Recovery Of Polyhydroxyalkanoates Research Articles

Advances in Microbial Biotechnology for Sustainable Alternatives to Petroleum-Based Plastics: A Comprehensive Review of Polyhydroxyalkanoate Production.

The industrial production of polyhydroxyalkanoates (PHAs) faces several limitations that hinder their competitiveness against traditional plastics, mainly due to high production costs and complex recovery processes. Innovations in microbial biotechnology offer promising solutions to overcome these challenges. The modification of the biosynthetic pathways is one of the main tactics; allowing for direct carbon flux toward PHA formation, increasing polymer accumulation and improving polymer properties. Additionally, techniques have been implemented to expand the range of renewable substrates used in PHA production. These feedstocks are inexpensive and plentiful but require costly and energy-intensive pretreatment. By removing the need for pretreatment and enabling the direct use of these raw materials, microbial biotechnology aims to reduce production costs. Furthermore, improving downstream processes to facilitate the separation of biomass from culture broth and the recovery of PHAs is critical. Genetic modifications that alter cell morphology and allow PHA secretion directly into the culture medium simplify the extraction and purification process, significantly reducing operating costs. These advances in microbial biotechnology not only enhance the efficient and sustainable production of PHAs, but also position these biopolymers as a viable and competitive alternative to petroleum-based plastics, contributing to a circular economy and reducing the dependence on fossil resources.

Evaluation of polyhydroxyalkanoate recovery from food waste by sequencing batch biofilm reactor with high mixed microbial biomass

In this study, the enrichment sequencing batch biofilm reactor (SBBR) and conventional enrichment reactor (SBR) were established, and a new strategy was developed to achieve higher biomass yield and polyhydroxyalkanoate (PHA) production from food waste than those achieved through the conventional method. After the 10th day of an expanded cultivation period, the respective active biomass concentrations in SBR and SBBR were 5.16 and 13.92 g/L, indicating a significant improvement in the biomass yield of mixed microbial culture in the SBBR. The PHA accumulation test revealed that maximum PHA concentration in SBR and SBBR reached 6.60 and 12.70 g/L, respectively, after 80 days of enrichment, indicating that compared to SBR, the SBBR could more effectively improve the PHA accumulation capacity. Meganema, Thauera, and Paracoccus were the dominant PHA-accumulating bacteria in the SBR and SBBR. Additionally, the volumetric productivity of the SBBR was 3.22 g COD/L/d, twice that of the SBR. This study provides new strategies and insights for improving PHA production and reducing the costs of PHA production and reactor investment.

Sustainable transformation: Unlocking energy positivity in municipal wastewater treatment through innovative advanced primary treatment configurations for maximum solids separation

Motivated by the growing need for sustainable wastewater management and environmentally friendly solutions for wastewater treatment in the future, the current study explored all possible configurations for advanced primary treatment (APT) on a long-term assessment, encompassing extensive lab and pilot-scale studies for removing particulate organic carbon (POC) in municipal wastewater treatment plants (WWTPs). This study primarily aimed to have a comprehensive insight into various APT configurations for municipal wastewater treatment. The physical-APT method employed a microsieve (MS) with an adjustable drum to 20 and 100 µm mesh. The flocculation process was integrated with one or two consecutive sedimentation tanks to simulate the chemical-APT method. Lastly, the chemical-physical-APT (ChPh-APT) approach was emulated by combining the flocculation process with the MS technique. Considering the best-case scenario to remove utmost POC, the results illuminated a substantial potential for biogas production enhancement by around 85 % (from 1.3 to 2.4 g (cap. d)-1 converted chemical oxygen demand (COD) to biogas) through ChPh-APT configuration. Furthermore, estimations showed that maximum POC removal can significantly reduce energy demand from 42 to 28 kWh (cap. a)-1 while simultaneously increasing green electricity generation from 11 to 33 kWh (cap. a)-1. This transformation will eliminate dependency on external energy sources and significantly increase by-product generation like volatile fatty acids (VFA), Polyhydroxyalkanoate (PHA), and hydrogen (H2) in the concept of a biorefinery. The possible increase in the production of H2 can be from roughly 32 to 58 L (cap. d)-1, PHA recovery from 1.3 to 2.4 g (cap. d)-1, and VFAs from about 12 to 22 g (cap. d)-1. Consequently, this transformation will enhance the efficiency of municipal WWTPs and turn them into energy-positive, sustainable, and resource-recovery entities.

Physicochemical cell disruption of Bacillus sp. for recovery of polyhydroxyalkanoates: future bioplastic for sustainability

Polyhydroxybutyrate (PHB) is known for wide applications, biocompatibility, and degradability; however, it cannot be commercialized due to conventional recovery using solvents. The present study employed mechanical cell-disruption methods, such as Pestle and mortar, sonication, and glass bead vortexing, for solvent-free extraction of PHA from Bacillus sp. Different time intervals were set for grinding (5, 10, 15 min), sonicating (1, 3 and 5 min), and vortexing (2, 5 and 8 g glass beads with 5, 10 and 15 min each) hence studying their effect on cell lysis to release PHA. Tris buffer containing phenylmethyl sulfonyl fluoride (PMSF) (20 mM Tris–HCl, pH 8.0, 1 mM PMSF) was employed as a lysis buffer to study its action over Bacillus cells. Its presence was checked with the above methods in cell lysis. Sonicating cells for 5 min in the presence of lysis buffer achieved a maximum PHA yield of 45%. Cell lysis using lysis buffer yielded 35% PHA when vortexing with 5 g glass beads for 15 min. Grinding cells for 15 min showed a maximum yield of 34% but lacked a lysis buffer. The overall results indicated that the action of lysis buffer and physical extraction methods improved PHA yield by %. Therefore, the study sought to evaluate the feasibility of applying laboratory methods for cell disruption. These methods can showcase possible opportunities in large-scale applications. The polymer yield results were compared with standard sodium hypochlorite extraction. Confirmation of obtained polymers as polyhydroxy butyrate (PHB) was made through FTIR and 1HNMR characterization.

Combined recovery of polyhydroxyalkanoates and reclaimed water in the mainstream of a WWTP for agro-food industrial wastewater valorisation by membrane bioreactor technology

The present study investigated the combined production of reclaimed water for reuse purposes and polyhydroxyalkanoates (PHA) from an agro-food industrial wastewater. A pilot plant implementing a two-stage process for PHA production was studied. It consisted of a mainstream sequencing batch membrane bioreactor (SBMBR) in which selection of PHA-accumulating organisms and wastewater treatment were carried out in, and a side-stream fed-batch reactor (FBR) where the excess sludge from the SBMBR was used for PHA accumulation. The performance of the SBMBR was compared with that of a conventional sequencing batch reactor (SBR) treating the same wastewater under different food to microorganisms’ ratios (F/M) ranging between 0.125 and 0.650 kgCOD kgTSS−3 d−1. The SBMBR enabled to obtain very high-quality effluent in compliance with the relevant national (Italy) and European regulations (Italian DM 185/03 and EU, 2020/741) in the field of wastewater reclamation, whereas the performances in the SBR collapsed at F/M higher than 0.50 kgCOD kgTSS−1d−1.A maximum intracellular storage of 45% (w/w) and a production yield of 0.63 gPHA L−1h−1 were achieved when the SBMBR system was operated with a F/M ratio close to 0.50 kgCOD kgTSS−1d−1. This resulted approximately 35% higher than those observed in the SBR, since the ultrafiltration membrane avoided the washout of dispersed and filamentous bacteria capable of storing PHA.Furthermore, while maximizing PHA productivity in conventional SBR systems led to process dysfunctions, in the SBMBR system it helped mitigate these issues by reducing membrane fouling behaviour. The results of this study supported the possibility to achieve combined recovery of reclaimed water and high-value added bioproducts using membrane technology, leading the way for agro-food industrial wastewater valorization in the frame of a circular economy model.

An efficient and eco-friendly approach for the sustainable recovery and properties characterization of polyhydroxyalkanoates produced by methanotrophs

Synthetic biodegradable and bio-based polymers have emerged as sustainable alternatives to nonrenewable petroleum-derived polymers which cause serious environmental issues. In particular, polyhydroxyalkanoates (PHA) are promising biopolymers owing to their outstanding biodegradability and biocompatibility. The production of the homopolymer poly(3-hydroxybutyrate) (PHB) and copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from type II methanotrophs via microbial fermentation was presented. For the efficient extraction and recovery of intracellular PHA from methanotrophs, different extraction approaches were investigated including solvent extraction using 1,3-dioxolane as a green solvent, integrated cell lysis and solvent extraction, and cell digestion without the use of organic solvents. Among various extraction approaches, the integrated method exhibited the highest extraction performance, with PHA recovery and purity exceeding 91 % and 93 %, respectively, even when the PHA content of the cells was low. Furthermore, the molecular weight, thermal stability, and mechanical properties of the recovered PHA were comprehensively analyzed to suggest its suitable practical applications. The obtained properties were comparable to that of the commercial PHA products and PHA produced from other microbial species, indicating an efficient recovery of high-quality PHA produced from methanotrophs.

Sustainable Opportunities in the Downstream Processing of the Intracellular Biopolymer Polyhydroxyalkanoate

AbstractBiopolymers have gained popularity as an alternative product to traditional plastics due to environmental concerns. Polyhydroxyalkanoates (PHAs), one of the most well‐known forms of biopolymers, are among the many that have been discovered so far. PHA has a wide range of applications, is biodegradable and compatible with living things, but its expensive extraction and production make it difficult to compete with traditional plastics. Solvents are the most popular extraction technique but have serious economic and environmental downsides. The “green method of extraction” has become a cutting‐edge remedy for addressing the shortcomings of downstream processing. However, developing the extraction technique for affordable and environmentally friendly biopolymer production is a difficult study topic. To make it easier to choose green recovery techniques for future study, this review combines the benefits and drawbacks of the numerous PHA recovery methods that are already in use. Also, advanced green downstream methods are scrutinized that undoubtedly makes PHA replace conventional plastics and reach a market globally promoting sustainability.

Simultaneous treatment of epichlorohydrin wastewater and polyhydroxyalkanoate recovery by halophilic aerobic granular sludge highly enriched by Halomonas sp.

The treatment of epichlorohydrin (ECH) wastewater exists chances for achieving cleaner production. This study initially employed moderately halophilic aerobic granular sludge (HAGS) to treat ECH wastewater, and the resulting HAGS was utilized to recover polyhydroxyalkanoate (PHA). During the acclimation process of HAGS, the chemical oxygen demand removal efficiency stabilized at 70 %. Moreover, due to the high enrichment of Halomonas sp. (relative abundance of 86 ± 0.50 %), the maximum PHA content of wasted HAGS was 52.67 wt% in the fermentation process. Simultaneously, the utilization of nuclear magnetic resonance spectroscopy (1H and 13C spectra) and fourier transform infrared spectroscopy for the structural analysis of polymers revealed that polyhydroxybutyrate was the predominant substance extracted from HAGS. In this study, the innovative use of highly enriched HAGS for treating ECH wastewater and simultaneously recovering PHA not only enables the efficient biological treatment of ECH wastewater but also realizes resource recovery of ECH wastewater.

Biotransformation of starch-based wastewater into bioplastics: Optimization of poly(3-hydroxybutyrate) production by Cupriavidus necator DSM 545 using potato wastewater hydrolysate

Biodegradable biopolymers, such as polyhydroxyalkanoates (PHAs), have emerged as an alternative to petrochemical-based plastics. The present work explores the production of PHAs based on the biotransformation of potato processing wastewater and addresses two different strategies for PHA recovery. To this end, culture conditions for PHA synthesis by Cupriavidus necator DSM 545 were optimized on a laboratory scale using a response surface methodology-based experimental design. Optimal conditions rendered a PHB, poly(3-hydroxybutyrate), accumulation of 83.74 ± 2.37 % (5.1 ± 0.2 gL−1), a 1.4-fold increase compared to the initial conditions. Moreover, polymer extraction with non-halogenated agent improved PHB recovery compared to chloroform method (PHB yield up to 78.78 ± 0.57 %), while maintaining PHB purity. (99.83 ± 4.95 %). Overall, the present work demonstrated the potential valorization of starch-based wastewater by biotransformation into PHBs, a high value-added product, and showed that recovery approaches more eco-friendly than the traditional treatments could be applied to PHB recovery to some extent.

Expression, thermal stability modification and application in PHB degradation of polyhydroxyalkanoate depolymerase from Thermomonospora umbrina

Polyhydroxyalkanoate depolymerase (PHAD) can be used for the degradation and recovery of polyhydroxyalkanoate (PHA). In order to develop a PHAD with good stability under high temperature, PHAD from Thermomonospora umbrina (TumPHAD) was heterelogously expressed in Escherichia coli BL21(DE3). At the same time, a mutant A190C/V240C with enhanced stability was obtained via rational design of disulfide bonds. Characterization of enzymatic properties showed that the mutant A190C/V240C had an optimum temperature of 60 ℃, which was 20 ℃ higher than that of the wild type. The half-life at 50 ℃ was 7 hours, at 50 ℃ which was 21 times longer than that of the wild type. The mutant A190C/V240C was used for the degradation of polyhydroxybutyrate (PHB), one of the typical PHA. At 50 ℃, the degradation rate of PHB being treated for 2 hours and 12 hours was 2.1 times and 3.8 times higher than that of the wild type, respectively. The TumPHAD mutant A190C/V240C obtained in this study shows tolerance to high temperature resistance, good thermal stability and strong PHB degradation ability, which may facilitate the degradation and recovery of PHB.

Visualization of Polyhydroxyalkanoate Accumulated in Waste Activated Sludge.

Polyhydroxyalkanoates (PHAs) can be produced with municipal waste activated sludge from biological wastewater treatment processes. Methods of selective fluorescent staining with confocal laser scanning microscopy (CLSM) were developed and optimized to evaluate the distribution of PHA storage activity in this mixed culture activated sludge microbial communities. Selective staining methods were applied to a municipal activated sludge during pilot scale PHA accumulation in replicate experiments. Visualization of stained flocs revealed that a significant but limited fraction of the biomass was engaged with PHA accumulation. Accumulated PHA granules were furthermore heterogeneously distributed within and between flocs. These observations suggested that the PHA content for the bacteria storing PHAs was significantly higher than the average PHA content measured for the biomass as a whole. Optimized staining methods provided high acuity for imaging of PHA distribution when compared to other methods reported in the literature. Selective staining methods were sufficient to resolve and distinguish between distinctly different morphotypes in the biomass, and these observations of distinctions have interpreted implications for PHA recovery methods. Visualization tools facilitate meaningful insights for advancements of activated sludge processes where systematic observations, as applied in the present work, can reveal underlying details of structure-function relationships.

Recent updates to microbial production and recovery of polyhydroxyalkanoates.

The increasing use of synthetic polymers and their disposal has raised concern due to their adverse effects on the environment. Thus, other sustainable alternatives to synthetic plastics have been sought, such as polyhydroxyalkanoates (PHAs), which are promising microbial polyesters, mainly due to their compostable nature, biocompatibility, thermostability, and resilience, making this biopolymer acceptable in several applications in the global market. The large-scale production of PHAs by microorganisms is still limited by the high cost of production compared to conventional plastics. This review reports some strategies mentioned in the literature aimed at production and recovery, paving the way for the bio-based economy. For this, some aspects of PHAs are addressed, such as synthesis, production systems, process control using by-products from industries, and advances and challenges in the downstream. The bioplastics properties made them a prime candidate for food, pharmaceutical, and chemical industrial applications. With this paper, it is possible to see that biodegradable polymers are promising materials, mainly for reducing the pollution produced by polymers derived from petroleum.

Upcycling of PHA-producing bacteria for biostimulants production and polyhydroxyalkanoates recovery

The use of petroleum-based plastic has led to its accumulation in the environment, with negative impacts on the ecosystem and the biota. Polyhydroxyalkanoates (PHAs), biobased and biodegradable plastics produced by microbes, have many commercial applications, however their high production cost means they cannot yet compete with traditional plastics. At the same time, the problem of the growing human population implies that improved crop production is needed to avoid malnutrition. Biostimulants enhance plant growth and have the potential to improve agricultural yields; they can be obtained from biological feedstock, including microbes. Therefore, there is scope for coupling the production of PHAs with that of biostimulants, making the process more cost-efficient and minimizing by-products generation. In this work, low-value agro-zoological residues were processed to obtain PHA-storing bacteria via acidogenic fermentation; PHAs destined for the bioplastic market were extracted, and the protein-rich by-products were turned into protein hydrolysates using different treatment methods, assessing their biostimulant effects in growth trials with tomato and cucumber plants. The results indicate that the best hydrolysis treatment, realizing the highest amount of organic nitrogen (6.8 gN-org/L) while achieving the best PHA recovery (63.2 % gPHA/gTS), is obtained with strong acids. All the protein hydrolysates were effective in improving either roots or leaf development, with various results, depending on the species and the growth method. The acid hydrolysate was the most effective treatment to enhance the development of shoots (21 % increase compared to the control) and roots (16 % increase for the dry weight and 17 % for main root length) of hydroponically-grown cucumber plants, while pot-grown tomatoes, biostimulated via foliar spray, developed bigger shoots (up to 41 %) with the hydrolysate obtained from the alkaline treatment. These preliminary results indicate that simultaneous production of PHAs and biostimulants is feasible, and that commercialization could be achievable given the expected reduction in production costs.

Chemical digestion method to promote activated sludge cell wall breaking and optimize the polyhydroxyalkanoate (PHA) extraction process

A novel protocol for the recovery of PHA from mixed-cultures proposed. In this experiment, activated sludge for PHA synthesis was investigated and a two-stage chemical digestion method was used for activated sludge to improve the yield of PHA. The highest PHA extraction combination that could be obtained in this experiment was sodium hypochlorite(NaClO) plus sodium dodecyl sulfate (SDS), and the optimal concentration of NaClO solution was 25 % (v/v), and the ratio of the dry weight of activated sludge to SDS was 1:2. The recovery and purity of PHA were 72.14 % and 54.47 %, respectively. The reaction time between NaClO and activated sludge affects the recovery of PHA, and the optimal reaction time of NaClO was experimentally obtained as 30 min. The purity of the PHA extract obtained after purification using methanol was improved.

Using inorganic acids to stop purple phototrophic bacteria metabolism improves PHA recovery at a large scale

Polyhydroxyalkanoate (PHA) production at a large scale by purple phototrophic bacteria (PPB) is hindered due to high production costs and limited recovery due to its consumption during starvation periods. The present study identified cost-effective inorganic acids as inactivation methods for PPB to obtain higher PHA recovery. The study was performed on reactors of different scales (10 L and 0.5 L) to grow PPB and recover PHA subsequently. The permanent feast strategy was adopted to obtain higher PHA in an anaerobic environment. As a result, the study achieved 33% (dry weight) PHA recovery using inorganic acid inactivation, while formaldehyde inactivation (traditional method) achieved significantly lower PHA recovery (20% only). The results from inorganic acid inactivation were further examined for their stability. The samples were stable even after day 14, and the PHA recovery was the same as on day 0. This pioneering study shows that inorganic acids can be used to inactivate the PPB metabolism to obtain higher PHA recovery; inorganic acid inactivation could be economical for large-scale PHA production.Graphical

Recovery of polyhydroxyalkanoates (PHAs) polymers from a mixed microbial culture through combined ultrasonic disruption and alkaline digestion

PHAs are a form of cellular storage polymers with diverse structural and material properties, and their biodegradable and renewable nature makes them a potential green alternative to fossil fuel-based plastics. PHAs are obtained through extraction via various mechanical, physical and chemical processes after their intracellular synthesis. Most studies have until now focused on pure cultures, while information on mixed microbial cultures (MMC) remains limited. In this study, ultrasonic (US) disruption and alkaline digestion by NaOH were applied individually and in combination to obtain PHAs products from an acclimated MMC using phenol as the carbon source. Various parameters were tested, including ultrasonic sound energy density, NaOH concentration, treatment time and temperature, and biomass density. US alone caused limited cell lysis and resulted in high energy consumption and low efficiency. NaOH of 0.05–0.2 M was more efficient in cell disruption, but led to PHAs degradation under elevated temperature and prolonged treatment. Combining US and NaOH significantly improved the overall process efficiency, which could reduce energy consumption by 2/3rds with only minimal PHAs degradation. The most significant factor was identified to be NaOH dosage and treatment time, with US sound energy density playing a minor role. Under the semi-optimized condition (0.2 M NaOH, 1300 W L-1, 10 min), over 70% recovery and 80% purity were achieved from a 3 g L−1 MMC slurry of approximately 50% PHAs fraction. The material and thermal properties of the products were analyzed, and the polymers obtained from US + NaOH treatments showed comparable or higher molecular weight to previously reported results. The products also exhibited good thermal stability and rheological properties, compared to the commercial standard. In conclusion, the combined US and NaOH method has the potential in real application as an efficient process to obtain high quality PHAs from MMC, and cost-effectiveness can be further optimized.

Improvement of the Polyhydroxyalkanoates Recovery from Mixed Microbial Cultures Using Sodium Hypochlorite Pre-Treatment Coupled with Solvent Extraction.

The use of mixed microbial cultures (MMC) and organic wastes and wastewaters as feed sources is considered an appealing approach to reduce the current polyhydroxyalkanoates (PHAs) production costs. However, this method entails an additional hurdle to the PHAs downstream processing (recovery and purification). In the current work, the effect of a sodium hypochlorite (NaClO) pre-treatment coupled with dimethyl carbonate (DMC) or chloroform (CF) as extraction solvents on the PHAs recovery efficiency (RE) from MMC was evaluated. MMC were harvested from a sequencing batch reactor (SBR) fed with a synthetic prefermented olive mill wastewaster. Two different carbon-sources (acetic acid and acetic/propionic acids) were employed during the batch accumulation of polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from MMC. Obtained PHAs were characterized by 1H and 13C nuclear magnetic resonance, gel-permeation chromatography, differential scanning calorimetry, and thermal gravimetric analysis. The results showed that when a NaClO pre-treatment is not added, the use of DMC allows to obtain higher RE of both biopolymers (PHB and PHBV), in comparison with CF. In contrast, the use of CF as extraction solvent required a pre-treatment step to improve the PHB and PHBV recovery. In all cases, RE values were higher for PHBV than for PHB.

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

Mixed microbial culture polyhydroxyalkanoates (MMC PHA) process combined with mainstream wastewater treatment is a competitive resource recovery integrated process in which economical non-oxygen electron acceptors can be used to enrich PHA-producer and reduce operating costs. Oxygen and three non-oxygen electron acceptors (endogenous nitrate, exogenous nitrate, and nitrite) were used and comprehensively compared in terms of PHA production ability and microbial community ecophysiology in this study. The results showed that the PHA production ability with exogenous nitrate was comparable to that of oxygen, which had a maximum PHA content of 64.0 wt% (versus 62.3% of oxygen), qPHA of 0.7 mg COD/mg Xa/h, as well as 99.1% denitrification efficiency. The non-oxygen acceptors delayed the time for Arcobacter to occupy the dominant niche in the community, and the environmental selection pressure produced was slightly lower than that of oxygen. The symbiotic community between denitrifying bacteria and PHA-producers ensured the high PHA production potential and denitrification efficiency of the non-oxygen system. An economic assessment showed that the integrated PHA production and nitrate removal process with the mainstream wastewater treatment process proposed in this study could reduce aeration energy by 64%, and PHA recovery yield was 0.1 kg CODPHA/kg CODWAS from waste activated sludge (WAS).

Polyhydroxyalkanoate recovery from newly screened Bacillus sp. LPPI-18 using various methods of extraction from Loktak Lake sediment sample

BackgroundNowadays, the conventional plastic wastes are very challenging to environments and its production cost also creates an economic crisis due to petrochemical-based plastic. In order to solve this problem, the current studies were aimed at screening and characterizing these polyhydroxyalkanoate (PHA)-producing isolates and evaluating the suitability of some carbon source for newly screened PHA-producing isolates. Material and methodsSome carbon sources such as D-fructose, glucose, molasses, D-ribose and sucrose were evaluated for PHA production. Data were analyzed using SPSS version 20. The 16SrRNA gene sequence of these isolates was performed. These newly isolated taxa were related to Bacillus species. It was designated as Bacillus sp. LPPI-18 and affiliated Bacillus cereus ATCC 14577T (AE01687) (99.10%). Paenibacillus sp. 172 (AF273740.1) was used as an outgroup. ResultsBacillus sp. LPPI-18 is a gram-positive, rod-shaped, endospore former, and citrate test positive. This isolate showed positive for amylase, catalase, pectinase, and protease test. They produced intracellular PHA granules when this isolate was stained with Sudan Black B (SBB) and Nile blue A (NBA) preliminary and specific staining dyes, respectively. Both temperature and pH used to affect polyhydroxyalkanoates (PHA) productivity. Bacteria are able to reserve PHA in the form of granules during stress conditions. This isolate produces only when supplied with carbon sources. More PHA contents (PCs) were obtained from glucose, molasses, and D-fructose. In this regard, the maximum mean value of PC was obtained from glucose (40.55±0.7%) and the minimum was obtained from D-ribose (12.4±1.4%). Great variations (P≤0.05) of PCs were observed among glucose and sucrose, molasses and sucrose, and D-fructose and sucrose carbon sources for PHA productivity (PP) of cell dry weight (CDW) g/L. After extraction, PHA film was produced for this typical isolate using glucose as a sole carbon source. Fourier transform infrared spectrum was performed for this isolate and showed the feature of polyester at 1719.64 to 1721.16 wavelengths for these extracted samples. The peak of fingerprinting (band of carboxylic acid group) at this wavelength is a characteristic feature of polyhydroxybutyrate (PHB) and corresponds to the ester functional group (C=O). ConclusionIn this study, newly identified Bacillus sp. LPPI-18 is found to be producing biodegradable polymers that are used to replace highly pollutant conventional plastic polymers. This isolate is also used to employ certain cost-effective carbon sources for the production of PHA polymers. Graphical Abstract▪

Subcritical Water as a Pre-Treatment of Mixed Microbial Biomass for the Extraction of Polyhydroxyalkanoates.

Polyhydroxyalkanoate (PHA) recovery from microbial cells relies on either solvent extraction (usually using halogenated solvents) and/or digestion of the non-PHA cell mass (NPCM) by the action of chemicals (e.g., hypochlorite) that raise environmental and health hazards. A greener alternative for PHA recovery, subcritical water (SBW), was evaluated as a method for the dissolution of the NPCM of a mixed microbial culture (MMC) biomass. A temperature of 150 °C was found as a compromise to reach NPCM solubilization while mostly preventing the degradation of the biopolymer during the procedure. Such conditions yielded a polymer with a purity of 77%. PHA purity was further improved by combining the SBW treatment with hypochlorite digestion, in which a significantly lower hypochlorite concentration (0.1%, v/v) was sufficient to achieve an overall polymer purity of 80%. During the procedure, the biopolymer suffered some depolymerization, as evidenced by the lower molecular weight (Mw) and higher polydispersity of the extracted samples. Although such changes in the biopolymer’s molecular mass distribution impact its mechanical properties, impairing its utilization in most conventional plastic uses, the obtained PHA can find use in several applications, for example as additives or for the preparation of graft or block co-polymers, in which low-Mw oligomers are sought.