Polyhydroxyalkanoates Accumulation Research Articles

Polyhydroxyalkanoates production from cheese whey under near-seawater salinity conditions

Treating saline streams presents considerable challenges due to their adverse effects on conventional biological processes, thereby leading to increased expenses in managing those side streams. With this in consideration, this study explores into the potential for valorizing fermented cheese whey (CW), a by-product of the dairy industry, into polyhydroxyalkanoates (PHA) using mixed microbial cultures (MMC) under conditions of near-seawater salinity (30 gNaCl/L). The selection of a PHA-accumulating MMC was successfully achieved using a sequential batch reactor operated under a feast and famine regime, with a hydraulic retention time of 14.5 h, a variable solids retention time of 3 and 4.5 days, and an organic loading rate (OLR) of 60 Cmmol/(L d). The selected culture demonstrated efficient PHA production rates and yields, maintaining robust performance even under high salinity conditions. During PHA accumulation, a maximum PHA content in biomass of 56.4 % wt. was achieved for a copolymer P(3HB-co-3HHx) with a 3HHx content of 7 %. Additionally, to asses the capacity of the culture to produce polymers with different compositions, valeric acid was supplemented to the real fermented feedstock which resulted in the production of terpolymers P(3HB-co-3HV-co-3HHx) with varied monomeric content and a higher maximum PHA content of 62 % wt. Additionally, this study highlights the potential utilization of seawater as alternative to freshwater for PHA production, thereby enhancing circular economy principles and promoting environmental sustainability.

Hints from nature for a PHA circular economy: Carbon synthesis and sharing by Pseudomonas solani GK13

Polyhydroxyalkanoates (PHAs) are a well-known group of biodegradable and biocompatible bioplastics that are synthesised and stored by microorganisms as carbon and energy reservoirs. Extracellular PHA depolymerases (ePhaZs), secreted by a limited range of microorganisms, are the main hydrolytic enzymes responsible for their environmental degradation. Pseudomonas sp. GK13, initially identified as P. fluorescens GK13, produces PHA and a prototypic ePhaZ that specifically degrades mcl-PHA. In this study, a comprehensive characterization of strain GK13 was performed. The whole genomic sequence of GK13 was consolidated into one complete chromosome, leading to its reclassification as P. solani GK13. We conducted a detailed in silico examination of the bacteria genomic sequence, specifically targeting PHA metabolic functions. From the different growth conditions explored, PHA accumulation occurred only under carbon/nitrogen (C/N) imbalance, whereas ePhaZ production was induced even at balanced C/N ratios in mineral media. We extend our study to other bacteria belonging to the Pseudomonas genus revealing that the ePhaZ production capacity is closely associated with mcl-PHA synthesis capacity, as also suggested by metagenomic samples. This finding suggests that these types of microorganisms could contribute to the carbon economy of the microbial community, by storing PHA in carbon-rich times, and sharing it with the rest of the population during times of carbon scarcity through PHA hydrolysis. The conclusion pointed that carbon cycle metabolism performed by P. solani GK13 may contribute to the environmental circular economy at a microscopic scale.

First evidence for temperature’s influence on the enrichment, assembly, and activity of polyhydroxyalkanoate-synthesizing mixed microbial communities

Polyhydroxyalkanoates (PHA) are popular biopolymers due to their potential use as biodegradable thermoplastics. In this study, three aerobic sequencing batch reactors were operated identically except for their temperatures, which were set at 15 °C, 35 °C, and 48 °C. The reactors were subjected to a feast–famine feeding regime, where carbon sources are supplied intermittently, to enrich PHA-accumulating microbial consortia. The biomass was sampled for 16S rRNA gene amplicon sequencing of both DNA (during the enrichment phase) and cDNA (during the enrichment and accumulation phases). All temperatures yielded highly enriched PHA-accumulating consortia. Thermophilic communities were significantly less diverse than those at low or mesophilic temperatures. In particular, Thauera was highly adaptable, abundant, and active at all temperatures. Low temperatures resulted in reduced PHA production rates and yields. Analysis of the microbial community revealed a collapse of community diversity during low-temperature PHA accumulation, suggesting that the substrate dosing strategy was unsuccessful at low temperatures. This points to future possibilities for optimizing low-temperature PHA accumulation.

Synthesis and characterization of bioplastics, Polyhydroxyalkanoates produced from sugarcane bagasse by using Bacillus cereus

Polyhydroxyalkanoates (PHA) are biodegradable bioplastics accumulated in microbial cells which have immense potential to replace the existing non-biodegradable plastics. This study focuses on optimizing Bacillus cereus growth conditions to enhance PHA formation, utilizing the sugarcane bagasse (SCB) as the primary feedstock. The SCB was subjected to enzymatic hydrolysis, resulting in 8.33 mg/g of total sugar production. Subsequently, Bacillus cereus was cultivated and the treated SCB was employed for PHA synthesis. An essential precursor to this synthesis involves an in-depth investigation into the characterization of the PHA produced using Bacillus cereus. A total of 20 experimental runs of different parameter conditions for Bacillus cereus culture to produce PHA were optimized by using the Response Surface Methodology (RSM). At an inoculum size of 8% (v/v), temperature of 25 °C, and incubation time of 48 h, the highest PHA was produced with 86.4 g/L or 62.1% (w/w). The optimal growth rate of Bacillus cereus was obtained at 7.71% (v/v), 35.1 °C for 48.3 h. The greatest PHA content and yield were found to be 57.9% (w/w) of PHA accumulation and 90.39 g/L, respectively. The synthesized PHA is characterized by using FTIR, XRD, FESEM-EDX, and soil burial test. From the analysis done, the properties of the produced sample were confirmed as the PHA. The soil burial test observed that the polymer was a PHA with a biodegradability rate of 41.73% in a week. The study successfully optimized the Bacillus cereus culture conditions for PHA production by using sugarcane bagasse hydrolysate at 8% (v/v) as the feedstock.

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.