The potential of Burkholderia thailandensis E264 for co-valorization of C5 and C6 sugars into multiple value-added bio-products.

Despite known metabolic versatility of Burkholderia spp., sugar metabolism and end-product synthesis patterns in Burkholderia thailandensis have been poorly characterized. This work has demonstrated that B. thailandensis is capable of simultaneously uptaking glucose and xylose and accumulating up to 64% of its dry mass as poly(3-hydroxyalkanoate) (PHA) biopolymers, resulting in a PHA titer of up to 3.8 g L-1 in shake flasks. Rhamnolipids - mainly (68-75%) in the form of Rha-Rha-C14-C14 - were produced concomitantly with a titer typically in the range of 0.2-0.4 g L-1. Gluconic and xylonic acids were also detected in titers of up to 5.3 g L-1, and while gluconic acid appeared to be back consumed, xylonic acid formed as a major end product. This first example of co-production of three products from mixed sugars using B. thailandensis paves the way for improving biorefinery economics.

Biosynthesis and properties of polyhydroxyalkanoates synthesized from mixed C5 and C6 sugars obtained from hardwood hydrolysis.

Glucose and xylose are fermentable sugars readily available from lignocellulosic biomass, and are a sustainable carbon substrate supporting industrial biotechnology. Three strains were assessed in this work - Paraburkholderia sacchari, Hydrogenophaga pseudoflava, and Bacillus megaterium - for their ability to uptake both C5 and C6 sugars contained in a hardwood hydrolysate produced via a thermomechanical pulping-based process with concomitant production of poly(3-hydroxyalkanoate) (PHA) biopolymers. In batch conditions, B. megaterium showed poor growth after 12 h, minimal uptake of xylose throughout the cultivation, and accumulated a maximum of only 25 % of the dry biomass as PHA. The other strains simultaneously utilized both sugars, although glucose uptake was faster than xylose. From hardwood hydrolysate, P. sacchari accumulated 57 % of its biomass as PHA within 24 h, whereas H. pseudoflava achieved an intracellular PHA content of 84 % by 72 h. The molecular weight of the PHA synthesized by H. pseudoflava (520.2 kDa) was higher than that of P. sacchari (265.5 kDa). When the medium was supplemented with propionic acid, the latter was rapidly consumed by both strains and incorporated as 3-hydroxyvalerate subunits into the polymer, demonstrating the potential for production of polymers with improved properties and value. H. pseudoflava incorporated 3-hydroxyvalerate subunits with at least a 3-fold higher yield, and produced polymers with higher 3-hydroxyvalerate content than P. sacchari. Overall, this work has shown that H. pseudoflava can be an excellent candidate for bioconversion of lignocellulosic sugars to PHA polymers or copolymers as part of an integrated biorefinery.

The Thermal and Mechanical Properties of Medium Chain-Length Polyhydroxyalkanoates Produced by Pseudomonas putida LS46 on Various Substrates.

Medium chain-length polyhydroxyalkanoates (mcl-PHA) were produced by Pseudomonas putida LS46 cultured with a variety of carbohydrate and fatty acid substrates. The monomer compositions and molecular weights of the polymers varied greatly and was dependent on whether the substrate was metabolized via the fatty acid degradation or the de novo fatty acid synthesis pathways. The highest molecular weights were obtained from medium chain-length fatty acids, whereas low molecular weights were obtained from longer chain-length and more unsaturated fatty acids or carbohydrates. The differences in monomer compositions and molecular weights due to the choice of substrate did not affect the polymer thermal degradation point. The glass transition temperatures varied from -39.4°C to -52.7°C. The melting points, when observed, ranged from 43.2°C to 51.2°C. However, a profound substrate effect was observed on the crystallinity of these polymers. Reduced crystallinity was observed when the monomer compositions deviated away from C8-C10 monomer lengths. The highest crystallinity was observed from medium chain-length fatty acids, which resulted in polymers with the highest tensile strength. The polymer produced from octanoic acid exhibited the highest tensile strength of 4.3 MPa with an elongation-at-break of 162%, whereas the polymers produced from unsaturated, long-chain fatty acids remained amorphous. A comparative analysis of the substrate effect on the physical-mechanical and thermal properties of mcl-PHAs better clarifies the relationship between the monomer composition and their potential applications, and also aids to direct future PHA synthesis research toward properties of interest.

Rheological Behavior of High Cell Density Pseudomonas putida LS46 Cultures during Production of Medium Chain Length Polyhydroxyalkanoate (PHA) Polymers.

The rheology of high-cell density (HCD) cultures is an important parameter for its impact on mixing and sparging, process scale-up, and downstream unit operations in bioprocess development. In this work, time-dependent rheological properties of HCD Pseudomonas putida LS46 cultures were monitored for microbial polyhydroxyalkanoate (PHA) production. As the cell density of the fed-batch cultivation increased (0 to 25 g·L-1 cell dry mass, CDM), the apparent viscosity increased nearly nine-fold throughout the fed-batch process. The medium behaved as a nearly Newtonian fluid at lower cell densities, and became increasingly shear-thinning as the cell density increased. However, shear-thickening behavior was observed at shearing rates of approximately 75 rad·s-1 or higher, and its onset increased with viscosity of the sample. The supernatant, which contained up to 9 g·L-1 soluble organic material, contributed more to the observed viscosity effect than did the presence of cells. Owing to this behavior, the oxygen transfer performance of the bioreactor, for otherwise constant operating conditions, was reduced by 50% over the cultivation time. This study has shown that the dynamic rheology of HCD cultures is an important engineering parameter that may impact the final outcome in PHA cultivations. Understanding and anticipating this behavior and its biochemical origins could be important for improving overall productivity, yield, process scalability, and the efficacy of downstream processing unit operations.

Development of High Cell Density Cultivation Strategies for Improved Medium Chain Length Polyhydroxyalkanoate Productivity Using Pseudomonas putida LS46.

High cell density (HCD) fed-batch cultures are widely perceived as a requisite for high-productivity polyhydroxyalkanoate (PHA) cultivation processes. In this work, a reactive pulse feed strategy (based on real-time CO2 or dissolved oxygen (DO) measurements as feedback variables) was used to control an oxygen-limited fed-batch process for improved productivity of medium chain length (mcl-) PHAs synthesized by Pseudomonas putida LS46. Despite the onset of oxygen limitation half-way through the process (14 h post inoculation), 28.8 ± 3.9 g L-1 total biomass (with PHA content up to 61 ± 8% cell dry mass) was reliably achieved within 27 h using octanoic acid as the carbon source in a bench-scale (7 L) bioreactor operated under atmospheric conditions. This resulted in a final volumetric productivity of 0.66 ± 0.14 g L-1 h-1. Delivering carbon to the bioreactor as a continuous drip feed process (a proactive feeding strategy compared to pulse feeding) made little difference on the final volumetric productivity of 0.60 ± 0.04 g L-1 h-1. However, the drip feed strategy favored production of non-PHA residual biomass during the growth phase, while pulse feeding favored a higher rate of mcl-PHA synthesis and yield during the storage phase. Overall, it was shown that the inherent O2-limitation brought about by HCD cultures can be used as a simple and effective control strategy for mcl-PHA synthesis from fatty acids. Furthermore, the pulse feed strategy appears to be a relatively easy and reliable method for rapid optimization of fed-batch processes, particularly when using toxic substrates like octanoic acid.

The role of dissolved oxygen content as a modulator of microbial polyhydroxyalkanoate synthesis.

Polyhydroxyalkanoates (PHAs) are a diverse class of bio-polymers synthesized by bacteria, usually during imbalanced growth conditions. Optimizing PHA productivity is highly dependent on the bioreactor oxygen transfer rate (OTR), which is an important consideration for process performance and economics, particularly with increasing scale. Relatively few in-depth studies are available regarding the effect of OTR and dissolved oxygen content (DOC) on PHA formation, synthesis rates, composition, and characteristics. This review examines past research studies on the effect of low DOC environments on production of short-chain length (scl-) PHAs, synthesized by both pure and mixed cultures, in order to identify opportunities and gaps concerning the effect of DOC on production of medium-chain length (mcl-) PHAs, an area that has not been studied in detail. The literature indicates that production of scl-PHA (a reductive process) acts as an electron sink allowing cells to maintain balanced redox state at low DOC. Conversely, production of mcl-PHA via fatty acid de novo synthesis (also a reductive process) does not occur to any significant extent in low DOC environments, while mcl-PHA synthesis from fatty acids (an oxidative process) can be promoted in low DOC environments. The monomer composition, molecular mass, as well as physical and thermal properties of the polymer can change in response to OTR, but further research in this area is required for both scl- and mcl-PHAs. Process design and management of bioreactor OTR in PHA production might therefore be directed by the final application of the polymer rather than cost considerations.

Carbon flux to growth or polyhydroxyalkanoate synthesis under microaerophilic conditions is affected by fatty acid chain-length in Pseudomonas putida LS46.

Economical production of medium-chain length polyhydroxyalkanoates (mcl-PHA) is dependent on efficient cultivation processes. This work describes growth and mcl-PHA synthesis characteristics of Pseudomonas putida LS46 when grown on medium-chain length fatty acids (octanoic acid) and lower-cost long-chain fatty acids (LCFAs, derived from hydrolyzed canola oil) in microaerophilic environments. Growth on octanoic acid ceased when the oxygen uptake rate was limited by the oxygen transfer rate, and mcl-PHA accumulated to 61.9% of the cell dry mass. From LCFAs, production of non-PHA cell mass continued at a rate of 0.36 g L-1 h-1 under oxygen-limited conditions, while mcl-PHA accumulated simultaneously to 31% of the cell dry mass. The titer of non-PHA cell mass from LCFAs at 14 h post-inoculation was double that obtained from octanoic acid in bioreactors operated with identical feeding and aeration conditions. While the productivity for octanoic acid was higher by 14 h, prolonged cultivation on LCFAs achieved similar productivity but with twice the PHA titer. Simultaneous co-feeding of each substrate demonstrated the continued cell growth under microaerophilic conditions characteristic of LCFAs, and the resulting polymer was dominant in C8 monomers. Furthermore, co-feeding resulted in improved PHA titer and volumetric productivity compared to either substrate individually. These results suggest that LCFAs improve growth of P. putida in oxygen-limited environments and could reduce production costs since more non-PHA cell mass, the cellular factories required to produce mcl-PHA and the most oxygen-intensive cellular process, can be produced for a given oxygen transfer rate.

Bioreactor Operating Strategies for Improved Polyhydroxyalkanoate (PHA) Productivity.

Microbial polyhydroxyalkanoates (PHAs) are promising biodegradable polymers that may alleviate some of the environmental burden of petroleum-derived polymers. The requirements for carbon substrates and energy for bioreactor operations are major factors contributing to the high production costs and environmental impact of PHAs. Improving the process productivity is an important aspect of cost reduction, which has been attempted using a variety of fed-batch, continuous, and semi-continuous bioreactor systems, with variable results. The purpose of this review is to summarize the bioreactor operations targeting high PHA productivity using pure cultures. The highest volumetric PHA productivity was reported more than 20 years ago for poly(3-hydroxybutryate) (PHB) production from sucrose (5.1 g L-1 h-1). In the time since, similar results have not been achieved on a scale of more than 100 L. More recently, a number fed-batch and semi-continuous (cyclic) bioreactor operation strategies have reported reasonably high productivities (1 g L-1 h-1 to 2 g L-1 h-1) under more realistic conditions for pilot or industrial-scale production, including the utilization of lower-cost waste carbon substrates and atmospheric air as the aeration medium, as well as cultivation under non-sterile conditions. Little development has occurred in the area of fully continuously fed bioreactor systems over the last eight years.

Transcriptomic and proteomic analyses of core metabolism in Clostridium termitidis CT1112 during growth on α-cellulose, xylan, cellobiose and xylose.

BACKGROUND: Clostridium termitidis CT1112 is an anaerobic, Gram-positive, mesophilic, spore-forming, cellulolytic bacterium, originally isolated from the gut of a wood feeding termite Nasusitermes lujae. It has the ability to hydrolyze both cellulose and hemicellulose, and ferment the degradation products to acetate, formate, ethanol, lactate, H2, and CO2. It is therefore ges in gene and gene product expression during growth of C. termitidis on cellobiose, xylose, xylan, and α-cellulose. RESULTS: Correlation of transcriptome and proteome data with growth and fermentation profiles identified putative carbon-catabolism pathways in C. termitidis. The majority of the proteins associated with central metabolism were detected in high abundance. While major differences were not observed in gene and gene-product expression for enzymes associated with metabolic pathways under the different substrate conditions, xylulokinase and xylose isomerase of the pentose phosphate pathway were found to be highly up-regulated on five carbon sugars compared to hexoses. In addition, genes and gene-products associated with a variety of cellulosome and non-cellulosome associated CAZymes were found to be differentially expressed. Specifically, genes for cellulosomal enzymes and components were highly expressed on α-cellulose, while xylanases and glucosidases were up-regulated on 5 carbon sugars with respect to cellobiose. Chitinase and cellobiophosphorylases were the predominant CAZymes expressed on cellobiose. In addition to growth on xylan, the simultaneous consumption of two important lignocellulose constituents, cellobiose and xylose was also demonstrated. CONCLUSION: There are little changes in core-metabolic pathways under the different carbon sources compared. The most significant differences were found to be associated with the CAZymes, as well as specific up regulation of some key components of the pentose phosphate pathway in the presence of xylose and xylan. This study has enhanced our understanding of the physiology and metabolism of C. termitidis, and provides a foundation for future studies on metabolic engineering to optimize biofuel production from natural biomass.