Polyhydroxyalkanoates Accumulation Research Articles

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.

Polyhydroxyalkanoate biosynthesis and optimisation of thermophilic Geobacillus stearothermophilus strain K4E3_SPR_NPP.

Polyhydroxyalkanoates (PHA) can be used to combat the challenges associated with plastic because it is biodegradable and can be produced from renewable resources. Extremophiles are considered to be potential PHA producers. An initial screening for the PHA synthesizing ability of a thermophilic bacteria Geobacillus stearothermophilus strain K4E3_SPR_NPP was carried out using Sudan black B staining. Nile red viable colony staining was used to further verify that the isolates produced PHA. Crotonic acid assays were used to determine the concentrations of PHA. The bacteria showed 31% PHA accumulation per dry cell weight (PHA/DCW) when glucose was used as a carbon source for growth. The molecule was identified to be medium chain length PHA, A copolymer of PHA containing poly(3-hydroxybutyrate)-poly(3-hydroxyvalerate)-poly(3-hydroxyhexanoate) (PHB-PHV-PHHX) using 1H-NMR. Six carbon sources and four nitrogen sources were screened for the synthesis of maximum PHA content, of which lactose and ammonium nitrate showed45% and 53% PHA/DCW respectively. The important factors in the experiment are identified using the Plackett-Burman design, and optimization is performed using the response surface method. Response surface methodology was used to optimize the three important factors, and the maximum biomass and PHA productions were discovered. Optimal concentrations yielded a maximum of 0.48g/l biomass and 0.32g/l PHA, measuring 66.66% PHA accumulation. Dairy industry effluent was employed for the synthesis of PHA, yielding 0.73g/l biomass and 0.33g/l PHA, measuring 45% PHA accumulation. These findings add credibility to the possibility of adopting thermophilic isolates for PHA production using low-cost substrates.

Cellular RNA levels define heterotrophic substrate-uptake rate sub-guilds in activated sludge microbial communities.

A heterotrophic-specialist model was proposed previously to divide wastewater treatment plant (WWTP) heterotrophs into sub-guilds of consumers of readily or slowly degradable substrates (RDS or SDS, respectively). The substrate degradation rate model coupled to metabolic considerations predicted that RNA and polyhydroxyalkanoate (PHA) levels would be positively correlated in the activated sludge communities with high RNA and PHA occurring in RDS-consumers, and low RNA with no PHA accumulation occurring in SDS-consumers because their external substrates are always present. This prediction was verified in previous studies and in the current one. Thus, RNA and PHA levels were used as biomarkers of the RDS- and SDS-consumer sub-guilds for cell sorting using flow cytometry of samples from three WWTPs. Subsequently, 16S rRNA gene amplicon sequencing revealed that the sorted groups were highly similar over time and among WWTPs, and demonstrated a clear segregation by RNA levels. Predicted ecophysiological traits based on 16S rRNA phylogeny suggested that the high-RNA population showed RDS-consumer traits such as higher rrn copy numbers per genome. Using a mass-flow immigration model, it appeared that the high-RNA populations exhibited high immigration rates more frequently than low-RNA populations, but the differences in frequencies were less with increasing solids residence times.

Polyhydroxyalkanoate synthesis from primitive components of organic solid waste: Comparison of dominant strains and improvement of metabolic pathways

The production of polyhydroxyalkanoate (PHA) from organic solid waste is a research hotspot. However, the allocation and conversion mechanism of different carbon resources between precursor routes and PHA metabolism remains unclear. We compared the PHA synthesis of dominant functional bacteria, Cupriavidus necator (C. necator) and Bacillus cereus (B. cereus), and replenished the metabolic pathways of primitive components (glucose, glycerin, lactic acid and propionic acid). The results showed that the PHA accumulation ability of C. necator (11.8%∼36.5%) was better than that of B. cereus (10.1%∼24.5%). C. necator had a faster rate of cell growth and PHA synthesis than B. cereus, which showed advantages in production efficiency. Most of the carbon of glucose flowed to the direct poly-3-hydroxybutyrate synthesis pathway. The carbon competition inhibited the PHA production from glycerin in C. necator, and cysteine metabolism was an effective pathway for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in B. cereus. There were active amino acid pathway and aminobutyric acid pathway in the utilization of lactic acid in C. necator and B. cereus, respectively. The yield of copolymer was limited by acetyl-CoA in propionic acid system. This study laid the foundation for exploring the PHA synthesis from the components of organic solid waste.

Rapid Enrichment of Cupriavidus necator in Mixed Microbial Cultures Using Autotrophic Growth: Mixed Microbial Cultures for Biodegradable Polymer Production Using CO2 and Organic Wastes

Polyhydroxyalkanoate (PHA) is a biodegradable polymer that can replace petroleum-based plastics. Cupriavidus necator has been extensively utilized in industrial PHA production because the bacterium can grow and accumulate high amounts of PHA in their cells under heterotrophic and/or autotrophic growth conditions. Microbial PHA production using mixed cultures reduces the high PHA production costs and allows for the utilization of waste organics as carbon sources. However, C. necator cultivation in mixed microbial cultures has not been investigated before. In this work, we developed innovative methods for rapid and selective enrichment of C. necator in mixed cultures, utilizing the unique metabolic characteristics of C. necator (i.e., the ability to grow heterotrophically and/or autotrophically). Three enrichment methods were proposed: autotrophic-only, alternating autotrophic–heterotrophic, and heterotrophic-only cycles. These methods were examined with two wastewater sources as initial mixed mixtures. In the autotrophic-only method, the fraction of C. necator exceeded 92% of the total microbial population in only 60 h, whereas the alternating autotrophic–heterotrophic cycles showed >81.3% selection of C. necator. However, the heterotrophic-only method resulted in practically no enrichment of C. necator after five repeated feast–famine cycles. When tested for PHA accumulation, enriched cultures using the autotrophic and alternating cycles accumulated significant amounts of PHA, confirming the selective and rapid enrichment of C. necator over other microorganisms. Based on the experimental results, the autotrophic-based enrichment is a potential approach for the cost-effective cultivation of C. necator, which can reduce PHA production costs.

Pilot-scale valorisation of salmon peptone into polyhydroxyalkanoates by mixed microbial cultures under conditions of high ammonia concentration

The aquaculture industry produces vast amounts of organic side streams that are often ensilaged for later use in low value applications. However, the potential of converting fish silage, particularly the proteinaceous fraction, into more economically valuable products such as biodegradable polyhydroxyalkanoates (PHA), has not been explored. This study investigates the valorisation of salmon peptone, the proteinaceous fraction obtained from salmon silage, into PHA by mixed microbial cultures (MMC) under conditions without nutrient limitation, where the high concentration of ammonia (up to 2.3 ± 0.2 gN/L) which the culture was subjected to has not been previously reported. Acidogenic fermentation of salmon peptone (SP) was attained in a continuous reactor, resulting in the production of a fermentate rich in acetate and butyrate (16.4 ± 1.6 gCODFP/L). The enriched MMC with PHA-accumulating organisms was selected in a sequential batch reactor (SBR) under nutrient-rich conditions (100 gCOD: 27.6 gN: 0.48 gP at OLR 7.1 gCODFP/(L.d)). The dominant microorganisms in the enriched culture were from the genus Brachymonas, which are described as capable of simultaneous cell growth and PHA accumulation, storing up to 53.0 ± 3.4% wt. of P(3HB-co-3HV) with a 3HV content of 34% wt.. Despite the high ammonia concentration and constant nutrient availability, a good overall PHA yield (189 gPHA/kgSP) and global volumetric PHA productivity (3.10 ± 0.11 gPHA/(L.h)) were achieved. The results demonstrate the technological feasibility of producing PHA from salmon peptone under conditions that are typically considered unfavorable for PHA accumulation, which represents a significant novelty of this work.

The Isolation, Screening, and Characterization of Polyhydroxyalkanoate-Producing Bacteria from Hypersaline Lakes in Kenya

Extremophilic microorganisms such as those that thrive in high-salt and high-alkaline environments are promising candidates for the recovery of useful biomaterials including polyhydroxyalkanoates (PHAs). PHAs are ideal alternatives to synthetic plastics because they are biodegradable, biocompatible, and environmentally friendly. This work was aimed at conducting a bioprospection of bacteria isolated from hypersaline-alkaliphilic lakes in Kenya for the potential production of PHAs. In the present study, 218 isolates were screened by Sudan Black B and Nile Red A staining. Of these isolates, 31 were positive for PHA production and were characterized using morphological, biochemical, and molecular methods. Through 16S rRNA sequencing, we found that the isolates belonged to the genera Arthrobacter spp., Bacillus spp., Exiguobacterium spp., Halomonas spp., Paracoccus spp., and Rhodobaca spp. Preliminary experiments revealed that Bacillus sp. JSM-1684023 isolated from Lake Magadi had the highest PHA accumulation ability, with an initial biomass-to-PHA conversion rate of 19.14% on a 2% glucose substrate. Under optimized fermentation conditions, MO22 had a maximum PHA concentration of 0.516 g/L from 1.99 g/L of cell dry weight and 25.9% PHA conversion, equivalent to a PHA yield of 0.02 g/g of biomass. The optimal PHA production media had an initial pH of 9.0, temperature of 35 °C, salinity of 3%, and an incubation period of 48 h with 2.5% sucrose and 0.1% peptone as carbon and nitrogen sources, respectively. This study suggests that bacteria isolated from hypersaline and alkaliphilic tropical lakes are promising candidates for the production of polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) from Endophytic Bacterial Strains as Potential Biocontrol Agents against Postharvest Diseases of Apples.

Due to the increasing use and accumulation of petrochemical plastics in the environment and the rapid depletion of natural resources, microbial polyhydroxyalkanoates have great potential to replace them. This study provides new insights in the field of obtaining of polyhydroxyalkanoates (PHAs) from endophytic bacterial strains and applying them as potential biocontrol agents against postharvest diseases of apples. Two strains-Pseudomonas flavescens D5 and Bacillus aerophilus A2-accumulated PHAs in amounts ranging from 2.77 to 5.9 g L-1. The potential to use low-cost substrates such as beet molasses and soapstock for PHA accumulation was shown. The PHAs produced by the Ps. flavescens D5 strain had pronounced antagonistic activity against Penicillium expansum (antifungal property = 62.98-73.08%). The use of PHAs as biocontrol agents significantly reduced the severity of apple blue mold, especially in the preventive treatment option.

Impact of phosphorus limitation on medium-chain-length polyhydroxyalkanoate production by activated sludge

For a sustainable economy, biodegradable biopolymers polyhydroxyalkanoates (PHA) are desirable substitutes to petroleum-based plastics that contaminate our environment. Medium-chain-length (MCL) PHA bioplastics are particularly interesting due to their thermoplastic properties. To hamper the high cost associated to PHA production, the use of bacterial mixed cultures cultivated in open systems and using cheap resources is a promising strategy. Here, we studied the operating conditions favouring direct MCL accumulation by activated sludge, using oleic acid as a model substrate and phosphorus limitation in fed-batch bioreactors. Our results confirm the presence of PHA-accumulating organisms (PHAAO) in activated sludge able to accumulate MCL from oleic acid. A positive correlation between phosphorus (P) limitation and PHA accumulation was demonstrated, allowing up to 26% PHA/total biomass accumulation, and highlighted its negative impact on the MCL/PHA fraction in the polymer. Diversity analysis through 16S rRNA amplicon sequencing revealed a differential selection of PHAAO according to the P-limitation level. A differential behaviour for the orders Pseudomonadales and Burkholderiales at increasing P-limitation levels was revealed, with a higher abundance of the latter at high levels of P-limitation. The PHA accumulation observed in activated sludge open new perspectives for MCL-PHA production system based on P-limitation strategy applied to mixed microbial communities. KEY POINTS: • Direct accumulation of MCL-PHA in activated sludge was demonstrated. • MCL-PHA content is negatively correlated with P-limitation. • Burkholderiales members discriminate the highest P-limitation levels.

Modeling polyhydroxyalkanoates production from sugarcane vinasse by mixed microbial cultures

Sugarcane vinasse is wastewater suitable for polyhydroxyalkanoates (PHA) production by mixed cultures in a three-stage process. In this work, models representing the selection of PHA-producing microorganisms in a sequencing batch reactor (SBR) submitted to a feast-famine process and uncoupled carbon/nitrogen feeding, as well as an accumulation of PHA in a fed-batch reactor from sugarcane vinasse were developed. Sensitivity analysis of the models allowed to rank the influence of parameters and calibration was carried out applying the Box-Draper method in conjunction with the Markov Chain Monte Carlo optimization method. The optimized selection model was able to describe the SBR under different operational conditions. Optimization for each condition of the accumulation showed that higher CODt/Nitrogen ratios in the SBR induced the selection of a culture with a growth response rather than storage, contributing to elucidate the impact that the CODt/Nitrogen ratio in the SBR has on PHA accumulation.

Sequential production of two biopolymers-polyhydroxyalkanoate and levan by microbial fermentation

The sequential production of polyhydroxyalkanoates (PHA) and levan was investigated by microbial fermentation using agro-industrial residues. PHA production was carried out by Cupriavidus necator DSMZ 545 in two steps: a) bacterial growth, using pure (MN) and hydrolyzed (MH) molasses and; b) accumulation of PHA, using standard glycerol (GP) and crude glycerol (GB). The fermented remaining from the C. necator growth stage was separated from the PHA-containing biomass and used in the subsequent production of levan by Bacillus subtilis (natto) Takahashi. The medium containing MH+GP generated a rate of 15 mg of PHA, while the medium based on MH+GB the rate of PHA was almost twice as high (28.4 mg). FTIR spectroscopic analysis of PHA indicated stretching vibrations characteristic for a PHB-like molecule. In turn, the levan produced in this study was precipitated with different volumes of ethanol, generating levans with different molecular weights. The results of the characterization of this fructan by chromatography showed that it was predominantly constituted by fructose units. In addition, the dynamic and kinematic viscosity values for levan were similar in the analyzed concentrations and, therefore, did not provide any clues about the molecular weight of this biopolymer. Finally, the sequential production process of PHA and levan, as a second bioproduct, represents an elegant alternative to reduce the total costs of PHA production.

Towards high-throughput screening (HTS) of polyhydroxyalkanoate (PHA) production via Fourier transform infrared (FTIR) spectroscopy of Halomonas sp. R5-57 and Pseudomonas sp. MR4-99.

High-throughput screening (HTS) methods for characterization of microbial production of polyhydroxyalkanoates (PHA) are currently under investigated, despite the advent of such systems in related fields. In this study, phenotypic microarray by Biolog PM1 screening of Halomonas sp. R5-57 and Pseudomonas sp. MR4-99 identified 49 and 54 carbon substrates to be metabolized by these bacteria, respectively. Growth on 15 (Halomonas sp. R5-57) and 14 (Pseudomonas sp. MR4-99) carbon substrates was subsequently characterized in 96-well plates using medium with low nitrogen concentration. Bacterial cells were then harvested and analyzed for putative PHA production using two different Fourier transform infrared spectroscopy (FTIR) systems. The FTIR spectra obtained from both strains contained carbonyl-ester peaks indicative of PHA production. Strain specific differences in the carbonyl-ester peak wavenumber indicated that the PHA side chain configuration differed between the two strains. Confirmation of short chain length PHA (scl-PHA) accumulation in Halomonas sp. R5-57 and medium chain length PHA (mcl-PHA) in Pseudomonas sp. MR4-99 was done using Gas Chromatography-Flame Ionization Detector (GC-FID) analysis after upscaling to 50 mL cultures supplemented with glycerol and gluconate. The strain specific PHA side chain configurations were also found in FTIR spectra of the 50 mL cultures. This supports the hypothesis that PHA was also produced in the cells cultivated in 96-well plates, and that the HTS approach is suitable for analysis of PHA production in bacteria. However, the carbonyl-ester peaks detected by FTIR are only indicative of PHA production in the small-scale cultures, and appropriate calibration and prediction models based on combining FTIR and GC-FID data needs to be developed and optimized by performing more extensive screenings and multivariate analyses.

β-oxidation–polyhydroxyalkanoates synthesis relationship in Pseudomonas putida KT2440 revisited

Pseudomonas putida KT2440 is a well-known model organism for the medium-chain-length (mcl) polyhydroxyalkanoate (PHA) accumulation. (R)-Specific enoyl-coenzyme A hydratase (PhaJ) was considered to be the main supplier of monomers for PHA synthesis by converting the β-oxidation intermediate, trans-2-enoyl-CoA to (R)-3-hydroxyacyl-CoA when fatty acids (FA) are used. Three PhaJ homologues, PhaJ1, PhaJ4 and MaoC, are annotated in P. putida KT2440. To investigate the relationship of fatty acids–PHA metabolism and the role of each PhaJ in PHA biosynthesis in P. putida KT2440, a series of P. putida KT2440 knockouts was obtained. PHA content and monomer composition in wild type (WT) and mutants under different growth conditions were analysed. PhaJ4 was the main monomer supplier for PHA synthesis with FA as sole carbon and energy source, with preference towards C8 and C10 substrate, whereas PhaJ1 showed preference for the C6 substrate. However, when all three PhaJ homologues were deleted, the mutant still accumulated PHA up to 10.7% of the cell dry weight (CDW). The deletion of (R)-3-hydroxydecanoyl-ACP:CoA transacylase (PhaG), which connects de novo FA and PHA synthesis pathways, while causing a further 1.8-fold decrease in PHA content, did not abolish PHA accumulation. Further proteome analysis revealed quinoprotein alcohol dehydrogenases PedE and PedH as potential monomer suppliers, but when these were deleted, the PHA level remained at 2.2–14.8% CDW depending on the fatty acid used and whether nitrogen limitation was applied. Therefore, it is likely that some other non-specific dehydrogenases supply monomers for PHA synthesis, demonstrating the redundancy of PHA metabolism.Key points• β-oxidation intermediates are converted to PHA monomers by hydratases PhaJ1, PhaJ4 and MaoC in Pseudomonas putida KT2440.• When these are deleted, the PHA level decreases, but it is not abolished.• PHA non-specific enzyme(s) also contributes to PHA metabolism in KT2440.

Biogeochemical properties of blue carbon sediments influence the distribution and monomer composition of bacterial polyhydroxyalkanoates (PHA)

Coastal wetlands are highly efficient ‘blue carbon’ sinks which contribute to mitigating climate change through the long-term removal of atmospheric CO2 and capture of carbon (C). Microorganisms are integral to C sequestration in blue carbon sediments and face a myriad of natural and anthropogenic pressures yet their adaptive responses are poorly understood. One such response in bacteria is the alteration of biomass lipids, specifically through the accumulation of polyhydroxyalkanoates (PHAs) and alteration of membrane phospholipid fatty acids (PLFA). PHAs are highly reduced bacterial storage polymers that increase bacterial fitness in changing environments. In this study, we investigated the distribution of microbial PHA, PLFA profiles, community structure and response to changes in sediment geochemistry along an elevation gradient from intertidal to vegetated supratidal sediments. We found highest PHA accumulation, monomer diversity and expression of lipid stress indices in elevated and vegetated sediments where C, nitrogen (N), PAH and heavy metals increased, and pH was significantly lower. This was accompanied by a reduction in bacterial diversity and a shift to higher abundances of microbial community members favouring complex C degradation. Results presented here describe a connection between bacterial PHA accumulation, membrane lipid adaptation, microbial community composition and polluted C rich sediments.Graphical Geochemical, microbiological and polyhydroxyalkanoate (PHA) gradient in a blue carbon zone.

Strategy for economical and enhanced polyhydroxyalkanoate production from synergistic utilization of palm oil and derived wastewater by activated sludge

The potential of palm oil and derived wastewater pretreated by enzyme as co-substrates to accumulate polyhydroxyalkanoate (PHA) rich in short and medium-chain-length monomers under two feeding strategies was evaluated batchwise through mixed microbial cultures (MMCs) in activated sludge. A terpolymer with the maximum PHA content of 30.5 wt%, volumetric yield of 0.372 g COD/g COD and composition of ca. 84.7 ∼ 97.4/0.5 ∼ 1.6/2.1 ∼ 13.7 (3-hydroxybutyrate/ 3-hydroxyvalerate/ 3-hydroxyoctanoate, %) was achieved as a result of co-substrate incorporation. From the perspective of economic benefits, PHA accumulated via adopting strategy of supplementing carbon source to the same initial concentration per cycle saved 42.7 % of carbon consumption, along with a reduction in culture time (72 h). The above discoveries signify that the combination of palm oil and derived wastewater plus MMCs provides an alternative to the plastics industries for a more sustainable and efficient utilization of biological resources and an economic PHA accumulation approach.

Aerobic granulation for polyhydroxyalkanoates accumulation using organic waste leachates

This work aimed to evaluate polyhydroxyalkanoates (PHA)-accumulating aerobic granular sludge (AGS) production using an inoculum of activated sludge and a prefermented medium consisting of leachates from the anaerobic hydrolysis of the organic fraction of municipal solid waste (OFMSW). Operating conditions induced the evolution in the biomass structure to obtain aerobic granules with an average diameter of 1 mm within the first 45 days. This process resulted in remarkable evolution in the settling properties, with a four-fold improvement in the sludge volume index (SVI) and the zone settling velocity (Vs), as well as a considerable increase in the density during the same period. Moreover, AGS exhibited a maximum PHA accumulation capacity of 0.215 g-PHA/g-VSS, which was two-fold higher than in the inoculum, indicating the enrichment of the biomass on PHA-accumulating microorganisms as a result of the application of the anaerobic feeding of the substrate and the operation with long famine periods; operating conditions reported as favorable for both, the cultivation of stable AGS and the selection biopolymers-accumulating microorganisms. However, the carbon balance revealed that VFA from the leachates was preferably used for EPS synthesis instead of PHA production, showing that microorganisms in AGS absorb organic carbon to convert it into EPS for internal structure maintenance.

Bioconversion of Used Transformer Oil into Polyhydroxyalkanoates by Acinetobacter sp. Strain AAAID-1.5

In this research, the utilisation of used transformer oil (UTO) as carbon feedstock for the production of polyhydroxyalkanoate (PHA) was targeted; with a view to reducing the environmental challenges associated with the disposal of the used oil and provision of an alternative to non-biodegradable synthetic plastic. Acinetobacter sp. strain AAAID-1.5 is a PHA-producing bacterium recently isolated from a soil sample collected in Penang, Malaysia. The PHA-producing capability of this bacterium was assessed through laboratory experiments in a shake flask biosynthesis under controlled culture conditions. The effect of some biosynthesis factors on growth and polyhydroxyalkanoate (PHA) accumulation was also investigated, the structural composition of the PHA produced by the organism was established, and the characteristics of the polymer were determined using standard analytical methods. The results indicated that the bacteria could effectively utilise UTO and produce PHA up to 34% of its cell dry weight. Analysis of the effect of some biosynthesis factors revealed that the concentration of carbon substrate, incubation time, the concentration of yeast extract and utilisation of additional carbon substrates could influence the growth and polymer accumulation in the test organism. Manipulation of culture conditions resulted in an enhanced accumulation of the PHA. The data obtained from GC-MS and NMR analyses indicated that the PHA produced might have been composed of 3-hydroxyoctadecanoate and 3-hydroxyhexadecanoate as the major monomers. The physicochemical analysis of a sample of the polymer revealed an amorphous elastomer with average molecular weight and polydispersity index (PDI) of 110 kDa and 2.01, respectively. The melting and thermal degradation temperatures were 88 °C and 268 °C, respectively. The findings of this work indicated that used transformer oil could be used as an alternative carbon substrate for PHA biosynthesis. Also, Acinetobacter sp. strain AAAID-1.5 could serve as an effective agent in the bioconversion of waste oils, especially UTO, to produce biodegradable plastics. These may undoubtedly provide a foundation for further exploration of UTO as an alternative carbon substrate in the biosynthesis of specific polyhydroxyalkanoates.

Modeling the growth of diverse microorganisms during feast‐famine enrichment

Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can decrease the severe environmental pollution of petroleum plastics. PHA production by mixed microbial communities has been extensively studied to lower the high PHA prices. However, the competition between distinct microbial communities during the enrichment of PHA accumulators in mixed cultures has not been widely investigated. Thus, in this work, we developed a mathematical model for the competition between PHA accumulators and non-PHA accumulators in the feast-famine enrichment strategy. The developed model successfully simulated published lab-scale experimental data for Plasticicumulans acidivorans, a well-studied PHA accumulator that can store PHA up to 90% of the cell weight. The growth kinetics for both PHA and non-PHA accumulators were estimated and compared to the values in the literature. The uncertainties in the model kinetics were studied by expanding the model to include additional sub-biomass components for each heterotrophic group. As a result, the microbial diversity of microbial communities was observed to influence the enrichment of PHA accumulators in mixed cultures. Additionally, the calibrated model was applied to investigate the cultivation conditions, such as cycle lengths, carbon-to-nitrogen ratio, and solids retention time for successful P. acidivorans enrichment in mixed cultures. The developed model can be applied to control the cultivation and enrichment of PHA accumulators in large-scale PHA production systems. PRACTITIONER POINTS: A new model for the enrichment of PHA accumulators was developed. The model can simulate PHA accumulation by enriched cultures. The model was calibrated and validated for Plasticicumulans acidivorans. The impact of microbial diversity on enriching PHA accumulators was investigated. Short cycles (<12 h) and SRT (<10 d) are suggested for successful enrichment.

Optimized pulse-feeding fed-batch fermentation for enhanced lignin to polyhydroxyalkanoate transformation.

With an anticipated growth of Bio-Circular-Green economy, the amount of lignin generated as by-product from biorefineries is increasing. Hence, lignin valorising strategies become a very interesting option to improve economic viability of the biorefineries. This study revealed the development of bioprocesses for upgrading lignin into bioplastic. Specifically, a novel strain of Pseudomonas fulva has been applied for microbial bioconversion of organosolv lignin to fermentative polyhydroxyalkanoate (PHA) production. Fed-batch fermentation of lignin-to-PHA with pulse-feeding approach was optimized using Design of Experiment. Effects of C:N ratio and feeding profiles on PHA accumulation in P. fulva were investigated to determine optimal operation. Under optimized fed-batch, the PHA concentration of 195.2 ± 6.6mg/L could be reached and the PHA content was more than 2 folds enhancement compared to batch process. Type of PHA produced was also characterized for chemical composition via GC-MS analysis. The established lignin to PHA conversion could provide platform for developing integrated lignin bioprocessing to promote cost-effective biorefineries.

Thauera sp. Sel9, a new bacterial strain for polyhydroxyalkanoates production from volatile fatty acids

Thauera is one of the main genera involved in polyhydroxyalkanoate (PHA) production in microbial mixed cultures (MMCs) from volatile fatty acids (VFAs). However, no Thauera strains involved in PHA accumulation have been obtained in pure culture so far. This study is the first report of the isolation and characterization of a Thauera sp. strain, namely Sel9, obtained from a sequencing batch reactor (S-SBR) set up for the selection of PHA storing biomass. The 16S rRNA gene evidenced a high sequence similarity with T. butanivorans species. Genome sequencing identified all genes involved in PHA synthesis, regulation and degradation. The strain Sel9 was able to grow with an optimum of chemical oxygen demand-to-nitrogen (COD:N) ratio ranging from 4.7 to 18.9. Acetate, propionate, butyrate and valerate were used as sole carbon and energy sources: a lag phase of 72 h was observed in presence of propionate. Final production of PHAs, achieved with a COD:N ratio of 75.5, was 60.12 ± 2.60 %, 49.31 ± 0.7 %, 37.31 ± 0.43 % and 18.06 ± 3.81 % (w/w) by using butyrate, acetate, valerate and propionate as substrates, respectively. Also, the 3-hydroxybutyrate/3-hydroxyvalerate ratio reflected the type of carbon sources used: 12.30 ± 0.82 for butyrate, 3.56 ± 0.02 for acetate, 0.93 ± 0.03 for valerate and 0.76 ± 0.02 for propionate. The results allow a better elucidation of the role of Thauera in MMCs and strongly suggest a possible exploitation of Thauera sp. Sel9 for a cost-effective and environmentally friendly synthesis of PHAs using VFAs as substrate.