PHB Production Research Articles

Biotransformation of d-Xylose-Rich Rice Husk Hydrolysate by a Rice Paddy Soil Bacterium, Priestia sp. Strain JY310, to Low Molecular Weight Poly(3-hydroxybutyrate).

Poly(3-hydroxybutyrate) (PHB) is a versatile thermoplastic with superior biodegradability and biocompatibility that is intracellularly accumulated by numerous bacterial and archaeal species. Priestia sp. strain JY310 that was able to efficiently biotransform reducing sugars in d-xylose-rich rice husk hydrolysate (reducing sugarRHH) to PHB was isolated from the soil of a rice paddy. Reducing sugarRHH including 12.5% d-glucose, 75.3% d-xylose, and 12.2% d-arabinose was simply prepared using thermochemical hydrolysis of 3% H2SO4-treated rice husk for 15 min at 121 °C. When cultured with 20 g/L reducing sugarRHH under optimized culture conditions in a batch bioreactor, Priestia sp. strain JY310 could produce PHB homopolymer up to 50.4% of cell dry weight (6.2 g/L). The melting temperature, heat of fusion, and thermal decomposition temperature of PHB were determined to be 167.9 °C, 92.1 J/g, and 268.1 °C, respectively. The number average and weight average molecular weights of PHB with a broad polydispersity index value (4.73) were estimated to be approximately 16.2 and 76.8 kg/mol, respectively. The findings of the present study suggest that Priestia sp. strain JY310 can be exploited as a good candidate for the low-cost production of low molecular weight PHB with improved biodegradability and reduced brittleness from inexpensive agricultural waste hydrolysates.

Circular PHB production via Paraburkholderia sacchari cultures using degradation monomers from PHB-based post-consumer bioplastics as carbon sources

The circular and sustainable production of biopolymers necessitates the development of efficient chemical and biological recycling of post-consumer bioplastics. Chemical or enzymatic degradation of poly(3-hydroxybutyrate) (PHB) based bioplastics will result into crotonic acid (CA), 3-hydoxybutyrate methyl ester (3HMB) and 3-hydroxybutyrate (3HB). These PHB-derived monomers should be subsequently used as carbon sources for circular PHB production. This study presents results of shake flask fermentations carried out by Paraburkholderia sacchari using CA, 3HB and 3HMB for PHB production. Sequential addition of 3HB led to 6.5 g/L total dry weight and 2.5 g/L PHB with 0.26 g/g yield, which was slightly higher than the respective values achieved on glucose. CA consumption was not observed neither when it was added at the beginning of fermentation nor when it was supplied together with glucose. CA was only consumed when bacterial growth was initially carried out on glucose. Bacterial growth and PHB production were lower in 3HMB. The BioSupPack project aims to develop circular biological recycling of PHB-based post-consumer bioplastics via enzymatic hydrolysis and sequential fermentation of monomers into new PHB. The production of PHB by P. sacchari shows that the development of such circular process is feasible.

Hybrid synthesis of polyhydroxybutyrate bioplastics from carbon dioxide

We report a hybrid chem-biological system, coupling photovoltaic hydrogen production, chemical CO2hydrogenation, an artificial carbon conversion pathway and the natural PHB synthesis pathway, which drove a completely closed-loop economy for PHB production.

Effect of acute vs chronic stress on Polyhydroxybutyrate production by indigenous cyanobacterium

Fossil-based plastic has become a global-threat due to its high stability and transformation into more lethal forms such as micro plastics with time. An alternative should be found to combat this global enemy. Polyhydroxybutyrate or PHB can be such an alternative to plastic. Present study explores the synthesis of PHB in Neowollea manoromense, using two different cultivation-approaches: acute and chronic stress. This study has used 6 carbon sources and 3 different level of phosphate to study the accumulation of PHB along with lipid, carbohydrate, and proteins. Highest PHB in chronic-stress was achieved under glucose supplementation without phosphate at 21st day (156.5 ± 22.5 μg/mg), whereas in acute-stress, it was achieved under acetate without phosphate (91.0 ± 2.7 μg/mg). Despite higher accumulation in chronic-stress, high PHB productivity was achieved in acute-stress. Principal Component Analysis suggests that all the variables were positively correlated with each other. Here we first report PHB accumulation in Neowollea manoromense. This study highlights that acute-stress can be a powerful tool in establishment of a sustainable cyanobacteria based bio refinery for PHB production.

Polyhydroxybutyrate/valerate production from methanol by Methylorubrum extorquens: Process limiting factors and polymer characterisation

This study investigates production of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Methylorubrum extorquens from methanol and pentanol in automated fed-batch cultures. An in-line methanol sensor was implemented to control methanol addition. A cell growth phase (solely using methanol as carbon source) was always followed by a PHB(V) production phase induced by nitrogen limitation. The highest biomass productivity and yield in the cell growth phase were 0.82 g.L−1.h−1 and 0.34 g.g−1, respectively. Different ratios of pentanol to methanol (from 0 % to 20 vol%) were applied in the PHB(V) production phases. The presence of pentanol significantly decreased the glass transition and melting temperature of the polymers and the ratio of pentanol to methanol in the feed had a significant effect on the PHV yield (ratio of the produced PHV polymer to the consumed pentanol). The highest PHV yield (63 %) was obtained at 2 vol% pentanol.

Optimization and production of holocellulosic enzyme cocktail from fungi Aspergillus nidulans under solid-state fermentation for the production of poly(3-hydroxybutyrate)

The production of petroleum-based plastics increased dramatically following industrialization. Because of multifaceted properties such as durability, thermostability, water resistance, and many others, these plastics have become an indispensable part of daily life. However, while improving people’s quality of life, indiscriminate use of plastics has caused pollution and raised environmental concerns. To address this situation and reduce environmental risks, microbially produced biopolymers such as poly-3-hydroxyalkanoates can be used to make bioplastics that are completely biodegradable under normal environmental conditions. At the moment, the cost of bioplastic production is high when compared to petroleum-based plastics, so alternate strategies for making the bioplastic process economical are urgently needed. Agricultural waste is abundant around the world and can be efficiently used as a low-cost renewable feedstock after pretreatment and enzymatic hydrolysis. Fungi are well known as primary degraders of lignocellulosic waste, and this property was used in the current study to enzymatically hydrolyze the pretreated paddy straw for the production of reducing sugars, which were then used in the microbial fermentation for the production of PHB. In this study, Aspergillus nidulans was used to advance a low-cost and efficient enzyme hydrolysis system for the generation of reducing sugars from lignocellulosic biomass. For the production of the holocellulosic enzyme complex, the fungus was grown on wheat straw with Reese mineral medium as a wetting agent. After 216 h of solid-state fermentation at 30 °C, pH 6.0, the enzyme extract from A. nidulans demonstrated the highest activity, CMCase 68.58 (± 0.55), FPase 12.0 (± 0.06), Xylanase 27.17 (± 0.83), and β-glucosidase 1.89 (± 0.037). The initial pH, incubation temperature, and time all had a significant impact on final enzyme activity. Enzymatic hydrolysis of pretreated paddy straw produced reducing sugars (8.484 to 30.91 gL−1) that were then used to produce poly(3-hydroxybutyrate) using halophilic bacterial isolates. Burkholderia gladioli 2S4R1 and Bacillus cereus LB7 accumulated 26.80% and 20.47% PHB of the cell dry weight, respectively. This suggests that the holocellulosic enzyme cocktail could play a role in the enzymatic hydrolysis of lignocellulosic materials and the production of PHA from less expensive feedstocks such as agricultural waste.

Co-production of polyhydroxyalkanoates and levan by Halomonas smyrnensis AAD6T

The simultaneous production of microbial polymers levan and poly[3-hydroxybutyrate] (PHB), a type of polyhydroxyalkanoates, was investigated in this work. The study involved the fermentation of sucrose and molasses by H. smyrnensis AAD6T (BAE2 strain) to produce PHB (intracellular) and levan (extracellular). Both polymers were isolated and characterized by FTIR. Levan was also characterized by thin-layer chromatography (TLC) and viscosimetric analysis. The amount of biomass was 25 g until the end of fermentation. The PHB rate was 0.015 g in both media and the average PHB productivity was 6.0 x 10-4 g PHB/g biomass. The highest rate of levan was 9 g/L in the range of 72–80 h, in the molasses-based medium. The FTIR spectra showed specific signals for each of the polymers, such as the peak at 1700 for the carbonyl group of esters for the PHB and signals at 900 and 800, which are typical signals for levan fructose rings. Furthermore, acid hydrolysis of levan revealed that it was formed only by fructose, as confirmed by TLC With this study, H. smyrnensis AAD6T BAE2 co-produced PHB and levan using a low-cost carbon source, showing great potential in reducing biopolymer manufacturing costs.

Biosynthesis and biodegradation of poly(3-hydroxybutyrate) from Priestia flexa; A promising mangrove halophyte towards the development of sustainable eco-friendly bioplastics

The protracted persistence of petrochemical plastics in the environment and their non-biodegradability impede the survival of living creatures. Recently, biopolymers are being thoroughly researched as a potential replacement for conventional plastics. This present study sought to locate Poly(3-hydroxybutyrate) synthesizing bacterial species prevalent in the mangrove ecosystem. Six halophilic bacterial isolates were obtained from the mangrove habitat, four isolates displayed superior cell dry weight as well as PHB accumulation. Isolate PMPHB5 showed the highest cell dry weight (4.92 ± 0.02 g/L), while the maximum PHA yield (80%) was found with PMPHB7. Hence, PMPHB7 was chosen for further optimization of carbon source wherein glucose demonstrated improved cell growth as well as PHB production. The characterization of the PHB granules was performed by FT-IR spectroscopy and FE-SEM EDX. The presence of characteristic elements in the sample was confirmed using EDX. Isolate PMPHB7 was further identified as Priestia flexa through 16S rRNA gene sequencing (GenBank accession number: ON362236) and a phylogenetic tree was constructed to reveal the molecular relationships of this organism with others. The solvent-cast biopolymer film was made to check the biodegradability of the extracted PHB. When buried in soil, it was found that the biopolymer film exhibited approximately 73% biodegradation after 21 days. Thus, the present study sheds light on the potential of mangrove-associated halophytes to efficiently produce PHB that is readily biodegradable in soil.

Polyhydroxybutyrate-Natural Fiber Reinforcement Biocomposite Production and Their Biological Recyclability through Anaerobic Digestion

The existing recycling methods of PHA-based material are ineffective in terms of increasing resource efficiency and the production of high value end-of-life products. Therefore, in this study, a novel approach of acidogenic fermentation was proposed to recycle PHB-based composites reinforced with natural fibers such as cellulose, chitin, chitosan, orange waste, sawdust, soy protein, and starch. The inclusion of cellulose, chitosan, and sawdust improved the impact properties of the composites while other fillers had various effects on the mechanical properties. These three composites and neat PHB were subsequently subjected to biological degradation via acidogenic digestion to determine the possibility of converting PHB-based composites into volatile fatty acids (VFAs). Two different pH levels of 6 and 10 were applied to assess the effect of pH on the bioconversion and inhibition of the methanogenesis. The results showed promising PHB degradation, contributing to considerable VFA production of 2.5 g/L at pH 6 after 47 days. At pH 6, the presence of the natural fibers in the biocomposites promoted the degradation rate. On the contrary, pH 10 proved to be more suitable for the degradation of the fibers. The VFA which is produced can be recirculated into PHB production, fitting with the concept of a circulating bioeconomy.

Process intensification of biopolymer polyhydroxybutyrate production by pseudomonas putida SS9: A statistical approach

There are numerous elements of daily life where plastic is employed, yet it is uncertain exactly when it will deteriorate. Poly-(3-hydroxybutyrate) (PHB), a biodegradable polymer, is viewed as a possible substitute for synthetic plastics made from petroleum. With Pseudomonas putida SS9, the current study sought to enhance operational conditions and nutritional factors to enhance PHB production. To maximize the impacts of operational factors, a combination of response surface modeling (RSM) and artificial neural networks (ANN) has been applied. PHB content was used as the response while the interaction effects of the factors were examined. The optimal parameters for PHB synthesis were further tested in a lab scale fermentor. Under optimal conditions, 13.83 g/L of C, 0.57 g/L of N, 0.59 g/L of P, the maximal productivity of PHB obtained with Pseudomonas putida SS9 is 12.89 g/L after 84 h. A mean square value of 15.7 with P < 0.0001 were obtained from the ANOVA results of quadratic polynomial model using RSM. The same construct was employed in MATLAB software to train a feed-forward ANN using the back-propagation approach, generating 12.88 g/L. The data indicated that a properly trained ANN model outperforms the RSM model in prediction. Furthermore, employing dairy waste (cheese whey) as a low-cost feedstock resulted in an equally proportionate PHB yield of 12.02 g/L. Therefore, cheese whey appeared to be a viable alternative carbon source over optimized synthetic media.

Polyhydroxyalkanoates (PHAs) Production from Residual Glycerol by Wild Type Cupriavidus necator

PurposeThe main objective of the present study is to assess and improve fermentation conditions to produce polyhydroxyalkanoates (PHAs), such as the homopolymer poly(3-hydroxybutyrate) (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), from renewable and inexpensive industrial waste by wild type Cupriavidus necator.MethodsUntreated residual glycerol, a byproduct generated by biodiesel industry, was used as the main carbon source by wild type C. necator. The bacterial adaptation stage, the glycerol concentration and other medium components were adjusted to enhance the PHB production. Fed batch fermentation with a pulse feeding strategy of propionic acid solution was employed for PHBV production.ResultsC. necator, previously adapted to glycerol utilization, was able to accumulate PHB up to 4.1 g/l under optimal substrate concentration conditions. The bacterium could also synthesize up to 3.4 g/l PHBV, with a mole fraction of 3-hydroxyvalerate (3HV) up to 12% by fed-batch fermentation with a pulsed propionic acid feed strategy.ConclusionThis is the first report on the use of residual glycerol as ideal feedstock for PHA production by wild type C. necator strain.Graphical Abstract

Characterization of Polyhydroxybutyrate, PHB, Synthesized by Newly Isolated Haloarchaea Halolamina spp.

This work aims to characterize the haloarchaeal diversity of unexplored environmental salty samples from a hypersaline environment on the southern coast of Jeddah, Saudi Arabia, looking for new isolates able to produce polyhydroxyalkanoates (PHAs). Thus, the list of PHA producers has been extended by describing two species of Halolamina; Halolamina sediminis sp. strain NRS_35 and unclassified Halolamina sp. strain NRS_38. The growth and PHA-production were investigated in the presence of different carbon sources, (glucose, sucrose, starch, carboxymethyl cellulose (CMC), and glycerol), pH values, (5-9), temperature ranges (4-65 °C), and NaCl concentrations (100-350 g L-1). Fourier-transform infra-red analysis (FT-IR) and Liquid chromatography-mass spectrometry (LC-MS) were used for qualitative identification of the biopolymer. The highest yield of PHB was 33.4% and 27.29% by NRS_35 and NRS_38, respectively, using starch as a carbon source at 37 °C, pH 7, and 25% NaCl (w/v). The FT-IR pattern indicated sharp peaks formed around 1628.98 and 1629.28 cm-1, which confirmed the presence of the carbonyl group (C=O) on amides and related to proteins, which is typical of PHB. LC-MS/MS analysis displayed peaks at retention times of 5.2, 7.3, and 8.1. This peak range indicates the occurrence of PHB and its synthetic products: Acetoacetyl-CoA and PHB synthase (PhaC). In summary, the two newly isolated Halolamina species showed a high capacity to produce PHB using different sources of carbon. Further research using other low-cost feedstocks is needed to improve both the quality and quantity of PHB production. With these results, the use of haloarchaea as cell factories to produce PHAs is reinforced, and light is shed on the global concern about replacing plastics with biodegradable polymers.

Thermodynamic limitations of PHB production from formate and fructose in Cupriavidus necator

The chemolithotroph Cupriavidus necator H16 is known as a natural producer of the bioplastic-polymer PHB, as well as for its metabolic versatility to utilize different substrates, including formate as the sole carbon and energy source. Depending on the entry point of the substrate, this versatility requires adjustment of the thermodynamic landscape to maintain sufficiently high driving forces for biological processes. Here we employed a model of the core metabolism of C. necator H16 to analyze the thermodynamic driving forces and PHB yields from formate for different metabolic engineering strategies. For this, we enumerated elementary flux modes (EFMs) of the network and evaluated their PHB yields as well as thermodynamics via Max-min driving force (MDF) analysis and random sampling of driving forces. A heterologous ATP:citrate lyase reaction was predicted to increase driving force for producing acetyl-CoA. A heterologous phosphoketolase reaction was predicted to increase maximal PHB yields as well as driving forces. These enzymes were then verified experimentally to enhance PHB titers between 60 and 300% in select conditions. The EFM analysis also revealed that PHB production from formate may be limited by low driving forces through citrate lyase and aconitase, as well as cofactor balancing, and identified additional reactions associated with low and high PHB yield. Proteomics analysis of the engineered strains confirmed an increased abundance of aconitase and cofactor balancing. The findings of this study aid in understanding metabolic adaptation. Furthermore, the outlined approach will be useful in designing metabolic engineering strategies in other non-model bacteria.

A Gene Circuit Combining the Endogenous I-E Type CRISPR-Cas System and a Light Sensor to Produce Poly-β-Hydroxybutyric Acid Efficiently

‘Metabolic burden,’ which arises when introducing exogenic synthesizing pathways into a host strain, remains a challenging issue in metabolic engineering. Redirecting metabolic flux from cell growth to product synthesis at an appropriate culture timepoint is ideal for resolving this issue. In this report, we introduce optogenetics—which is capable of precise temporal and spatial control—as a genetic switch, accompanied by the endogenous type I-E CRISPRi system in Escherichia coli (E. coli) to generate a metabolic platform that redirects metabolic flux. Poly-β-hydroxybutyric acid (PHB) production was taken as an example to demonstrate the performance of this platform. A two-to-three-fold increase in PHB content was observed under green light when compared with the production of PHB under red light, confirming the regulatory activity of this platform and its potential to redirect metabolic flux to synthesize target products.

Biogas bioconversion into poly(3-hydroxybutyrate) by a mixed microbial culture in a novel Taylor flow bioreactor

Biogas-based biopolymer production represents an alternative biogas valorization route with potential to cut down plastic pollution and greenhouse gas emissions. This study investigated for the first time the continuous bioconversion of methane, contained in biogas, into poly(3-hydroxybutyrate) (PHB) by a mixed methanotrophic culture using an innovative high mass-transfer Taylor flow bioreactor. Following a hydrodynamic flow regime mapping, the influence of the gas residence time and the internal gas recirculation on CH4 abatement was assessed under non nutrient limiting conditions. Under optimal operational conditions (gas residence time of 60 min and internal gas recycling ratio of 17), the bioreactor was able to support a CH4 removal efficiency of 63.3%, a robust CH4 elimination capacity (17.2 g-CH4 m-3h-1) and a stable biomass concentration (1.0 g L-1). The simultaneous CH4 abatement and PHB synthesis was investigated under 24-h:24-h nitrogen feast/famine continuous operation. The cyclic nitrogen starvation and the Taylor flow imposed in the bioreactor resulted in a relatively constant biomass concentration of 0.6 g L-1 with PHB contents ranging from 11 to 32% w w-1 (on a dry weight basis), entailing an average PHB productivity of 5.9 g-PHB m-3 d-1 with an associated PHB yield of 19.8 mg-PHB g-CH4-1. Finally, the molecular analysis of the microbial population structure indicated that type II methanotrophs outcompeted non-PHB accumulating type I methanotrophs, with a heterotrophic-methanotrophic consortium enriched in Methylocystis, Hyphomicrobium, Rubinisphaeraceae SH PL14 and Pseudonocardia.

Production and optimization of bioplastic (Polyhydroxybutyrate) from Bacillus cereus strain SH-02 using response surface methodology

BackgroundPolyhydroxybutyrate (PHB) is a biopolymer formed by some microbes in response to excess carbon sources or essential nutrient depletion. PHBs are entirely biodegradable into CO2 and H2O under aerobic and anaerobic conditions. It has several applications in various fields such as medicine, pharmacy, agriculture, and food packaging due to its biocompatibility and nontoxicity nature.ResultIn the present study, PHB-producing bacterium was isolated from the Dirout channel at Assiut Governorate. This isolate was characterized phenotypically and genetically as Bacillus cereus SH-02 (OM992297). According to one-way ANOVA test, the maximum PHB content was observed after 72 h of incubation at 35 °C using glucose and peptone as carbon and nitrogen source. Response surface methodology (RSM) was used to study the interactive effects of glucose concentration, peptone concentration, and pH on PHB production. This result proved that all variables have a significant effect on PHB production either independently or in the interaction with each other. The optimized medium conditions with the constraint to maximize PHB content and concentration were 22.315 g/L glucose, and 15.625 g/L peptone at pH 7.048. The maximum PHB content and concentration were 3100.799 mg/L and 28.799% which was close to the actual value (3051 mg/l and 28.7%). The polymer was identified as PHB using FTIR, NMR, and mass spectrometry. FT-IR analysis showed a strong band at 1724 cm− 1 which attributed to the ester group’s carbonyl while NMR analysis has different peaks at 169.15, 67.6, 40.77, and 19.75 ppm that were corresponding to carbonyl, methine, methylene, and methyl resonance. Mass spectroscopy exhibited molecular weight for methyl 3- hydroxybutyric acid.ConclusionPHB–producing strain was identified as Bacillus cereus SH-02 (OM992297). Under optimum conditions from RSM analysis, the maximum PHB content and concentration of this strain can reach (3100.799 mg/L and 28.799%); respectively. FTIR, NMR, and Mass spectrometry were used to confirm the polymer as PHB. Our results demonstrated that optimization using RSM is one of the strategies used for reducing the production cost. RSM can determine the optimal factors to produce the polymer in a better way and in a larger quantity without consuming time.

A Review on Enhancing Cupriavidus necator Fermentation for Poly(3-hydroxybutyrate) (PHB) Production From Low-Cost Carbon Sources

In the context of a circular economy, bioplastic production using biodegradable materials such as poly(3-hydroxybutyrate) (PHB) has been proposed as a promising solution to fundamentally solve the disposal issue of plastic waste. PHB production techniques through fermentation of PHB-accumulating microbes such as Cupriavidus necator have been revolutionized over the past several years with the development of new strategies such as metabolic engineering. This review comprehensively summarizes the latest PHB production technologies via Cupriavidus necator fermentation. The mechanism of the biosynthesis pathway for PHB production was first assessed. PHB production efficiencies of common carbon sources, including food waste, lignocellulosic materials, glycerol, and carbon dioxide, were then summarized and critically analyzed. The key findings in enhancing strategies for PHB production in recent years, including pre-treatment methods, nutrient limitations, feeding optimization strategies, and metabolism engineering strategies, were summarized. Furthermore, technical challenges and future prospects of strategies for enhanced production efficiencies of PHB were also highlighted. Based on the overview of the current enhancing technologies, more pilot-scale and larger-scale tests are essential for future implementation of enhancing strategies in full-scale biogas plants. Critical analyses of various enhancing strategies would facilitate the establishment of more sustainable microbial fermentation systems for better waste management and greater efficiency of PHB production.

Hyper production of polyhydroxyalkanoates by a novel bacterium Salinivibrio sp. TGB11

Strains of Salinivibrio genus are able to grow under wide range of NaCl concentration. Salinivibrio sp. TGB11, a novel bacterium screened from saltworks, was found capable of producing polyhydroxyalkanoates from sugars and volatile fatty acids. In shake flask cultures, the use of glucose, sucrose, acetate, and butyrate achieved poly(3-hydroxybutyrate) (PHB) titers of 5.00, 3.23, 0.77, and 0.88 g/L, respectively. The addition of 0.5 g/L propionate with glucose yielded 4.38 g/L poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), with a 3-hydroxyvalerate monomer content of 8.57 mol%, yet propionate concentration higher than 1.5 g/L severely inhibited cell growth. In fed-batch bioreactor studies, PHB titer of 100.22 g/L and PHBV titer of 59.30 g/L were achieved. The PHB production yield was 0.42 g/g, reaching 88% of the theoretical value. Salinivibrio sp. TGB11 showed the highest PHB titer and yield in Salinivibrio genus and could be a promising host for industrial purposes.

PHB production from cellobiose with Saccharomyces cerevisiae

Replacement of petrochemical-based materials with microbially produced biodegradable alternatives calls for industrially attractive fermentation processes. Lignocellulosic materials offer non-edible alternatives for cultivated sugars, but require often use of expensive sugar releasing enzymes, such as β-glucosidases. These cellulose treatment costs could be reduced if microbial production hosts could use short cellodextrins such as cellobiose directly as their substrates. In this study, we demonstrate production of poly(hydroxybutyrate) (PHB) in yeast Saccharomyces cerevisiae using cellobiose as a sole carbon source. Yeast strains expressing PHB pathway genes from Cupriavidus necator and cellodextrin transporter gene CDT-1 from Neurospora crassa were complemented either with β-glucosidase gene GH1-1 from N. crassa or with cellobiose phosphorylase gene cbp from Ruminococcus flavefaciens. These cellobiose utilization routes either with Gh1-1 or Cbp enzymes differ in energetics and dynamics. However, both routes enabled higher PHB production per consumed sugar and higher PHB accumulation % of cell dry weight (CDW) than use of glucose as a carbon source. As expected, the strains with Gh1-1 consumed cellobiose faster than the strains with Cbp, both in flask and bioreactor batch cultures. In shake flasks, higher final PHB accumulation % of CDW was reached with Cbp route (10.0 ± 0.3%) than with Gh1-1 route (8.1 ± 0.2%). However, a higher PHB accumulation was achieved in better aerated and pH-controlled bioreactors, in comparison to shake flasks, and the relative performance of strains switched. In bioreactors, notable PHB accumulation levels per CDW of 13.4 ± 0.9% and 18.5 ± 3.9% were achieved with Cbp and Gh1-1 routes, respectively. The average molecular weights of accumulated PHB were similar using both routes; approximately 500 kDa and 450 kDa for strains expressing either cbp or GH1-1 genes, respectively. The formation of PHB with high molecular weights, combined with efficient cellobiose conversion, demonstrates a highly potential solution for improving attractiveness of sustainable polymer production using microbial cells.

Production of glycogen, PHB, biohydrogen, NAD(P)H, and proteins in Synechocystis sp. PCC 6803 disrupted in metabolically linked biosynthetic pathway(s)

Production of glycogen, PHB, biohydrogen, NAD(P)H, and proteins in Synechocystis sp. PCC 6803 disrupted in metabolically linked biosynthetic pathway(s)