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

Impact of phosphate limitation on PHA production in a feast-famine process

In the growing polymer industry, the interest of researchers is captivated by bioplastics production with biodegradable and biocompatible properties. This study examines the polyhydroxyalkanoates (PHA) production performance of individual Lysinibacillus sp. RGS and Ralstonia eutropha ATCC 17699 and their co-culture by utilizing sugarcane bagasse (SCB) hydrolysates. Initially, acidic (H2SO4) and acidified sodium chlorite pretreatment was employed for the hydrolysis of SCB. The effects of chemical pretreatment on the SCB biomass assembly and its chemical constituents were studied by employing numerous analytical methods. Acidic pretreatment under optimal conditions showed effective delignification (60%) of the SCB biomass, leading to a maximum hydrolysis yield of 74.9 ± 1.65% and a saccharification yield of 569.0 ± 5.65 mg/g of SCB after enzymatic hydrolysis. The resulting SCB enzymatic hydrolysates were harnessed for PHA synthesis using individual microbial culture and their defined co-culture. Co-culture strategy was found to be effective in sugar assimilation, bacterial growth, and PHA production kinetic parameters relative to the individual strains. Furthermore, the effects of increasing acid pretreated SCB hydrolysates (20, 30, and 40 g/L) on cell density and PHA synthesis were studied. The effects of different cost-effective nutrient supplements and volatile fatty acids (VFAs) with acid pretreated SCB hydrolysates on cell growth and PHA production were studied. By employing optimal conditions and supplementation of corn steep liquor (CSL) and spent coffee waste extracted oil (SCGO), the co-culture produced maximum cell growth (DCW: 11.68 and 11.0 g/L), PHA accumulation (76% and 76%), and PHA titer (8.87 and 8.36 g/L), respectively. The findings collectively suggest that the development of a microbial co-culture strategy is a promising route for the efficient production of high-value bioplastics using different agricultural waste biomass.

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

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.

Polyhydroxyalkanoate production during electroactive biofilm formation and stabilization in wetland microbial fuel cells for petroleum hydrocarbon bioconversion.

Effect of composition of volatile fatty acids on yield of polyhydroxyalkanoates and mechanisms of bioconversion from activated sludge

New plants

Optimal production of polyhydroxyalkanoates (PHA) in activated sludge fed by volatile fatty acids (VFAs) generated from alkaline excess sludge fermentation

Bio-oil upgrading strategies to improve PHA production from selected aerobic mixed cultures

Photosynthetic mixed culture polyhydroxyalkanoate (PHA) production from individual and mixed volatile fatty acids (VFAs): Substrate preferences and co-substrate uptake

Synthetic plastics are in great demand in society due to their diversified properties, but they cause environmental pollution due to their non-biodegradable nature. Therefore, synthetic plastics are in need to be replaced with biodegradable plastics. Polyhydroxyalkanoates (PHAs), bacterial biopolymers are natural alternative to synthetic plastics. These are present inside the bacterial cytoplasm in granular form. Presently, the production cost of PHA is high due to expensive carbon substrates used in its biosynthesis. Therefore, this study focuses on the cost-effective production of PHA using waste carbon sources. Rice bran and sugarcane molasses were used as the carbon source for PHA production from Bacillus subtilis, Bacillus cereus, Alcaligenes sp. and Pseudomonas aeruginosa. PHA production from these bacterial strains was confirmed through Sudan Black-B screening. With rice bran, as carbon source, the highest PHA yield obtained was for P. aeruginosa, which yielded 93.7% and lowest was 35.5% for B. cereus. Surprisingly, B. cereus produced the highest cell dry mass (0.045 g/L) but its extracted PHA contents were lowest being only 0.02 g/L. Alcaligenes sp. with 0.031 g/L CDM yielded 87.1% PHA. B. subtilis had a CDM 0.029 g/L, 0.02 g/L PHA content and a yield of 69.10%. In the case of sugarcane molasses, P. aeruginosa produced 95% PHA yield, 0.02 g/L CDM, and 0.019 g/L PHA content. Alcaligenes sp. yielded 90.9% PHA, 0.011 g/L CDM, and 0.01 g/L PHA content. B. subtilis produced 91.6% PHA yield, 0.012 g/L CDM, 0.011 g/L PHA content; B. cereus produced 80% PHA yield, 0.015 g/L CDM, 0.012 g/L PHA content at 37°C, pH 7. Higher concentrations of carbon sources increased the CDM and decreased the PHA yield. The maximum yield of PHA was obtained from sugarcane molasses. 24-48 h of incubation was optimal for B. subtilis and B. cereus, while for Alcaligenes and P. aeruginosa incubation time of 48-96 h was desirable for higher PHA yield. The extracted biopolymers were analyzed by Fourier transform infrared spectroscopy (FTIR), which identified the extracted biopolymers as poly-3-hydroxybutyrate P(3HB). The thermal properties of the extracted biopolymers, such as melting temperatures, were analyzed by differential scanning calorimetry (DSC), which confirmed the thermal stability.

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.

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