Polyhydroxyalkanoates Accumulation Research Articles

Influence of Aeration Rate on Uncoupled Fed Mixed Microbial Cultures for Polyhydroxybutyrate Production

The use of residual streams as feedstock for the production of polyhydroxyalkanoates (PHAs) is growing steadily, as it allows the valorization of waste and nutrients otherwise disposed of and the potential production of a biodegradable bioplastic. To date, the environmental and economic costs associated with this process limit its scale-up, which is why it is important to identify possible solutions and optimize the costliest steps. With this in mind, a laboratory-scale sequenced batch reactor (SBR, 5 L) was constructed to allow the selection of a mixed microbial culture able to convert volatile fatty acids (VFAs) into PHA. The reactor is fed with synthetic water containing VFAs, ammonium, phosphate, and micronutrients, typical compounds of fermented streams of certain wastes, such as cheese whey, food waste, or wastewater sludge. The biomass selected and produced by this first reactor is sent to an accumulation reactor, which is fed with a solution rich in VFAs, allowing the accumulation of PHAs. The role of aeration and its impacts on the main process parameters were analyzed. Three scenarios corresponding to different aeration rates were analyzed: 0.08, 0.16, and 0.32 vvm. The SBR was operated at an organic load rate of 600 mgCOD L−1d−1, under a dynamic feeding regime (feast–famine) and a short hydraulic retention time (HRT; 1 day). The results obtained showed that a value of 0.32 enabled better selection and better settling of the sludge. Furthermore, a potential correlation between aeration rate and VFA and NH4+ consumption rates was identified. The resulting biomass was able to accumulate up to 0.15 ± 0.02 g PHAgVSS−1.

The fundamental role of pH in CH4 bioconversion into polyhydroxybutyrate in mixed methanotrophic cultures

Climate change and plastic pollution are likely the most relevant challenges for the environment in the 21st century. Developing cost-effective technologies for the bioconversion of methane (CH4) into polyhydroxyalkanoates (PHAs) could simultaneously mitigate CH4 emissions and boost the commercialization of biodegradable polymers. Despite the fact that the role of temperature, nitrogen deprivation, CH4:O2 ratio or micronutrients availability on the PHA accumulation capacity of methanotrophs has been carefully explored, there is still a need for optimization of the CH4-to-PHA bioconversion process prior to becoming a feasible platform in future biorefineries. In this study, the influence of different cultivation broth pH values (5.5, 7, 8.5 and 10) on bacterial biomass growth, CH4 bioconversion rate, PHA accumulation capacity and bacterial community structure was investigated in a stirred tank bioreactor under nitrogen deprivation conditions. Higher CH4 elimination rates were obtained at increasing pH, with a maximum value of 50.4 ± 2.7 g CH4·m−3·h−1 observed at pH 8.5. This was likely mediated by an increased ionic strength in the mineral medium, which enhanced the gas-liquid mass transfer. Interestingly, higher PHB accumulations were observed at decreasing pH, with the highest PHB contents recorded at a pH 5.5 (43.7 ± 3.4 %w·w−1). The strong selective pressure of low pH towards the growth of Type II methanotrophic bacteria could explain this finding. The genus Methylocystis increased its abundance from 34 % up to 85 and 90 % at pH 5.5 and 7, respectively. On the contrary, Methylocystis was less abundant in the community enriched at pH 8.5 (14 %). The accumulation of intracellular PHB as energy and carbon storage material allowed the maintenance of high CH4 biodegradation rates during 48 h after complete nitrogen deprivation. The results here obtained demonstrated for the first time a crucial and multifactorial role of pH on the bioconversion performance of CH4 into PHA.

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.

N-terminal truncation of PhaCBP-M-CPF4 and its effect on PHA production

BackgroundAmong the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaCBP-M-CPF4) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaCBP-M-CPF4 through N-terminal truncation.ResultsThe N-terminal truncated mutants of PhaCBP-M-CPF4 were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaCBP-M-CPF4 mutants were evaluated in C. necator mutant PHB−4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaCBP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaCBP-M-CPF4 G8. PhaCBP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (Mw) of PHA produced due to lower expression of the truncated PhaCBP-M-CPF4. Transformants harbouring PhaCBP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaCBP-M-CPF4 G8 produced higher Mw PHA in mostly single larger PHA granules with comparable production as the full-length PhaCBP-M-CPF4.ConclusionThis research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, Mw, and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher Mw in mostly single larger granules.

Establishing CRISPRi for Programmable Gene Repression and Genome Evolution in Cupriavidus necator.

Cupriavidus necator H16 is a "Knallgas" bacterium with the ability to utilize various carbon sources and has been employed as a versatile microbial cell factory to produce a wide range of value-added compounds. However, limited genome engineering, especially gene regulation methods, has constrained its full potential as a microbial production platform. The advent of CRISPR/Cas9 technology has shown promise in addressing this limitation. Here, we developed an optimized CRISPR interference (CRISPRi) system for gene repression in C. necator by expressing a codon-optimized deactivated Cas9 (dCas9) and appropriate single guide RNAs (sgRNAs). CRISPRi was proven to be a programmable and controllable tool and could successfully repress both exogenous and endogenous genes. As a case study, we decreased the accumulation of polyhydroxyalkanoate (PHB) via CRISPRi and rewired the carbon fluxes to the synthesis of lycopene. Additionally, by disturbing the expression of DNA mismatch repair gene mutS with CRISPRi, we established CRISPRi-Mutator for genome evolution, rapidly generating mutant strains with enhanced hydrogen peroxide tolerance and robustness in microbial electrosynthesis (MES) system. Our work provides an efficient CRISPRi toolkit for advanced genetic manipulation and optimization of C. necator cell factories for diverse biotechnology applications.

Optimized Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) Production by Moderately Haloalkaliphilic Bacterium Halomonas alkalicola Ext

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polymers that are produced by microorganisms as storage materials under limited nutrition and excess carbon. These PHAs have been found to be ideal for replacing synthetic plastics for use in packaging and biomedical applications. In this study, an alkaliphilic and moderately halophilic bacterium Halomonas alkalicola Ext was isolated from Lake Simbi Nyaima in western Kenya and investigated for PHA production. Sudan Black B and Nile Red A staining showed that bacterium had distinct ability for accumulation of PHAs. To optimize PHA production, the bacterium was grown in submerged fermentation under varying culture conditions and different sources and concentrations of carbon and nitrogen. With one-factor-at-a-time (OFTA) approach, optimal PHA yields were obtained after 72 hours at a pH of 10.0, temperature of 35°C, and 2.5% (w/v) NaCl. The bacterium yielded the highest biomass, and PHA amounts on 2% galactose and 0.1% ammonium sulfate as sources of carbon and nitrogen, respectively. A record PHA yield of 0.071 g g-1 with a titer of 1.419±0.09 g/L was achieved from 3.397 g/L of biomass, equivalent to 41.8% PHA content. Using response surface methodology, PHA titer was increased by 1.5% to 1.44 g/L, while PHA content was improved 1.1-fold to 45.57%. Polymer analysis revealed that the extracted PHA was a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) (3−HB:3−HV=92:8) with two copolymer subunits of 3-hydroxyvaryrate (3-HB) and 3-hydroxybutyrate (3-HV). Halomonas alkalicola Ext attained efficient galactose conversion into PHBV under high salinity and alkalinity conditions.

Microaerobic insights into production of polyhydroxyalkanoates containing 3-hydroxyhexanoate via native reverse β-oxidation from glucose in Ralstonia eutropha H16

BackgroundRalstonia eutropha H16, a facultative chemolitoautotroph, is an important workhorse for bioindustrial production of useful compounds such as polyhydroxyalkanoates (PHAs). Despite the extensive studies to date, some of its physiological properties remain not fully understood.ResultsThis study demonstrated that the knallgas bacterium exhibited altered PHA production behaviors under slow-shaking condition, as compared to its usual aerobic condition. One of them was a notable increase in PHA accumulation, ranging from 3.0 to 4.5-fold in the mutants lacking of at least two NADPH-acetoacetyl-CoA reductases (PhaB1, PhaB3 and/or phaB2) when compared to their respective aerobic counterpart, suggesting the probable existence of (R)-3HB-CoA-providing route(s) independent on PhaBs. Interestingly, PHA production was still considerably high even with an excess nitrogen source under this regime. The present study further uncovered the conditional activation of native reverse β-oxidation (rBOX) allowing formation of (R)-3HHx-CoA, a crucial precursor for poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)], solely from glucose. This native rBOX led to the natural incorporation of 3.9 mol% 3HHx in a triple phaB-deleted mutant (∆phaB1∆phaB1∆phaB2-C2). Gene deletion experiments elucidated that the native rBOX was mediated by previously characterized (S)-3HB-CoA dehydrogenases (PaaH1/Had), β-ketothiolase (BktB), (R)-2-enoyl-CoA hydratase (PhaJ4a), and unknown crotonase(s) and reductase(s) for crotonyl-CoA to butyryl-CoA conversion prior to elongation. The introduction of heterologous enzymes, crotonyl-CoA carboxylase/reductase (Ccr) and ethylmalonyl-CoA decarboxylase (Emd) along with (R)-2-enoyl-CoA hydratase (PhaJ) aided the native rBOX, resulting in remarkably high 3HHx composition (up to 37.9 mol%) in the polyester chains under the low-aerated condition.ConclusionThese findings shed new light on the robust characteristics of Ralstonia eutropha H16 and have the potential for the development of new strategies for practical P(3HB-co-3HHx) copolyesters production from sugars under low-aerated conditions.Graphical

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.

Assessment of polyhydroxyalkanoates and polysaccharides production in native phototrophic consortia under nitrogen and phosphorous-starved conditions

Growing demand for sustainable and eco-friendly alternatives to petroleum-based polymers has increased the interest in the microalgae-based production of polymers, specifically polyhydroxyalkanoates and polysaccharides. While most studies in microbial polymer production have primarily focused on axenic or genetically engineered cultures of cyanobacteria and eukaryotic algae, little is known about the potential of mixed phototrophic consortia. This study aimed to obtain and evaluate mixed photosynthetic consortia of different origins (natural and residual) as a novel approach for polyhydroxyalkanoates and polysaccharides accumulation. Activated sludge and freshwater samples were collected and inoculated in lab-scale photobioreactors to generate mixed photosynthetic consortia. After a preliminary screening for polymer-accumulating strains under nutrient-unbalanced conditions, the selected strains were subjected to a biphasic strategy (biomass accumulation and nutrient stress) to evaluate their polyhydroxyalkanoates and polysaccharide accumulation. First, cultures were subjected to a nutrient-rich phase to increase the biomass content and then deprived of nutrients (known as the polymer accumulation phase) to evaluate polyhydroxyalkanoates and polysaccharide yield. Findings in this study revealed that the highest polysaccharide yield for activated sludge biomass and freshwater consortia was 460 ± 16 and 320 ± 24 mg glucose g dried biomass−1, respectively. In contrast, the highest polyhydroxyalkanoates accumulation levels for both cultures were calculated at 5 mg polyhydroxyalkanoates g dried biomass−1. The efficacy of nutrient stress as a selective pressure strategy to develop mostly polysaccharides-accumulating consortia was demonstrated.

Enhancing poly(3-hydroxybutyrate) production in halophilic bacteria through improved salt tolerance

Polyhydroxyalkanoates (PHA) have emerged as a promising bio-compound in the industrial application due to their potential to replace conventional petroleum-based plastics with sustainable bioplastics. This study focuses on Halomonas sp. YJPS3-3, a halophilic bacterium, and presents a novel approach to enhance PHA production by exploiting its salt tolerance toward PHA biosynthesis. Through gamma irradiation-induced mutants with enhanced salt tolerance from 15% NaCl to 20% NaCl, mutant halo6 showing a significant 11% increase in PHA yield, was achieved. Moreover, the mutants displayed not only higher PHA content but also remarkable cell morphology with elongation. In addition, this research unravels the genetic determinants behind the elevated PHA content and identifies a corresponding shift in fatty acid composition favoring PHA accumulation. This novel mutant obtained from gamma irradiation with enhanced salt tolerance in halophilic bacteria opens up new avenues not only for the bioplastic industry but also for applications in the production of high-value metabolites.

How temperature shapes the biosynthesis of polyhydroxyalkanoates in mixed microbial cultures.

Three sequential batch reactors were operated for the enrichment in microbial communities able to store polyhydroxyalkanoates (PHAs) using activated sludge as inoculum. They ran simultaneously under the same operational conditions (organic loading rate, hydraulic and solids retention time, cycle length, C/N ratio) just with the solely difference of the working temperature: psychrophilic (15°C), mesophilic (30°C), and thermophilic (48°C). The microbial communities enriched showed different behaviors in terms of consumption and production rates. In terms of PHA accumulation, the psychrophilic community was able to accumulate an average amount of 17.7 ± 5.7wt% poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the mesophilic 40.3 ± 7.0wt% PHBV, and the thermophilic 14.8 ± 0.3wt% PHBV in dry weight over total solids. The average PHBV production yields for each selected community were 0.41 ± 0.12 CmmolPHBV /CmmolVFA at 15°C, 0.64 ± 0.05 CmmolPHBV /CmmolVFA at 30°C, and 0.39 ± 0.14 CmmolPHBV /CmmolVFA at 48°C. The overall performance of the mesophilic reactor was better than the other two, and the copolymers obtained at this temperature contained a higher PHV fraction. The physico-chemical properties of the obtained biopolymers at each temperature were also measured, and major differences were found in the molecular weight, following an increasing trend with temperature. PRACTITIONER POINTS: PHBV molecular weight is influenced by the operational temperature increasing with it. Increasing temperatures promote the production of HB over HV. The best accumulation performance was found at 30°C for the tested operational conditions.

Soft sensor based on Raman spectroscopy for the in-line monitoring of metabolites and polymer quality in the biomanufacturing of polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHA) are among the most promising bio-based alternatives to conventional petroleum-based plastics. These biodegradable polyesters can in fact be produced by fermentation from bacteria like Cupriavidus necator, thus reducing the environmental footprint of the manufacturing process. However, ensuring consistent product quality attributes is a major challenge of biomanufacturing. To address this issue, the implementation of real-time monitoring tools is essential to increase process understanding, enable a prompt response to possible process deviations and realize on-line process optimization. In this work, a soft sensor based on in situ Raman spectroscopy was developed and applied to the in-line monitoring of PHA biomanufacturing. This strategy allows the collection of quantitative information directly from the culture broth, without the need for sampling, and at high frequency. In fact, through an optimized multivariate data analysis pipeline, this soft sensor allows monitoring cell dry weight, as well as carbon and nitrogen source concentrations with root mean squared errors (RMSE) equal to 3.71, 7 and 0.03 g/L, respectively. In addition, this tool allows the in-line monitoring of intracellular PHA accumulation, with an RMSE of 14 gPHA/gCells. For the first time, also the number and weight average molecular weights of the polymer produced could be monitored, with RMSE of 8.7E4 and 11.6E4 g/mol, respectively. Overall, this work demonstrates the potential of Raman spectroscopy in the in-line monitoring of biotechnology processes, leading to the simultaneous measurement of several process variables in real time without the need of sampling and labor-intensive sample preparations.

Resource availability governs polyhydroxyalkanoate (PHA) accumulation and diversity of methanotrophic enrichments from wetlands.

Aquatic environments account for half of global CH4 emissions, with freshwater wetlands being the most significant contributors. These CH4 fluxes can be partially offset by aerobic CH4 oxidation driven by methanotrophs. Additionally, some methanotrophs can convert CH4 into polyhydroxyalkanoate (PHA), an energy storage molecule as well as a promising bioplastic polymer. In this study, we investigate how PHA-accumulating methanotrophic communities enriched from wetlands were shaped by varying resource availability (i.e., C and N concentrations) at a fixed C/N ratio. Cell yields, PHA accumulation, and community composition were evaluated in high (20% CH4 and 10mM NH4 +) and low resource (0.2% CH4 and 0.1mM NH4 +) conditions simulating engineered and environmental settings, respectively. High resource availability decreased C-based cell yields, while N-based cell yields remained stable, suggesting nutrient exchange patterns differed between methanotrophic communities at different resource concentrations. PHA accumulation was only observed in high resource enrichments, producing approximately 12.6% ± 2.4% (m/m) PHA, while PHA in low resource enrichments remained below detection. High resource enrichments were dominated by Methylocystis methanotrophs, while low resource enrichments remained significantly more diverse and contained only a minor population of methanotrophs. This study demonstrates that resource concentration shapes PHA-accumulating methanotrophic communities. Together, this provides useful information to leverage such communities in engineering settings as well as to begin understanding their role in the environment.

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.