in determining the final mol% composition of PHA copolymers produced by the peroxisome in plants.     

High-Efficiency Production of Bioplastics from Biodegradable Organic Solids

Microbial polyhydroxyalkanoates (PHAs) have been extensively studied as environmentally friendly biodegradable thermoplastics. The major obstacle to wide acceptance of PHAs is their high price, mainly attributed to the costs of raw materials and polymer recovery. A large amount of organic solids are discarded from food production and consumption and may be used as carbonaceous raw materials for production of PHAs. A novel technology was investigated at bench-top scale to produce PHAs from food scraps. The harvested cell mass had a high PHA content (72.6% of dry cell mass), the same as obtained from pure glucose and organic acids. The organic solid was first digested in an acidogenic reactor in which about 60% solid was converted to fermentative products, including short-chain fatty acids. The four major acids were acetic, propionic, butyric, and lactic acids at concentrations of 6, 2, 27, and 33 g/L, respectively. The acids were transported through a membrane barrier via molecular diffusion to an airlift bioreactor, where the acids were utilized by an enriched culture of Ralstonia eutropha for PHA synthesis. Purification of fermentative acids was not performed in this molecular diffusion–based integration of acidogenesis and polymerization. By using a dialysis membrane as the barrier, the dry cell mass concentration and PHA content reached 22.7 g/L and 72.6%, respectively. The PHA was a copolymer of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with 2.8 mole % of hydroxyvalerate.     

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.     

Microbial fuel cell (MFC) for bioelectricity generation from organic wastes

Microbial fuel cells (MFCs) have gained a lot of attention recently as a mode of converting organic matter into electricity. In this study, a compost-based microbial fuel cell that generates bioelectricity by biodegradation of organic matter is developed. Grass cuttings, along with leaf mold, rice bran, oil cake (from mustard plants) and chicken droppings (waste from chickens) were used as organic waste. The electric properties of the MFC under anaerobic fermentation condition were investigated along with the influence of different types of membranes, the mixing of fly ash, and different types of electrode materials. It is observed that the maximum voltage was increased by mixing fly ash. Cellophane showed the highest value of voltage (around 350 mV). Bamboo charcoal is good for anode material; however carbon fiber is better for the cathode material in terms of optimization of power generated. This developed MFC is a simple cell to generate electricity from organic waste.     

Characteristics of gas and residues produced from electric arc pyrolysis of waste lubricating oil

An attempted has been made to recover high-calorific fuel gas and useful carbonaceous residue by the electric arc pyrolysis of waste lubricating oil. The characteristics of gas and residues produced from electric arc pyrolysis of waste lubricating oil were investigated in this study. The produced gas was mainly composed of hydrogen (35–40%), acetylene (13–20%), ethylene (3–4%) and other hydrocarbons, whereas the concentration of CO was very low. Calorific values of gas ranged from 11,000 to 13,000 kcal kg^?1 and the concentrations of toxic gases, such as NO_x, HCl and HF, were below the regulatory emissions limit. Gas chromatography–mass spectrometry (GC/MS) analysis of liquid-phase residues showed that high molecular-weight hydrocarbons in waste lubricating oil were pyrolyzed into low molecular-weight hydrocarbons and hydrogen. Dehydrogenation was found to be the main pyrolysis mechanism due to the high reaction temperature induced by electric arc. The average particle size of soot as carbonaceous residue was about 10 μm. The carbon content and heavy metals in soot were above 60% and below 0.01 ppm, respectively. The utilization of soot as industrial material resources such as carbon black seems to be feasible after refining and grinding.     

Study of the VOC emissions from a municipal solid waste storage pilot-scale cell: comparison with biogases from municipal waste landfill site

The emission of volatile organic compounds (VOCs) from municipal solid waste stored in a pilot-scale cell containing 6.4 tonnes of waste (storage facility which is left open during the first period (40 days) and then closed with recirculation of leachates during a second period (100 days)) was followed by dynamic sampling on activated carbon and analysed by GC–MS after solvent extraction. This was done in order to know the VOC emissions before the installation of a methanogenesis process for the entire waste mass. The results, expressed in reference to toluene, were exploited during the whole study on all the analyzable VOCs: alcohols, ketones and esters, alkanes, benzenic and cyclic compounds, chlorinated compounds, terpene, and organic sulphides.The results of this study on the pilot-scale cell are then compared with those concerning three biogases from a municipal waste landfill: biogas (1) coming from waste cells being filled or recently closed, biogas (2) from all the waste storage cells on site, and biogas (3) which is a residual gas from old storage cells without aspiration of the gas. The analysis of the results obtained revealed: (i) a high emission of VOCs, principally alcohols, ketones and esters during the acidogenesis; (ii) a decrease in the alkane content and an increase in the terpene content were observed in the VOCs emitted during the production of methane; (iii) the production of heavier alkanes and an increase in the average number of carbon atoms per molecule of alkane with the progression of the stabilisation/maturation process were also observed.Previous studies have concentrated almost on the analysis of biogases from landfills. Our research aimed at gaining a more complete understanding of the decomposition/degradation of municipal solid waste by measuring the VOCs emitted from the very start of the landfill process i.e. during the acidogenesis and acetogenesis phases.     

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.     

Thermo-chemical extraction of fuel oil from waste lubricating grease

This study investigated the recovery of oil from waste grease through the process of thermal degradation in an aqueous solution of potassium hydroxide (KOH) followed by solvent extraction. Waste high temperature metal bearing grease was dissolved in a 15 w/w% KOH solution at 80 °C while being agitated at 2000 rpm using a shear action agitator for a period of 15 min. Two distinct layers were observed after 8 min of settling time. The top layer being of dark brown oil and the bottom layer was a heterogeneous mixture. The two layers were separated by decantation. The bottom layer was cooled down to 45 °C followed by slow addition of toluene (C₇H₈) while agitating at 1200 rpm for 15 min to prevent solids settling and minimise rapid volatilisation of the organic compounds in the mixture. Two distinct layers were also formed, the top homogeneous mixture of light brown oil–toluene mixture and the bottom sludge layer. The solvent was recovered from the oil for re-use by fractional distillation of the homogenous mixture. It was observed that 15 w/w% potassium hydroxide solution can chemically degrade the soap matrix in the grease and extract up to 49 w/w% of the fuel oil when subjected to high shear stress at a temperature of 80 °C. The 26 w/w% extraction of oil in the remaining sludge was obtained by solvent extraction process with mass ratios of sludge to solvent of 2:1. Solvent recovery of 88% by mass was obtained via fractional distillation method. The combined extraction processes brought an overall oil yield of 75 w/w% from the waste grease. The fuel oil obtained from this process has similar properties to paraffin oil and can be blended with other oils as an alternative energy source.     

Bacteria Degrading PCBs and CBs Isolated from Long-Term PCB Contaminated Soil

Bacteria able to degrade polychlorinated biphenyls (PCBs) and chlorobenzoic acids (CBs) were isolated from soil that had been contaminated with PCBs for 15–30 years. Contaminated soil in which PCB content ranged between 10–470 mg/kg was naturally vegetated with different plants including ash (Fraxinus excelsior), birch (Betula pendula), black locust (Robinia pseudoacacia), Austrian pine (Pinus nigra) and goat willow (Salix caprea) trees as well as a variety of grasses and forbs. Bacteria able to use biphenyl as a sole source of carbon and energy were found in the root zone of all plants, but occurred in the largest numbers beneath pine and black locust. Bacteria able to degrade chlorobenzoic acids were isolated from the same location contaminated with PCBs. Strains that were taxonomically identified by 16S rDNA as Pandoraea were able to use 2-CB, 3-CB, 2,3-CB, 2,5-CB as sole carbon sources, and the strain Arthrobacter utilised 4-CB.     

Removal of Oil from Water by Calcium Alginate Hydrogel Modified with Maleic Anhydride

Calcium alginate hydrogel was prepared and used as a biosorbent for the removal of oil from aqueous solutions. Calcium alginate hydrogel was further chemically modified by esterification with maleic anhydride. The changes in the physicochemical properties of maleic anhydride modified calcium alginate were investigated via multiple techniques (FTIR, SEM, BET and DSC/TGA). Adsorption batch experiments were carried out to compare the oil adsorption capacities of native and modified calcium alginates. Adsorption experiments were carried out as a function of solution pH, temperature and ionic strength to determine the optimal conditions for the adsorption of oil. Equilibrium and kinetic studies were conducted for the modified alginate. Results revealed that the maleic anhydride modification of calcium alginate improved its adsorption capacity towards oil. Higher adsorption capacities of modified alginate were attained at lower temperatures (20 °C), higher ionic strengths (1.0 M NaCl) and within the pH range of 5–9. The oil adsorption data obtained for modified alginate could be better described by the first order kinetic model (R²?=?0.981) and the BET equilibrium isotherm (R²?=?0.984). The maximum monolayer adsorption capacity predicted by the BET model for the modified calcium alginate was found to be 143.0 mg/g.     

Effects of physicochemical factors on PAH degradation by Planomicrobium alkanoclasticum

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that are mutagenic, carcinogenic, and toxic to living organisms. Here, the ability and effectiveness of selected bacteria isolated from an oil‐contaminated area in biodegrading PAHs were evaluated, and the optimal conditions conducive to bacterial PAH biodegradation were determined. Of six bacterial isolates identified based on their 16S rRNA sequences, Planomicrobium alkanoclasticum could subsist on and consume nearly all hydrocarbons according to the 2,6‐dichlorophenolindophenol assay. The efficacy of this isolate at PAH biodegradation was then empirically confirmed. After 30 days of incubation, P. alkanoclasticum degraded 90.8% of the 16 PAH compounds analyzed and fully degraded eight of them. The optimum P. alkanoclasticum growth conditions were 35°C, pH 7.5, and NaNO₃ as the nitrogen source. Under these biostimulant conditions, P. alkanoclasticum degraded 91.4% of the total PAH concentration and completely decomposed seven PAHs after 15 days incubation. Hence, P. alkanoclasticum is an apt candidate for the biodegradation of PAHs and the bioremediation of sites contaminated by them.     

Application of tracer ratio and inverse dispersion methods with boat-based plume measurements to estimate ammonia emissions from seabird colonies

Ammonia emissions from two contrasting seabird colonies in Scotland were measured, based on the determination of atmospheric concentrations downwind of the colonies. Atmospheric concentrations of ammonia (NH₃) across the downwind plume were compared with the inverse application of a Gaussian dispersion model (ID) to calculate the modelled NH₃ emission that would generate the measured cross-wind-integrated plume concentration. In parallel, a tracer gas (sulphur hexafluoride, SF₆) was released from the colonies with air samples taken to allow determination of SF₆ concentrations. On the basis of the known emission rate of SF₆, the magnitude of ammonia emissions was estimated by the cross-wind-integrated tracer ratio (TR) of NH₃/SF₆ concentrations. Coupled with data on annual bird attendance, the measurements indicate annual emissions from the Isle of May and the Bass Rock of 18 and 132 tonnes NH₃-N year^–1, respectively. The measured NH₃ emissions were compared with estimates of seabird nitrogen excretion to estimate the proportion of excreted N that is volatilised as NH₃ (F_Nr). The emission estimates of the two methods compared favourably, giving 4 and 6 kg NH₃-N h^–1 (F_Nr = 15%) for the Isle of May for the ID and TR methods, respectively, and 21 and 25 kg NH₃-N h^–1 (F_Nr = 50%) for the Bass Rock for the ID and TR methods, respectively. The results provide the first measurement-based estimates to allow regional up scaling of ammonia emissions from seabirds.