Developing Microbial Co-Culture System for Enhanced Polyhydroxyalkanoates (PHA) Production Using Acid Pretreated Lignocellulosic Biomass.

Process optimization for polyhydroxyalkanoate (PHA) production from waste via microbial enrichment cultures

Reducing the effect of non-volatile fatty acids (non-VFAs) on polyhydroxyalkanoates (PHA) production from fermented thermal-hydrolyzed sludge

Synthesis and characterization of bioplastics, Polyhydroxyalkanoates produced from sugarcane bagasse by using Bacillus cereus

Genetic engineering strategies for sustainable polyhydroxyalkanoate (PHA) production from carbon-rich wastes

Cell growth and polyhydroxyalkanoate (PHA) biosynthesis are two key traits in PHA production from lignin or its derivatives. However, the links between them remain poorly understood. Here, the transcription levels of key genes involved in PHA biosynthesis were tracked in Pseudomonas putida strain A514 grown on vanillic acid as the sole carbon source under different levels of nutrient availability. First, enoyl-coenzyme A (CoA) hydratase (encoded by phaJ4) is stress induced and likely to contribute to PHA synthesis under nitrogen starvation conditions. Second, much higher expression levels of 3-hydroxyacyl-acyl carrier protein (ACP) thioesterase (encoded by phaG) and long-chain fatty acid-CoA ligase (encoded by alkK) under both high and low nitrogen (N) led to the hypothesis that they likely not only have a role in PHA biosynthesis but are also essential to cell growth. Third, 40 mg/liter PHA was synthesized by strain AphaJ4C1 (overexpression of phaJ4 and phaC1 in strain A514) under low-N conditions, in contrast to 23 mg/liter PHA synthesized under high-N conditions. Under high-N conditions, strain AalkKphaGC1 (overexpression of phaG, alkK, and phaC1 in A514) produced 90 mg/liter PHA with a cell dry weight of 667 mg/liter, experimentally validating our hypothesis. Finally, further enhancement in cell growth (714 mg/liter) and PHA titer (246 mg/liter) was achieved in strain Axyl_alkKphaGC1 via transcription level optimization, which was regulated by an inducible strong promoter with its regulator, XylR-PxylA, from the xylose catabolic gene cluster of the A514 genome. This study reveals genetic features of genes involved in PHA synthesis from a lignin derivative and provides a novel strategy for rational engineering of these two traits, laying the foundation for lignin-consolidated bioprocessing.IMPORTANCE With the recent advances in processing carbohydrates in lignocellulosics for bioproducts, almost all biological conversion platforms result in the formation of a significant amount of lignin by-products, representing the second most abundant feedstock on earth. However, this resource is greatly underutilized due to its heterogeneity and recalcitrant chemical structure. Thus, exploiting lignin valorization routes would achieve the complete utilization of lignocellulosic biomass and improve cost-effectiveness. The culture conditions that encourage cell growth and polyhydroxyalkanoate (PHA) accumulation are different. Such an inconsistency represents a major hurdle in lignin-to-PHA bioconversion. In this study, we traced and compared transcription levels of key genes involved in PHA biosynthesis pathways in Pseudomonas putida A514 under different nitrogen concentrations to unveil the unusual features of PHA synthesis. Furthermore, an inducible strong promoter was identified. Thus, the molecular features and new genetic tools reveal a strategy to coenhance PHA production and cell growth from a lignin derivative.

Effect of syngas fermentation effluents containing volatile fatty acids on polyhydroxyalkanoates production.

Polyhydroxyalkanoates (PHA) are bioplastics produced by few bacteria as intracellular lipid inclusions under excess carbon source and nutrient-deprived conditions. These polymers are biodegradable and resemble petroleum-based plastics. The rising environmental concerns have increased the demand for PHA, but the low yield in wild-type bacterial strains limits large-scale production. An improvement in the PHA production can be achieved by genetically engineering the wild-type bacterial strains by removing competitive pathways that divert the metabolites away from PHA biosynthesis, cloning strong promotors to overexpress the genes involved in PHA biosynthesis and constructing non-native metabolic pathways that feed the metabolites for PHA production. The desired monomers in the PHA polymers were obtained by elimination of genes involved in PHA biosynthetic pathway. The chain length degradation specific-gene deletion of β-oxidation pathway resulted in the accumulation of PHA monomers having high carbon chain length. A controlled accumulation of monomers in the PHA polymer was achieved by constructing novel pathways in the bacteria and deleting native genes of competitive pathways from the genome of non-PHA producers. The present review attempts to showcase the novel genetic modification approaches conducted so far to enhance the PHA production with a special focus on metabolic pathway gene deletion in various bacteria.

Co-feeding phenolic acids with fatty acids for polyhydroxyalkanoate (PHA) synthesis in Pseudomonas putida KT2440 using continuous culture conditions.

Polyhydroxyalkanoates (PHAs) could be used to make sustainable, biodegradable plastics. However, the precise and accurate mechanistic modeling of PHA biosynthesis, especially medium-chain-length PHA (mcl-PHA), for yield improvement remains a challenge to biology. PHA biosynthesis is typically triggered by nitrogen limitation and tends to peak at an optimal carbon-to-nitrogen (C/N) ratio. Specifically, simulation of the underlying dynamic regulation mechanisms for PHA bioprocess is a bottleneck owing to surfeit model complexity and current modeling philosophies for uncertainty. To address this issue, we proposed a quantum-like decision-making model to encode gene expression and regulation events as hidden layers by the general transformation of a density matrix, which uses the interference of probability amplitudes to provide an empirical-level description for PHA biosynthesis. We implemented our framework modeling the biosynthesis of mcl-PHA in Pseudomonas putida with respect to external C/N ratios, showing its optimization production at maximum PHA production of 13.81% cell dry mass (CDM) at the C/N ratio of 40:1. The results also suggest the degree of P. putida’s preference in channeling carbon towards PHA production as part of the bacterium’s adaptative behavior to nutrient stress using quantum formalism. Generic parameters (kD, kN and theta θ) obtained based on such quantum formulation, representing P. putida’s PHA biosynthesis with respect to external C/N ratios, was discussed. This work offers a new perspective on the use of quantum theory for PHA production, demonstrating its application potential for other bioprocesses.

Biovalorization of lignin derived compounds with molasses as co-substrate for polyhydroxyalkanoate production

Background and objective: Polyhydroxyalkanoates are biodegradable polyesters synthesized by some prokaryotic organisms from renewable sources. Medium-chain-length Polyhydroxyalkanoates show interesting properties as elastic and adhesive specialty polymers. Medium-chain-length Polyhydroxyalkanoates producers such as Pseudomonas sp. have demonstrated high yields on fats and oils. Camelina sativa is non-food chain competing crop, whose seed contain about 43% (w w -1 ) oil in dry matter with about 90% (w w -1 ) of unsaturated fatty acids. Camelina oil was for the first time tested for the production of medium-chainlength Polyhydroxyalkanoates by different Pseudomonas strains. Material and methods: The production of Polyhydroxyalkanoate was evaluated in a nitrogen-limited minimal medium supplemented with crude Camelina oil or saponified oil to compare the production capability of Pseudomonas sp. strains. A phosphates-limited medium was used to optimize polyhydroxyalkanoate production in fed-batch assays. Experiments were carried out by duplicates. Results and conclusion: Pseudomonas resinovorans was used for direct fermentation of Camelina oil without prior hydrolysis. A first approach to process development in bioreactor has provided up to 40% (w w -1 ) polymer content, matching highest medium-chain-length polyhydroxyalkanoates titer reported from plant oils (13.2 g l -1 ). Camelina oil was shown to be a suitable substrate for production of medium-chain-length polyhydroxyalkanoates. This non-food vegetable oil gave good results for Pseudomonas resinovorans DSM 21078 without any pre-treatment. Conflict of interest : The authors declare no conflict of interest.

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters that a variety of microorganisms accumulate as carbon and energy storage molecules under starvation conditions in the presence of excess carbon. Anoxygenic photosynthetic bacteria exhibit a variety of growth styles and high PHA production activity. Here, we characterized PHA production by four marine purple non-sulfur bacteria strains (Rhodovulum sulfidophilum, Rhodovulum euryhalinum, Rhodovulum imhoffii, and Rhodovulum visakhapatnamense) under different growth conditions. Unlike the well-studied PHA-producing bacteria, nutrient limitation is not appropriate for PHA production in marine purple non-sulfur bacteria. We found that marine purple non-sulfur bacteria did not accumulate PHA under aerobic conditions in the presence of malate and pyruvate. Interestingly, PHA accumulation was observed upon the addition of acetate under aerobic conditions but was not observed upon the addition of reductants, suggesting that an acetate-dependent pathway is involved in PHA accumulation. Gene expression analysis revealed that the expression of isocitrate dehydrogenase in the tricarboxylic acid (TCA) cycle decreased under aerobic conditions and increased with the addition of acetate, indicating that TCA cycle activity is involved in PHA production under aerobic conditions. We also found that expression of PdhRrs, which belongs to the GntR family of transcription regulators, in Rhodovulum sulfidophilum was upregulated upon the addition of acetate. Taken together, the results show that the changes in the metabolic state upon the addition of acetate, possibly regulated by PdhR, are important for PHA production under aerobic conditions in marine purple non-sulfur bacteria.

Valorization of ricotta cheese exhausted whey (RCEW), a dairy by-product generated in large quantities worldwide, is essential to mitigate its environmental impact and unlock its economic potential. This study explores the use of RCEW as a substrate for polyhydroxyalkanoate (PHA) production by Azohydromonas lata DSM 1123. The substrate was characterized by low protein and fat contents and a relevant lactose concentration (3.81%, w/v). Due to A. lata's inability to directly metabolize lactose, β-galactosidase supplementation was necessary. Mineral supplementation of pasteurized RCEW significantly improved both microbial biomass and PHA synthesis, achieving up to 25.94% intracellular PHA content, whereas pre-adaptation trials failed to enhance strain performance. Moderate nitrogen limitation in the substrate (C/N ratio 44) favored PHA synthesis (0.55 g/L) and 32.74% intracellular accumulation. Thermal treatments decreased initial microbial contamination, hence a balanced mixture of pasteurized-sterilized (75:25) substrate was used to modulate RCEW protein content without the inclusion of additional technological or chemical processing steps and without lactose loss or dilution. Bioreactor trials using optimized RCEW pre-treatment conditions led to a further increase in biomass (2.36 g/L) and PHA production (0.88 g/L), especially under fed-batch conditions. The extracted polymer was confirmed to be polyhydroxybutyrate (PHB), with high thermal stability and a molecular weight of 5.9 KDa.

Lack of space for disposal and growing environmental concerns regarding non-biodegradable synthetic plastics has fuelled research into the development of eco-friendly biopolymer materials as sustainable alternatives to traditional petroleum derived polymers (Defra et al., 2006; Barnes et al., 2009; Hopewell et al., 2009). The cost of producing bioplastic has also been falling thanks to improved processes. Combining this with the increasing cost of crude oil has made bioplastic prices more competitive with regular plastics, although the cost is still a barrier to market uptake. The global demand for bioplastics is predicted to triple by 2015 to reach >1 million tons per year to represent a $2.9 billion market for opportunity and advancements (Adkins et al., 2012). Considerable research has therefore been carried out into the production of sustainable, biodegradable polymers, amenable to microbial catabolism to CO2 and H2O. A key group of microbial polyesters, widely regarded as potential replacement biopolymers, are the polyhydroxyalkaonates (PHAs) (Koller et al., 2008). In this thesis, PHA production by microbial populations in mixed cultures was investigated in order to obtain a better understanding of the process. A central theme of the thesis is examining the ways in which different organisms within a mixed culture contribute to the net PHA that has been produced by these organisms. We want to answer the question: do all the sub-populations in a mixed culture accumulate PHA in similar ways, so that the PHA content and PHA composition in all the organisms is similar? Secondly, we would like to address the question: in mixed cultures, how do the populations respond to various substrates and feeding strategies with regards to community dynamics and the accumulation of PHA? A prominent aspect of the PhD was to assess the potential for using a buoyant density method to separate populations of PHA accumulating organisms in mixed cultures. The purpose was to try to understand why PHA in mixed cultures has such a broad chemical compositional distribution, containing broad blends of different poly(hydroxybutyrate-co-valerate) (PHBV) copolymers, as discovered and published recently by the UQ research group (Laycock et al., 2013b). The possible reasons are: (1) different populations in mixed cultures accumulate different copolymers, and/or (2) single populations accumulate a broad range of copolymers and/or (3) a single cell can accumulate a range of copolymers intracellularly. Following a long process of optimisation, Percoll buoyant density gradient separation was used to separate mixed cultures into two fractions. It was found that the PHA content in one of the III fractions was higher (24±0.6%) than the other (16±4.9%). It was also found that the intracellular PolyP granules assessed using transmission electron microscopies (TEM) were on average larger and that there were more of them per unit area of cell in the fraction with the higher PHA content. The community population was also found to be different in the separated fractions. But while differences in content and microbial composition were observed, the PHA composition in both fractions was found to be essentially the same (43 – 44 mol% HV), which shows that groups of microbial populations within mixed cultures don’t necessarily generate PHA with unique composition. However, it was also found that biomass separation in Percoll was influenced by factors other than simple PHA content, and that this separation technique was not a generically applicable approach to biomass fractionation based on PHA content. The second aspect of this PhD was addressed through analysing the responses of biomass enriched on fermented whey permeate to different feeding strategies for PHA accumulation. It was found that the microbial community in the enrichment system was in a state of flux. Despite this flux in community, PHA accumulation in the biomass was very consistent, with very similar yields, final polymer contents and biomass growth rates. As expected, the polymer composition (mol% HV content) was primarily controlled by feed composition (ratio of acetic to propionic acids).

Polyhydroxyalkanoates (PHAs) can be used to replace petrochemical plastics in many applications.However, their production has limitation due to production cost.This research was conducted using alternative carbon source from waste to synthesize PHAs by Cupriavidus necator H16.In this study, PHAs were produced from used cooking oil (UCO) and compared with production PHAs from palm oil.Two UCO storage times (4 and 10 weeks) and three UCO concentrations (10, 20 and 30 g/L) were used to determine the most optimum condition for PHA synthesis.The best optimum condition for PHA synthesis was cultivated in the medium containing 30 g/L of UCO at 4 weeks storage.The cell dry weight (CDW) and PHA content were 5.26±0.61g/L and 27.36±2.04wt.%, respectively.These results were similar to cell cultivation using 20 g/L palm oil and 1% of fructose (5.93±0.33 g/L of CDW and 26.96±6.14wt.% of PHA contents).In addition, PHA content from the culture with 10 g/L of UCO stored for 10 weeks was higher than PHA content from the culture with 20 and 30 g/L of UCO.Thus, it can be concluded that UCO could be used in PHA production.