Substrate For Polyhydroxyalkanoates Production Research Articles

Polyhydroxyalkanoate production from rice straw hydrolysate: Insights into feast-famine dynamics and microbial community shifts

Activated sludge contains a versatile microbiome capable of converting wastes into valuable chemicals like polyhydroxyalkanoates (PHA). This study investigated the influence of repeated feast and famine phases on PHA production as well as the corresponding microbial population dynamics using waste activated sludge (WAS) as inoculum. Hydrolysate derived from rice straw was employed as a substrate for PHA production. The 16sRNA analysis results revealed that Corynebacteriaceae (40%), Bacillaceae (23%), and Pseudomonas (5%) were the primary contributors to PHA synthesis. Notably, Bacillaceae and Pseudomonas thrived in all the feast and famine phases. The achieved PHA concentration was 3.5 ± 0.2 g/L, and its structure and composition were assessed using Fourier Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR). The analysis revealed that the PHA consists of a copolymer of hydroxybutyrate (HB) and hydroxyvalerate (HV), specifically identified as Poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV).

Recent trends of biotechnological production of polyhydroxyalkanoates from C1 carbon sources.

Growing concerns over the use of limited fossil fuels and their negative impacts on the ecological niches have facilitated the exploration of alternative routes. The use of conventional plastic material also negatively impacts the environment. One such green alternative is polyhydroxyalkanoates, which are biodegradable, biocompatible, and environmentally friendly. Recently, researchers have focused on the utilization of waste gases particularly those belonging to C1 sources derived directly from industries and anthropogenic activities, such as carbon dioxide, methane, and methanol as the substrate for polyhydroxyalkanoates production. Consequently, several microorganisms have been exploited to utilize waste gases for their growth and biopolymer accumulation. Methylotrophs such as Methylobacterium organophilum produced highest amount of PHA up to 88% using CH4 as the sole carbon source and 52-56% with CH3OH. On the other hand Cupriavidus necator, produced 71-81% of PHA by utilizing CO and CO2 as a substrate. The present review shows the potential of waste gas valorization as a promising solution for the sustainable production of polyhydroxyalkanoates. Key bottlenecks towards the usage of gaseous substrates obstructing their realization on a large scale and the possible technological solutions were also highlighted. Several strategies for PHA production using C1 gases through fermentation and metabolic engineering approaches are discussed. Microbes such as autotrophs, acetogens, and methanotrophs can produce PHA from CO2, CO, and CH4. Therefore, this article presents a vision of C1 gas into bioplastics are prospective strategies with promising potential application, and aspects related to the sustainability of the system.

Finding of Novel Galactose Utilizing Halomonas sp. YK44 for Polyhydroxybutyrate (PHB) Production.

Polyhydroxybutyrate (PHB) is a biodegradable bioplastic with potential applications as an alternative to petroleum-based plastics. However, efficient PHB production remains difficult. The main cost of PHB production is attributed to carbon sources; hence, finding inexpensive sources is important. Galactose is a possible substrate for polyhydroxyalkanoate production as it is abundant in marine environments. Marine bacteria that produce PHB from galactose could be an effective resource that can be used for efficient PHB production. In this study, to identify a galactose utilizing PHB producer, we examined 16 Halomonas strains. We demonstrated that Halomonas cerina (Halomonas sp. YK44) has the highest growth and PHB production using a culture media containing 2% galactose, final 4% NaCl, and 0.1% yeast extract. These culture conditions yielded 8.98 g/L PHB (78.1% PHB content (w/w)). When galactose-containing red algae (Eucheuma spinosum) hydrolysates were used as a carbon source, 5.2 g/L PHB was produced with 1.425% galactose after treatment with activated carbon. Since high salt conditions can be used to avoid sterilization, we examined whether Halomonas sp. YK44 could produce PHB in non-sterilized conditions. Culture media in these conditions yielded 72.41% PHB content. Thus, Halomonas sp. YK44 is robust against contamination, allowing for long-term culture and economical PHB production.

Exploitation of wasted bread as substrate for polyhydroxyalkanoates production through the use of Haloferax mediterranei and seawater.

The use of the halophile microorganism Haloferax mediterranei, able to synthesize poly(hydroxybutyrate-hydroxyvalerate) (PHBV), is considered as a promising tool for the industrial production of bioplastic through bioprocessing. A consistent supplementation of the growth substrate in carbohydrates and minerals is overall necessary to allow its PHBV production. In this work, wasted bread was used as substrate for bioplastic production by microbial fermentation. Instead of the consistent and expensive minerals supplement required for Hfx. mediterranei DSM1411 growth, microfiltered seawater was added to the wasted bread-derived substrate. The suitable ratio of wasted bread homogenate and seawater, corresponding to 40:60, was selected. The addition of proteases and amylase to the bread homogenate promoted the microbial growth but it did not correspond to the increase of bioplastic production by the microorganism, that reach, under the experimental conditions, 1.53 g/L. An extraction procedure of the PHBV from cells, based on repeated washing with water, followed or not by a purification through ethanol precipitation, was applied instead of the conventional extraction with chloroform. Yield of PHBV obtained using the different extraction methods were 21.6 ± 3.6 (standard extraction/purification procedure with CHCl3:H2O mixture), 24.8 ± 3.0 (water-based extraction), and 19.8 ± 3.3 mg PHAs/g of wasted bread (water-based extraction followed by ethanol purification). Slightly higher hydroxyvalerate content (12.95 vs 10.78%, w/w) was found in PHBV obtained through the water-based extraction compared to the conventional one, moreover, the former was characterized by purity of 100% (w/w). Results demonstrated the suitability of wasted bread, supplemented with seawater, to be used as substrate for bioplastic production through fermentation. Results moreover demonstrated that a solvent-free extraction, exclusively based on osmotic shock, could be used to recover the bioplastic from cells.

Production of a newly discovered PHA family member with an isobutyrate-fed enrichment culture

Using microbial enrichment cultures for the production of waste-derived polyhydroxyalkanoates (PHAs) is a promising technology to recover secondary resources. Volatile fatty acids (VFAs) form the preferred substrate for PHA production. Isobutyrate is a VFA appearing in multiple waste valorization routes, such as anaerobic fermentation, chain elongation, and microbial electrosynthesis, but has never been assessed individually on its PHA production potential. This research investigates isobutyrate as sole carbon source for a microbial enrichment culture in comparison to its structural isomer butyrate. The results reveal that the enrichment of isobutyrate has a very distinct character regarding microbial community development, PHA productivity, and even PHA composition. Although butyrate is a superior substrate in almost every aspect, this research shows that isobutyrate-rich waste streams have a noteworthy PHA-producing potential. The main finding is that the dominant microorganism, a Comamonas sp., is linked to the production of a unique PHA family member, poly(3-hydroxyisobutyrate) (PHiB), up to 37% of the cell dry weight. This is the first scientific report identifying microbial PHiB production, demonstrating that mixed microbial communities can be a powerful tool for discovery of new metabolic pathways and new types of polymers.Key points• PHiB production is a successful storage strategy in an isobutyrate-fed SBR• Isomers isobutyrate and butyrate reveal a very distinct PHA production behavior• Enrichments can be a tool for discovery of new metabolic pathways and polymersGraphical abstract

Tung Oil-Based Production of High 3-Hydroxyhexanoate-Containing Terpolymer Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate-co-3-Hydroxyhexanoate) Using Engineered Ralstonia eutropha.

Polyhydroxyalkanoates (PHAs) are attractive new bioplastics for the replacement of plastics derived from fossil fuels. With their biodegradable properties, they have also recently been applied to the medical field. As poly(3-hydroxybutyrate) produced by wild-type Ralstonia eutropha has limitations with regard to its physical properties, it is advantageous to synthesize co- or terpolymers with medium-chain-length monomers. In this study, tung oil, which has antioxidant activity due to its 80% α-eleostearic acid content, was used as a carbon source and terpolymer P(53 mol% 3-hydroxybytyrate-co-2 mol% 3-hydroxyvalerate-co-45 mol% 3-hydroxyhexanoate) with a high proportion of 3-hydroxyhexanoate was produced in R. eutropha Re2133/pCB81. To avail the benefits of α-eleostearic acid in the tung oil-based medium, we performed partial harvesting of PHA by using a mild water wash to recover PHA and residual tung oil on the PHA film. This resulted in a film coated with residual tung oil, showing antioxidant activity. Here, we report the first application of tung oil as a substrate for PHA production, introducing a high proportion of hydroxyhexanoate monomer into the terpolymer. Additionally, the residual tung oil was used as an antioxidant coating, resulting in the production of bioactive PHA, expanding the applicability to the medical field.

Identification and Quantification of Polycyclic Aromatic Hydrocarbons in Polyhydroxyalkanoates Produced from Mixed Microbial Cultures and Municipal Organic Wastes at Pilot Scale.

Polyhydroxyalkanoates (PHAs) are well-known biodegradable plastics produced by various bacterial strains, whose major drawback is constituted by the high cost of their synthesis. Producing PHAs from mixed microbial cultures and employing organic wastes as a carbon source allows us to both reduce cost and valorize available renewable resources, such as food waste and sewage sludge. However, different types of pollutants, originally contained in organic matrices, could persist into the final product, thus compromising their safety. In this work, the exploitation of municipal wastes for PHA production is evaluated from the environmental and health safety aspect by determining the presence of polycyclic aromatic hydrocarbons (PAHs) in both commercial and waste-based PHA samples. Quantification of PAHs by gas chromatography-mass spectrometry on 24 PHA samples obtained in different conditions showed very low contamination levels, in the range of ppb to a few ppm. Moreover, the contaminant content seems to be dependent on the type of PHA stabilization and extraction, but independent from the type of feedstock. Commercial PHA derived from crops, selected for comparison, showed PAH content comparable to that detected in PHAs derived from organic fraction of municipal solid waste. Although there is no specific regulation on PAH maximum levels in PHAs, detected concentrations were consistently lower than threshold limit values set by regulation and guidelines for similar materials and/or applications. This suggests that the use of organic waste as substrate for PHA production is safe for both the human health and the environment.

Utilisation of tuna condensate waste from the canning industry as a novel substrate for polyhydroxyalkanoate production

Tuna condensate, an organic-rich by-product from the tuna canning industry, was assessed as a substrate for polyhydroxyalkanoate (PHA) production using Cupriavidus necator TISTR 1095. The effect of cultivation parameters on PHA accumulation was studied, including substrate concentration, carbon to nitrogen (C/N) ratio, initial pH-value control and fermentation strategies. For the bacterium, a biomass of 3.8 ± 0.1 g/L, PHA of 1.64 ± 0.1 g/L with PHA productivity of 0.027 g/L.h were obtained under batch cultivation using 100% tuna condensate with a C/N ratio of 88:1 and no control of pH. However, the PHA production was increased 1.3-fold when repeated-batch cultivation was applied. The highest biomass (7.5 ± 0.1 g/L) and PHA (3.8 ± 0.1 g/L) with 0.063 g/L.h of PHA productivity were achieved after the third cycle of repeated-batch cultivation. High chemical oxygen demand (COD) removal efficiency of 70% under the optimal condition was also demonstrated. The polymers generated by C. necator TISTR 1095 were characterised. The size of polymer granules was in the range of 0.7-0.8 μm. The polymer produced in the optimal medium under batch and repeated-batch cultivation was identified as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with a 20 mol% of 3-hydroxyvalerate. The molecular mass (Mn) and polydispersity of the polymer were 2 × 106 Da and 2.5, respectively. The results demonstrated that tuna condensate could be used as a cheap substrate for PHA production on an industrial scale.

Improving polyhydroxyalkanoates production in phototrophic mixed cultures by optimizing accumulator reactor operating conditions

Polyhydroxyalkanoates (PHAs) production with phototrophic mixed cultures (PMCs) has been recently proposed. These cultures can be selected under the permanent presence of carbon and the PHA production can be enhanced in subsequent accumulation steps. To optimize the PHA production in accumulator reactors, this work evaluated the impact of 1) initial acetate concentration, 2) light intensity, 3) removal of residual nitrogen on the culture performance. Results indicate that low acetate concentration (<30 CmM) and specific light intensities around 20 W/gX are optimal operating conditions that lead to high polyhydroxybutyrate (PHB) storage yields (0.83 ± 0.07 Cmol-PHB/Cmol-Acet) and specific PHB production rates of 2.21 ± 0.07 Cmol-PHB/Cmol X d. This rate is three times higher than previously registered in non-optimized accumulation tests and enabled a PHA content increase from 15 to 30% in <4 h. Also, it was shown for the first time, the capability of a PMC to use a real waste, fermented cheese whey, to produce PHA with a hydroxyvalerate (HV) content of 12%. These results confirm that fermented wastes can be used as substrates for PHA production with PMCs and that the energy levels in sunlight that lead to specific light intensities from 10 to 20 W/gX are sufficient to drive phototrophic PHA production processes.

Evaluation of Polyhydroxyalkanoates Production by Cupriavidus necator and Burkholderia cepacia Strains Using Sunflower Seed and Oil

Polyhydroxyalkanoates (PHAs) produced from alternative substrates are a promising option for petroleum based plastics substitution, once the synthetic materials exhibit low degradability resulting in their accumulation in natural environments, which causes serious damaging consequences to the planet ecosystems. PHAs are natural polyesters stored intracellularly by some bacteria strains as an energy source under the limitation of essential nutrients for their cellular growth. In this sense, the present study aimed to evaluate the potential of sunflower seed and sunflower oil as substrates for PHA production by Cupriavidus necator IPT 026 and IPT 027, and Burkholderia cepacia IPT 400 and IPT 119 (15 g L−1 of substrate, pH 7.0, 180 rpm, 72 h shake flask cultivation). All strains evaluated were able to produce PHAs using the substrates offered. Among the four analyzed strains, C. necator IPT 026 presented the highest productivity consuming sunflower seed, synthesizing 4.38 ± 0.17 g L−1 of polymer in the cultivation medium. All biopolymers produced were characterized by Fourier Transform Infrared, displaying the typical functional groups of PHAs. All biomaterials revealed to have high thermal stability (Ton set ≥ 238 °C), and presented low indexes of crystallinity (CI < 40%), important parameters for their industrial applicability.

Production of polyhydroxyalkanoates (PHA) from a non‐lignocellulosic component of sugarcane bagasse: fueling a biobased economy

AbstractThe present study examines the use of residual sugar from sugarcane bagasse for the production of polyhydroxyalkanoates (PHA), a class of biodegradable microbial polymers. To achieve this, 26 bacterial isolates were screened for PHA production. From the strains isolated, ART_MKT2E was found to produce PHA and was selected to discover its capability to use a bagasse‐based medium (BGM). To date, the lignocellulosic component of bagasse has been used as a substrate by treating bagasse with acids. This study demonstrates the use of residual sugar from bagasse as a substrate for PHA production. The concentration of bagasse filtrate was found to be optimum at 60%, along with yeast extract and additional salts (pH 7.0 ± 0.5). The maximum PHA production (w/w) was 55%. These results indicate the successful use of non‐lignocellulosic BGM. The remaining lignocellulosic component of bagasse could be fed as a substrate for another production process, promoting a sustainable, biobased economy. © 2018 Society of Chemical Industry and John Wiley &amp; Sons, Ltd

Recovery and concentration of thermally hydrolysed waste activated sludge derived volatile fatty acids and nutrients by microfiltration, electrodialysis and struvite precipitation for polyhydroxyalkanoates production

A novel chain of processes was proposed for the first time to convert thermally hydrolysed waste activated sludge (WAS) into concentrated volatile fatty acids (VFAs) and nutrient effluent stream for the production of biodegradable polyhydroxyalkanoates (PHAs). The integrated process involved a sequence of anaerobic fermentation followed by microfiltration (MF) and conventional electrodialysis (CED). The VFAs produced were primarily acetic and n-butyric acids, with concentrations of 3.27 and 4.37g/L, respectively, within a total of 11.73g/L of short chain organic acids produced from an organic loading rate of 20g VS/Lday and 2days hydraulic retention time anaerobic fermentation. MF achieved over 80% recovery rates of VFAs and ammonium. CED concentrated effectively the MF recovered stream with 92% of VFAs and ammonium transferred to the concentrated stream i.e.19.82g VFAs/L (∼32g CODVFAs/L) and ammonium of 3.02g/L. The excessive ammonium was removed through struvite precipitation before usage as substrate for PHAs production. This novel upfront process resulted in a 5-fold increase in PHA%/cell dry matter compared with the untreated thermally hydrolysed anaerobically acidified WAS stream. The results illustrate a promising market for the use of WAS through membrane and precipitation separation techniques for biomaterials production. The novel engineered processing system has much wider applications within the chemical industry and the potential for a very effective high rate methanogenesis.

Acidogenic spent wash valorization through polyhydroxyalkanoate (PHA) synthesis coupled with fermentative biohydrogen production

The production of polyhydroxyalkanoates (PHAs) by Bacillus tequilensis biocatalyst using spent wash effluents as substrate was evaluated to increase the versatility of the existing PHA production process and reduce production cost. In this study, spent wash was used as a substrate for biohydrogen (H2) production and the resulting acidogenic effluents were subsequently employed as substrate for PHA production. Maximum H2 production of 39.8L and maximum PHA accumulation of 40% dry cell weight was attained. Good substrate removal associated with decrement in acidification (53% to 15%) indicates that the VFA generated were effectively utilized for PHA production. The PHA composition showed presence of copolymer [P (3HB-co-3HV)] with varying contents of hydroxybutyrate and hydroxyvalerate. The results obtained suggest that the use of spent wash effluents as substrate can considerably reduce the production cost of PHA with simultaneous waste valorization. PHA synthesis with B. tequilensis and spent wash effluents is reported for the first time.

The initial metabolic conversion of levulinic acid in Cupriavidus necator

Levulinic acid or 4-ketovaleric acid is a potential renewable substrate for production of polyhydroxyalkanoates. In this work, the initial reactions of LA metabolism by Cupriavidus necator were examined in vitro. The organic acid was converted by membrane-bound crude enzymes obtained from the cells pre-grown on LA, while no LA activity was detected from cells pre-grown on acetic acid. Acetyl-CoA and propionyl-CoA were two major intermediates in the initial reactions of LA conversion. A mass balance on propionyl-CoA accounts for 84 mol% of LA added in vitro. It explains an interesting phenomenon that 3-hydroxbutyrate and 3-hydroxyvalerate are two major monomers of the biopolyester formed from LA, instead of 4-hydroxvalerate that has the similar chemical structure of LA as the precursor. A Monod model was used to describe the kinetics of LA utilization as a sole carbon source or a co-substrate of glucose and fructose. The μ(max) and K(m) of LA alone were 0.26 h⁻¹ and 0.01 g/L, respectively. The content and composition of PHA are also dependent on the culture conditions such as carbon to nitrogen ratio. The in vitro observation is supported by the high utilization rate of LA and the high molar percentage of 3HB and 3HV in the PHA derived from LA.

Feasibility study on the utilization of rubber latex effluent for producing bacterial biopolymers.

Rubber latex effluent is a polluting source that has a high biochemical oxygen demand (BOD). It is estimated that about 100 million liters of effluent are discharged daily from rubber processing factories. Utilization of this effluent such as the use of a coupled system not only can reduce the cost of treatment but also yield a fermentation feedstock for the production of bioplastic. This study initially was carried out to increase the production of organic acids by anaerobic treatment of rubber latex effluent. It was found that through anaerobic treatment the concentration of organic acids did not increase. Consequently, separation of organic acids from rubber latex effluent by anion exchange resin was examined as a preliminary study of recovering acetic and propionic acids. However, the suspended solids (SS) content in the raw effluent was rather high which partially blocked the ion-exchange columns. Lime was used to remove the SS in the rubber latex effluent. After the lime precipitation process, organic acids were found to adsorb strongly onto the anion exchange resin. Less adsorption of organic acids onto the resin was observed before the lime precipitation. This was probably due to more sites being occupied by colloidal particles on the resin thus inhibiting the adsorption of organic acids. The initial concentration of organic acids in the raw effluent was 3.9 g/L. After ion exchange, the concentration of the organic acids increased to 27 g/L, which could be utilized for production of polyhydroxyalkanoates (PHA). For PHA accumulation stage, concentrated rubber latex effluent obtained from ion exchange resins and synthetic acetic acid were used as the carbon source. Quantitative analyses from fed batch culture via HPLC showed that the accumulation of PHA in Alcaligenes eutrophus was maximum with a concentration of 1.182 g/L when cultivated on synthetic acetic acid, corresponding to a yield of 87% based on its cell dry weight. The dry cell weight increased from 0.71 to 1.67 g/L. On the other hand, using concentrated rubber latex effluent containing acetic and propionic acids resulted in reduced PHA content by dry weight (14%) but the dry cell weight increased from 0.49 to 1.30 g/L. The results clearly indicated that the cells grow well in rubber latex effluent but no PHA was accumulated. This could be due to the high concentration of propionic acid in culture broth or other factors such as heavy metals. Thus further work is required before rubber latex effluent can be utilized as a substrate for PHA production industrially.