Challenges, Progress, and Future Perspectives for Cyanobacterial Polyhydroxybutyrate Production

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

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

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

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.

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.

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.

It is urgent to expand the market of biodegradable alternatives to oil-derived plastics owing to (i) increasingly limited oil availability/accessibility, and (ii) the dramatic impact of traditional plastics on aquatic life, the food chain, all Earth ecosystems, and ultimately, human health. Polyhydroxyalkanoates (PHAs) are promising biodegradable polymers that can be obtained through microbial fermentation of agro-industrial byproducts, e.g., milk and cheese whey. Here, the PHA-accumulating efficiency of a mixed microbial culture (MMC, derived from activated sludges) grown on dairy byproducts (cheese and scotta whey) was measured. Bioreactor tests featuring temperature and pH control showed that both scotta and pre-treated Toma cheese whey could be used for PHA production by MMC, although scotta cheese whey supported higher PHA yield and productivity. The advantages of open MMCs include their plasticity and versatility to fast changing conditions; furthermore, no growth-medium sterilization is needed prior to fermentation. However, the use of pure cultures of efficient PHA producers may support better metabolic performances. Therefore, PHA-producing strains were isolated from a MMC, leading to the satisfactory identification of two bacterial strains, Citrobacter freundii and Leuconostoc spp., whose ability to accumulate PHAs in synthetic media was confirmed. A more detailed investigation by mass spectrometry revealed that the strain was L. mesenteroides. Although the validation of L. mesenteroides potential to produce PHA through fermentation of agro-industrial byproducts requires further investigations, this is the first study reporting PHA production with the Leuconostoc genus.

During the two years that have passed since the first volume of “Advances in Polyhydroxyalkanoate (PHA) production” was published, the progress in PHA-related research was indeed tremendous, calling for the next, highly bioprocess- and bioengineering-oriented volume. This editorial paper summarizes and puts into context the contributions to this second volume of the Bioengineering Special Issue; it covers highly topical fields of PHA-related R&D activities, covering, beside the pronounced bioengineering-related articles, the fields of the microbiology of underexplored, but probably emerging, PHA production strains from the groups of Pseudomonas, cyanobacteria, methanotrophs, and from the extremophilic domain of haloarchaea. Moreover, novel second-generation lignocellulose feedstocks for PHA production from agriculture to be used in biorefinery concepts, new approaches for fine-tuning the composition of PHA co- and terpolyesters, process simulation for PHA production from methane-rich natural gas, the challenges associated with rheology-governed oxygen transfer in high cell density cultivations, rapid spectroscopic in-line analytics for process monitoring, and the biomedical application of PHA biopolyesters after appropriate advanced processing are the subjects of the presented studies.

Insights into integration of polyhydroxyalkanoates (PHAs) production into wastewater treatment: Comparison of different electron acceptors on system function and PHA-producer enrichment

The literature contains a number of articles about microbially mediated production of polyhydroxyalkanoates (PHAs). These polyesters constitute biodegradable polymers possessing physical properties of thermoplastics or elastomers. Therefore, they can be applied as alternatives to common plastic materials originating from mineral oils. The most common representative of these biopolymers is poly-3-hydroxybutyrate (PHB). PHB constitutes a rather stiff and brittle material with a low extension to break. These mechanical properties can be enormously enhanced by incorporation of different co-monomers into the PHB matrix. This results in co- and terpolyesters possessing mechanical properties similar to high-tech materials. PHAs are synthesized in the cytoplasm of various prokaryotic strains, usually from carbohydrates, but also from other renewable resources. Under unfavorable growth conditions (surplus of carbon source plus limitation of an essential substrate, i.e. nitrogen, phosphate, or oxygen), some strains are able to divert the usual carbon flux (acetyl-CoA synthesized in the central metabolic pathways of the microorganism) from biosynthesis of protein (biomass) constituents to the formation of compounds acting as precursors for the production of PHA, mainly PHB. This typical feature is valid for most PHA producers, e.g., Wautersia eutropha, the production strain of BIOPOL^® (Metabolix Inc., Cambridge, MA) that was commercialized in the 1990s by Zeneca Bio Products and later by Monsanto. Some organisms exist that accumulate PHA during balanced growth at substantial rates. This was first found for strains of Alcaligenes latus. The outstanding properties of these growth-associated PHA producers enabled the development of an industrial polyester production process by the Austrian company Chemie Linz in the late 1980s. Due to a high degree of polymerization, the average molecular weights of PHAs can reach several millions. These polymers are organized in granules and serve as intracellular reserve carbon and energy sources that normally are degraded when external carbon sources are depleted. Because of their outstanding property of complete degradability to water and CO_2, PHAs are embedded in nature’ s closed cycle of carbon. If PHAs are applied instead of fossil-oil-originated polymers, the mass balance of carbon from biomass will be closed, but its durability will be prolonged if compared with the usual biological life cycle of PHA carbon. The fossil fuels energy demand during the span of life of PHAs will not exceed the amount necessary for the industrial production and processing of PHAs itself. The negative effects of CO_2 accumulation in the atmosphere, i.e., the greenhouse effect, will be related merely to the amount originated from energy production and not to the carbon in the biopolymer itself. Therefore, substituting common plastic materials by PHAs significantly lowers the amount of fossil-oil-originated carbon in the cycle of nature. Hence, it can be concluded that there are ecologically sound reasons for using PHAs instead of mineral-oil-originated plastics.

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.

To decrease the polyhydroxyalkanoate (PHA) production cost by supplying renewable carbon sources has been an important aspect in terms of commercializing this biodegradable polymer. The production of biodegradable poly(3-hydroxyalkanoates) (PHA) from raw potato starch by the Bacillus cereus 64-INS strain isolated from domestic sludge has been studied in a lab-scale fermenter. The bacterium was screened for the degradation of raw potato starch by a starch hydrolysis method and for PHA production by Nile blue A and Sudan black B staining. Shake-flask cultures of the bacterium with glucose [2% (w/v)] or raw potato starch [2% (w/v)] produced PHA of 64.35% and 34.68% of dry cell weight (DCW), respectively. PHA production was also carried out in a 5-L fermenter under control conditions that produced 2.78 g/L of PHA and PHA content of 60.53% after 21 hr of fermentation using potato starch as the sole carbon source. Gas chromatography–mass spectroscopy (GC-MS) analyses confirmed that the extracted PHA contained poly(3-hydroxybutyrate) (PHB) as its major constituent (>99.99%) irrespective of the carbon source used. The article describes, for what we believe to be the first time, PHB production being carried out without any enzymatic or chemical treatment of potato starch at higher levels by fermentation. More work is required to optimize the PHB yield with respect to starch feeding strategies.

Polyhydroxyalkanoates (PHA) are the biomaterials isolated naturally from bacterial strains. These are present in granules and accumulated intracellularly for storage and energy uptake in stressed conditions. This work was based on the extraction of polyhydroxyalkanoates from haloarchaeal strains isolated from samples of a salt mine and Halocin activity screening of these isolates. For the screening of polyhydroxyalkanoates, Nile Blue and Sudan Black Staining were performed. After confirmation and theoretical determination, polyhydroxyalkanoates extraction was done by sodium hypochlorite digestion and solvent extraction by chloroform method in combination. Polyhydroxyalkanoates production was calculated along with the determination of biomass. Halocin activity of these strains was also screened at different intervals. Isolated strains were identified by 16S RNA gene sequencing. Polyhydroxyalkanoates polymer was produced in form of biofilms and brittle crystals. Halocin activity was exhibited by four strains, among which confirmed halocin activity was shown by strain K7. The remarkable results showed that polyhydroxyalkanoates can replace synthetic plastics which are not environment friendly as they cause environmental pollution – a major threat to Earth rising gradually. Therefore, by switching to the use of biodegradable bioplastics from the use of synthetic plastics, it would be beneficial to the ecosphere.

Chapter 9 - Production and application of bacterial polyhydroxyalkanoates