Industrial Production Of Polyhydroxyalkanoates Research Articles

Dynamic modeling of Rhodospirillum rubrum PHA production triggered by redox stress during VFA photoheterotrophic assimilations

Polyhydroxyalkanoates (PHA) represent an environmentally friendly alternative to petroleum based plastics for a broad range of applications from packaging to biomedical devices. In the prospect of an industrial PHA production, it is highly valuable to accurately control the incorporation of different repeating units into the polymer, to produce a polyester with specific material characteristics. In this study, we develop macroscopic dynamic models predicting the polymer production and composition when mixtures containing up to four volatile fatty acids (VFA) are used as substrates. These models successfully reproduce the sequential (and preferential) substrate consumption and polymer production/reconsumption patterns, experimentally observed during biomass growth, thanks to simple kinetic structures based on Monod and inhibition factors. These models can serve as a basis for numerical simulation and process analysis, as well as process intensification through model-based optimization and control.

Novel thermophilic polyhydroxyalkanoates producing strain Aneurinibacillus thermoaerophilus CCM 8960.

Aneurinibacillus thermoaerophilus CCM 8960 is a thermophilic bacterium isolated from compost in Brno. The bacterium accumulates polyhydroxyalkanoates (PHAs), a biodegradable and renewable alternative to petrochemical polymers. The bacterium reveals several features that make it a very interesting candidate for the industrial production of PHA. At first, due to its thermophilic character, the bacterium can be utilized in agreement with the concept of next-generation industrial biotechnology (NGIB), which relies on extremophiles. Second, the bacterium is capable of producing PHA copolymers containing a very high portion of 4-hydroxybutyrate (4HB). Such materials possess unique properties and can be advantageously used in multiple applications, including but not limited to medicine and healthcare. Therefore, this work focuses on the in-depth characterization of A. thermoaerophilus CCM 8960. In particular, we sequenced and assembled the genome of the bacterium and identified its most important genetic features, such as the presence of plasmids, prophages, CRISPR arrays, antibiotic-resistant genes, and restriction-modification (R-M) systems, which might be crucial for the development of genome editing tools. Furthermore, we focused on genes directly involved in PHA metabolism. We also experimentally studied the kinetics of glycerol and 1,4-butanediol (1,4BD) utilization as well as biomass growth and PHA production during cultivation. Based on these data, we constructed a metabolic model to reveal metabolic fluxes and nodes of glycerol and 1,4BD concerning their incorporation into the poly(3-hydroxybutyrate-co-4-hydroxybutyrate (P(3HB-co-4HB)) structure. KEY POINTS: • Aneurinibacillus sp. H1 was identified as Aneurinibacillus thermoaerophilus. • PHA metabolism pathway with associated genes was presented. • Unique monomer composition of produced PHAs was reported.

Utilization of sugarcane molasses as the main carbon source for the production of polyhydroxyalkanoates from Enterobacter cloacae

Polyhydroxyalkanoates (PHAs) are biodegradable polymers that otherwise exhibit many properties similar to that of conventional, non-biodegradable plastics. Hence, they are considered as potential substitutes for non-biodegradable plastics. But industrial production and commercialization of PHA have been hindered greatly due to the high cost requirements, especially those involved in the provision of suitable carbon sources. In this study, sugarcane molasses was used as a cheap carbon source for PHA production using a wild strain of Enterobacter cloacae isolated from sugarcane extract. This isolate was selected for PHA production based on Sudan Black B staining, and it was subsequently identified by performing BLASTn using its 16S rRNA sequence (GenBank accession number: ON314993). The PHA yield was studied under varying conditions of initial pH, molasses concentration and inoculum concentration. The highest yield of 4.13 - 4.98 g/L or 48 - 56% PHA was obtained after 48-60 h incubation at initial pH 7, molasses concentration 4% and inoculum concentration 2%. After downstream processing, the extracted PHA was characterized by FTIR spectroscopy. It is concluded that E. cloacae has the potential to utilize sugarcane molasses as a cheap carbon source to yield impressive amounts of PHA, and that it should be considered a candidate for further studies and for the cheaper industrial production of PHA.

Enhancing the biological reductive dechlorination of trichloroethylene with PHA from mixed microbial cultures (MMC)

The biological reductive dechlorination (BRD) process is often limited by the lack of suitable electron donors, requiring the supply of long-lasting substrates to enhance the biological metabolism. Among the others, polyhydroxyalkanoates (PHA) are a particularly interesting slow-release source of electron-donors, being entirely biodegradable polyesters. Generally, industrial PHA production processes are based on pure culture fermentation with high related costs. In recent years, innovative and cost-effective PHA production from mixed microbial cultures (MMC) and waste feedstocks is attracting considerable attention. This research, for the first time, investigated the effect of distinctive types of PHA on the BRD process of trichloroethylene (TCE), in a continuous-flow lab-scale system using two PHA materials with different purity grade produced from MMC at pilot scale in comparison with a commercial PHA produced from pure culture. Promising results have been obtained with non-extracted MMC-PHA, a material consisting of both PHA (56%, w/w) and microbial cells, with constant production of acids over 110 days, which stimulated a nearly complete TCE dechlorination with the non-toxic ethene as the main byproduct (approximately 92%). Dehalococcoides mccartyi was the main microorganism responsible for TCE dechlorination process, representing ≥ 54.05% of the bacterial population, mainly carrying the reductive dehalogenase genes tceA and vcrA (≥9.17E+08 and ≥2.01E+07 gene copies/g of PHA or sand, respectively). This finding suggests the possibility to directly use PHA-rich biomass deriving from MMC production process, which does not require any polluting and expensive extraction procedures, as a novel material in the field of groundwater remediation with noticeable economic and environmental advantages.

Microbial cell factories for the production of polyhydroxyalkanoates.

Pollution caused by persistent petro-plastics is the most pressing problem currently, with 8 million tons of plastic waste dumped annually in the oceans. Plastic waste management is not systematized in many countries, because it is laborious and expensive with secondary pollution hazards. Bioplastics, synthesized by microorganisms, are viable alternatives to petrochemical-based thermoplastics due to their biodegradable nature. Polyhydroxyalkanoates (PHAs) are a structurally and functionally diverse group of storage polymers synthesized by many microorganisms, including bacteria and Archaea. Some of the most important PHA accumulating bacteria include Cupriavidus necator, Burkholderia sacchari, Pseudomonas sp., Bacillus sp., recombinant Escherichia coli, and certain halophilic extremophiles. PHAs are synthesized by specialized PHA polymerases with assorted monomers derived from the cellular metabolite pool. In the natural cycle of cellular growth, PHAs are depolymerized by the native host for carbon and energy. The presence of these microbial PHA depolymerases in natural niches is responsible for the degradation of bioplastics. Polyhydroxybutyrate (PHB) is the most common PHA with desirable thermoplastic-like properties. PHAs have widespread applications in various industries including biomedicine, fine chemicals production, drug delivery, packaging, and agriculture. This review provides the updated knowledge on the metabolic pathways for PHAs synthesis in bacteria, and the major microbial hosts for PHAs production. Yeasts are presented as a potential candidate for industrial PHAs production, with their high amenability to genetic engineering and the availability of industrial-scale technology. The major bottlenecks in the commercialization of PHAs as an alternative for plastics and future perspectives are also critically discussed.

The First Insight into Polyhydroxyalkanoates Accumulation in Multi-Extremophilic Rubrobacter xylanophilus and Rubrobacter spartanus.

Actinobacteria belonging to the genus Rubrobacter are known for their multi-extremophilic growth conditions—they are highly radiation-resistant, halotolerant, thermotolerant or even thermophilic. This work demonstrates that the members of the genus are capable of accumulating polyhydroxyalkanoates (PHA) since PHA-related genes are widely distributed among Rubrobacter spp. whose complete genome sequences are available in public databases. Interestingly, all Rubrobacter strains possess both class I and class III synthases (PhaC). We have experimentally investigated the PHA accumulation in two thermophilic species, R. xylanophilus and R. spartanus. The PHA content in both strains reached up to 50% of the cell dry mass, both bacteria were able to accumulate PHA consisting of 3-hydroxybutyrate and 3-hydroxyvalerate monomeric units, none other monomers were incorporated into the polymer chain. The capability of PHA accumulation likely contributes to the multi-extremophilic characteristics since it is known that PHA substantially enhances the stress robustness of bacteria. Hence, PHA can be considered as extremolytes enabling adaptation to extreme conditions. Furthermore, due to the high PHA content in biomass, a wide range of utilizable substrates, Gram-stain positivity, and thermophilic features, the Rubrobacter species, in particular Rubrobacter xylanophilus, could be also interesting candidates for industrial production of PHA within the concept of Next-Generation Industrial Biotechnology.

Recombinant DNA technology as a tool for improving production of polyhydroxyalkanoates by the natural producers

Polyhydroxyalkanoates (PHAs) are a group of the biodegradable polyesters, and represent an alternative to conventionally used petroleum-based plastics resistant to biodegradation. The production is not cost-competitive compared to conventional plastics, although, there are several bacterial producers capable of PHA accumulating up to 80 % of their cells dry weight using low-cost substrates. PHA production can be improved by transferring specific enzymes or entire metabolic pathways from the most efficient producers to other natural producers. Therefore, the review is focused on genetic modification of bacterial producers, namely the genera Cupriavidus, Pseudomonas, Halomonas, Aeromonas and Bacillus, for efficient industrial production of PHAs. Recombinant PHA producer can use non-traditional substrates like agro-industrial wastes, namely whey, lignocellulose or glycerol. It is possible to influence the shape and size of the producer's cell by over-expression or knockout of selected genes or to affect its preference for a specific component of a culture medium by modulation of a producer's basal metabolism. The costs of PHA production still be reduced by simplifying the downstream process by enzymatic hydrolysis of selected parts of the cell or blocking the protective mechanisms of the cell against its autolysis caused by the ionic strength of the solution.

Photoheterotrophic Assimilation of Valerate and Associated Polyhydroxyalkanoate Production by Rhodospirillum rubrum.

Purple nonsulfur bacteria are increasingly recognized for industrial applications in bioplastics, pigment, and biomass production. In order to optimize the yield of future biotechnological processes, the assimilation of different carbon sources by Rhodospirillum rubrum has to be understood. As they are released from several fermentation processes, volatile fatty acids (VFAs) represent a promising carbon source in the development of circular industrial applications. To obtain an exhaustive characterization of the photoheterotrophic metabolism of R. rubrum in the presence of valerate, we combined phenotypic, proteomic, and genomic approaches. We obtained evidence that valerate is cleaved into acetyl coenzyme A (acetyl-CoA) and propionyl-CoA and depends on the presence of bicarbonate ions. Genomic and enzyme inhibition data showed that a functional methylmalonyl-CoA pathway is essential. Our proteomic data showed that the photoheterotrophic assimilation of valerate induces an intracellular redox stress which is accompanied by an increased abundance of phasins (the main proteins present in polyhydroxyalkanoate [PHA] granules). Finally, we observed a significant increase in the production of the copolymer P(HB-co-HV), accounting for a very high (>80%) percentage of HV monomer. Moreover, an increase in the PHA content was obtained when bicarbonate ions were progressively added to the medium. The experimental conditions used in this study suggest that the redox imbalance is responsible for PHA production. These findings also reinforce the idea that purple nonsulfur bacteria are suitable for PHA production through a strategy other than the well-known feast-and-famine process.IMPORTANCE The use and the littering of plastics represent major issues that humanity has to face. Polyhydroxyalkanoates (PHAs) are good candidates for the replacement of oil-based plastics, as they exhibit comparable physicochemical properties but are biobased and biodegradable. However, the current industrial production of PHAs is curbed by the production costs, which are mainly linked to the carbon source. Volatile fatty acids issued from the fermentation processes constitute interesting carbon sources, since they are inexpensive and readily available. Among them, valerate is gaining interest regarding the ability of many bacteria to produce a copolymer of PHAs. Here, we describe the photoheterotrophic assimilation of valerate by Rhodospirillum rubrum, a purple nonsulfur bacterium mainly known for its metabolic versatility. Using a knowledge-based optimization process, we present a new strategy for the improvement of PHA production, paving the way for the use of R. rubrum in industrial processes.

In silico analysis of phag-like protein in Ralstonia Euthropa H16, potentially involved in polyhydroxyalkanoates synthesis

Polyhydroxyalkanoates (PHA) are synthesised by bacteria as carbon storage material. The protein PhaG directs carbon from non-related carbon sources such as glycerol, metabolised through fatty acid de novo synthesis (FAS) pathway, with PHA synthesis. The gene that codifies for this protein has not yet been found in the genome of Ralstonia eutropha H16, a model organism. By bioinformatic comparison to already known PhaG proteins, a PhaG-like protein was found codified by gene H16_A0147 and presence of the gene was preliminary confirmed by PCR. This is the first study that shows the presence and characteristics of a PhaG-like protein in R. eutropha H16 and represents the first step for the identification of a connection between FAS and PHA pathways in this model bacterium. Further gene deletion and enzymatic activity studies are necessary to confirm this potential relationship, which could improve industrial PHA production and utilisation of agro-industrial residues such as glycerol.

Polyhydroxyalkanoate Producing Potential of Saccharomyces cerevisiae

Polyhydroxyalkanoates (PHAs) are inclusion bodies accumulated by some microorganisms as reserve material under unbalanced growth condition such as limited oxygen, nitrogen, phosphorous or sulphur and excess carbon source. Saccharomyces cerevisiae strains were screened for Polyhydroxyalkanoates (PHA) producing potential using plate assay technique with Sudan Black B stain. Saccharomyces cerevisiae efficiency for PHA production was analysed utilizing sawdust as carbon source and the production rate was compared with glucose (control). Thus, PHA synthesising enzymes, PHA carbon chain distributions and concentrations were determined using gas chromatography flame ionization detector (GCFID) while microbial growth (biomass yield) during production was determined using Uv-Visible spectrophotometric technique at 600 nm. PHA was extracted from Saccharomyces cerevisiae cells using solvent extraction technique and the extract was further dissolved using acetone derivatisation process. PHA-synthesising enzymes such as Glyceraldehyde-3-phosphate dehydrogenase, Pyruvate dehydrogenase, 3-Ketothiolase, Acetoacetyl-CoA reductase, 3-Ketoacyl reductase and Polyhydroxyalkanoate synthase were extracted from cell-free supernatant obtained from glucose and sawdust media. Saccharomyces cerevisiae cells grown in sawdust medium had the highest PHA yield of 75.12 % while 64.66 % PHA was obtained from cells grown in glucose medium. Consequently, utilization of Saccharomyces cerevisiae in limiting condition using agricultural residue could reduce the cost of industrial production of PHA required for plastic production due to its ability to accumulate the polymer intracellularly.

Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique.

BackgroundRalstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO2 fixation, which makes it a potential strain for industrial PHA production and attractive host for CO2 conversion. Although the bacterium is not recalcitrant to genetic manipulation, current methods for genome editing based on group II introns or single crossover integration of a suicide plasmid are inefficient and time-consuming, which limits the genetic engineering of this organism. Thus, developing an efficient and convenient method for R. eutropha genome editing is imperative.ResultsAn efficient genome editing method for R. eutropha was developed using an electroporation-based CRISPR-Cas9 technique. In our study, the electroporation efficiency of R. eutropha was found to be limited by its restriction-modification (RM) systems. By searching the putative RM systems in R. eutropha H16 using REBASE database and comparing with that in E. coli MG1655, five putative restriction endonuclease genes which are related to the RM systems in R. eutropha were predicated and disrupted. It was found that deletion of H16_A0006 and H16_A0008-9 increased the electroporation efficiency 1658 and 4 times, respectively. Fructose was found to reduce the leaky expression of the arabinose-inducible pBAD promoter, which was used to optimize the expression of cas9, enabling genome editing via homologous recombination based on CRISPR-Cas9 in R. eutropha. A total of five genes were edited with efficiencies ranging from 78.3 to 100%. The CRISPR-Cpf1 system and the non-homologous end joining mechanism were also investigated, but failed to yield edited strains.ConclusionsWe present the first genome editing method for R. eutropha using an electroporation-based CRISPR-Cas9 approach, which significantly increased the efficiency and decreased time to manipulate this facultative chemolithoautotrophic microbe. The novel technique will facilitate more advanced researches and applications of R. eutropha for PHA production and CO2 conversion.

EVALUATION OF POLYHYDROXYALKANOATES PRODUCING BACTERIA ISOLATED FROM SOILS WITH WASTES OF Cocos nucifera

Polyhydroxyalkanoates (PHAs) are polyesters produced and accumulated in prokaryotes as carbon and energy storage materials. These polymers can be used to produce biodegradable plastics. Actually, the industrial production of PHAs for the manufacture of biodegradable plastics has a high production cost due to the use of expensive carbon substrates. An alternative to reduce these costs is the use of wastes from renewable resources. Coconut biomass, which is the most abundant agroindustrial waste in the state of Guerrero, Mexico, has a promising potential as a primary or secondary feedstock for PHAs production. In this research, it is identified and evaluated the PHAs producing bacteria isolated from soil where Cocos nucifera waste and fiber in decomposition are accumulated. It was found six bacteria capable of producing PHAs in different concentrations (0.06 to 0.422 g/L). These strains belong to the genus Staphylococcus and Bacillus . The production of PHAs of Bacillus megaterium isolate S15 was characterized, and turned out to be the best PHAs accumulating bacterium. A bank of strains capable of producing polyhydroxyalkanoates was obtained for future studies.

Producing PHAs in the bioeconomy — Towards a sustainable bioplastic

Biodegradable polymers such as polyhydroxyalkanoates (PHAs) can reduce pollution caused by the increasing global polymer demand. Although industrial production of PHAs grew rapidly in the past years, their total market share is still marginal. While this is often attributed to their higher price, which is mainly caused by high production costs, the industrial success of PHAs can also depend on policy framework. Environmental assessment tools such as life cycle analysis and the product environmental footprint showed that PHAs can contribute to greenhouse gas emission reduction targets, waste reduction as well as green jobs and innovation in the biotechnology sector. As many countries aspire to these targets under the umbrella of bioeconomy concepts, inclusion into the respective policies can stimulate industrial PHA production. With a high variability in the industrial production of PHAs in terms of feedstock, energy source, polymer properties etc., the choice of optimization criteria influences the design of new production processes. Considering the political targets for bioeconomy products is therefore useful to direct the technical design of sustainable PHA production, for example in integrated lignocellulose biorefineries.

Production of poly(3-hydroxybutyrate) by Halomonas boliviensis in an air-lift reactor.

BackgroundMicrobial polyesters, also known as polyhydroxyalkanoates (PHAs), closely resemble physical and mechanical features of petroleum derived plastics. Recombinant Escherichia coli strains are being used in industrial production of PHAs in Stirred Tank Bioreactors (STRs). However, use of Air-Lift Reactors (ALRs) has been known to offer numerous technical operating options over STRs, and as such has been successfully implemented in many bioprocesses. Halomonas boliviensis is a halophilic bacterium that is known to assimilate various carbohydrates and convert them into a particular type of PHA known as poly(3-hydroxybutyrate) (PHB). Owing to this capability, it has been used to synthesize the polyester using hydrolysates of starch or wheat bran in stirred tank bioreactors.ResultsThis research article firstly describes the production of PHB in shake flasks by H. boliviensis using different combinations of carbohydrates and partially hydrolyzed starch as carbon sources. The highest PHB yields, between 56 and 61 % (wt.), were achieved when either starch hydrolysate or a mixture of glucose and xylose were used as carbon sources. The starch hydrolysate obtained in this study was then used as carbon source in an ALR. The largest amount of PHB, 41 % (wt.), was attained after 24 hrs of cultivation during which maltose in the hydrolysate was assimilated more rapidly than glucose during active cell growth; however, the rate of assimilation of both the carbohydrates was found to be similar during synthesis of PHB. An incomplete pentose phosphate pathway, which lacks 6-phosphogluconate dehydrogenase, was deduced from the genome sequence of this bacterium and may result in the characteristic assimilation of glucose and maltose by the cells.ConclusionsThis study showed that the production of PHB by H. boliviensis using cheap substrates such as starch hydrolysate in a simple production system involving an ALR is feasible. Both maltose and glucose in the hydrolysate induce cell growth and PHB synthesis; most likely the cells balance adequately CoA and NAD(P)H during the assimilation of these carbohydrates. The combination of cheap substrates, simple production systems and the use of non-strict sterile conditions by the halophile H. boliviensis are desirable traits for large scale production of PHB, and should lead to a competitive bioprocess.Electronic supplementary materialThe online version of this article (doi:10.1186/s40709-015-0031-6) contains supplementary material, which is available to authorized users.

Reduced peroxisomal citrate synthase activity increases substrate availability for polyhydroxyalkanoate biosynthesis in plant peroxisomes.

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers used as renewable, biodegradable plastics. PHA production in plants may be a way to reduce industrial PHA production costs. We recently demonstrated a promising level of peroxisomal PHA production in the high biomass crop species sugarcane. However, further production strategies are needed to boost PHA accumulation closer to commercial targets. Through exogenous fatty acid feeding of Arabidopsis thaliana plants that contain peroxisome-targeted PhaA, PhaB and PhaC enzymes from Cupriavidus necator, we show here that the availability of substrates derived from the β-oxidation cycle limits peroxisomal polyhydroxybutyrate (PHB) biosynthesis. Knockdown of peroxisomal citrate synthase activity using artificial microRNA increased PHB production levels approximately threefold. This work demonstrates that reduction of peroxisomal citrate synthase activity may be a valid metabolic engineering strategy for increasing PHA production in other plant species.

The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: Highlights from a multi-level omics approach

BackgroundPseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures.ResultsWe found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g·(g·h)-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system.ConclusionThis is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.

Comparative genomics study of polyhydroxyalkanoates (PHA) and ectoine relevant genes from Halomonas sp. TD01 revealed extensive horizontal gene transfer events and co-evolutionary relationships

BackgroundHalophilic bacteria have shown their significance in industrial production of polyhydroxyalkanoates (PHA) and are gaining more attention for genetic engineering modification. Yet, little information on the genomics and PHA related genes from halophilic bacteria have been disclosed so far.ResultsThe draft genome of moderately halophilic bacterium, Halomonas sp. TD01, a strain of great potential for industrial production of short-chain-length polyhydroxyalkanoates (PHA), was analyzed through computational methods to reveal the osmoregulation mechanism and the evolutionary relationship of the enzymes relevant to PHA and ectoine syntheses. Genes involved in the metabolism of PHA and osmolytes were annotated and studied in silico. Although PHA synthase, depolymerase, regulator/repressor and phasin were all involved in PHA metabolic pathways, they demonstrated different horizontal gene transfer (HGT) events between the genomes of different strains. In contrast, co-occurrence of ectoine genes in the same genome was more frequently observed, and ectoine genes were more likely under coincidental horizontal gene transfer than PHA related genes. In addition, the adjacent organization of the homologues of PHA synthase phaC1 and PHA granule binding protein phaP was conserved in the strain TD01, which was also observed in some halophiles and non-halophiles exclusively from γ-proteobacteria. In contrast to haloarchaea, the proteome of Halomonas sp. TD01 did not show obvious inclination towards acidity relative to non-halophilic Escherichia coli MG1655, which signified that Halomonas sp. TD01 preferred the accumulation of organic osmolytes to ions in order to balance the intracellular osmotic pressure with the environment.ConclusionsThe accessibility of genome information would facilitate research on the genetic engineering of halophilic bacteria including Halomonas sp. TD01.

Peroxisomal polyhydroxyalkanoate biosynthesis is a promising strategy for bioplastic production in high biomass crops

Polyhydroxyalkanoates (PHAs) are bacterial carbon storage polymers with diverse plastic-like properties. PHA biosynthesis in transgenic plants is being developed as a way to reduce the cost and increase the sustainability of industrial PHA production. The homopolymer polyhydroxybutyrate (PHB) is the simplest form of these biodegradable polyesters. Plant peroxisomes contain the substrate molecules and necessary reducing power for PHB biosynthesis, but peroxisomal PHB production has not been explored in whole soil-grown transgenic plants to date. We generated transgenic sugarcane (Saccharum sp.) with the three-enzyme Ralstonia eutropha PHA biosynthetic pathway targeted to peroxisomes. We also introduced the pathway into Arabidopsis thaliana, as a model system for studying and manipulating peroxisomal PHB production. PHB, at levels up to 1.6%-1.8% dry weight, accumulated in sugarcane leaves and A.thaliana seedlings, respectively. In sugarcane, PHB accumulated throughout most leaf cell types in both peroxisomes and vacuoles. A small percentage of total polymer was also identified as the copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) in both plant species. No obvious deleterious effect was observed on plant growth because of peroxisomal PHA biosynthesis at these levels. This study highlights how using peroxisomal metabolism for PHA biosynthesis could significantly contribute to reaching commercial production levels of PHAs in crop plants.

Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium

Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.

Improved detergent-based recovery of polyhydroxyalkanoates (PHAs)

Extracting polyhydroxyalkanoate (PHA) polymer from bacterial cells often involves harsh conditions, including use of environmentally harmful solvents. We evaluated different detergents under various conditions to extract PHA from Ralstonia eutropha and Escherichia coli cells. Most detergents tested recovered highly pure PHA polymer from cells in amounts that depended on the percentage of polymer present in the cell. Detergents such as linear alkylbenzene sulfonic acid (LAS-99) produced a high yield of high purity polymer, and less detergent was needed compared to the amount of SDS to produce comparable yields. LAS-99 also has the advantage of being biodegradable and environmentally safe. Chemical extraction of PHA with detergents could potentially minimize or eliminate the need to use harsh organic solvents, thus making industrial PHA production a cleaner technology process.