Application of liquid-based colorimetric method for high throughput screening of bioplastic-degrading strains using esterase assay.

To alleviate environmental problems caused by using conventional plastics, bioplastics have garnered significant interest as alternatives to petroleum-based plastics. Despite possessing better degradability traits compared to traditional plastics, the degradation of bioplastics still demands a longer duration than initially anticipated. This necessitates the utilization of degradation strains or enzymes to enhance degradation efficiency, ensuring timely degradation. In this study, a novel screening method to identify bioplastic degraders faster was suggested to circumvent the time-consuming and laborious characteristics of solid-based plate assays. This liquid-based colorimetric method confirmed the extracellular esterase activity with p-nitrophenyl esters. It eliminated the needs to prepare plastic emulsion plates at the initial screening system, shortening the time for the overall screening process and providing more quantitative data. p-nitrophenyl hexanoate (C6) was considered the best substrate among the various p-nitrophenyl esters as substrates. The screening was performed in liquid-based 96-well plates, resulting in the discovery of a novel strain, Bacillus sp. SH09, with a similarity of 97.4% with Bacillus licheniformis. Furthermore, clear zone assays, degradation investigations, scanning electron microscopy, and gel permeation chromatography were conducted to characterize the biodegradation capabilities of the new strain, the liquid-based approach offered a swift and less labor-intensive option during the initial stages.

Enhanced tolerance of Cupriavidus necator NCIMB 11599 to lignocellulosic derived inhibitors by inserting NAD salvage pathway genes.

Polyhydroxybutyrate (PHB) is a bio-based, biodegradable and biocompatible plastic that has the potential to replace petroleum-based plastics. Lignocellulosic biomass is a promising feedstock for industrial fermentation to produce bioproducts such as polyhydroxybutyrate (PHB). However, the pretreatment processes of lignocellulosic biomass lead to the generation of toxic byproducts, such as furfural, 5-HMF, vanillin, and acetate, which affect microbial growth and productivity. In this study, to reduce furfural toxicity during PHB production from lignocellulosic hydrolysates, we genetically engineered Cupriavidus necator NCIMB 11599, by inserting the nicotine amide salvage pathway genes pncB and nadE to increase the NAD(P)H pool. We found that the expression of pncB was the most effective in improving tolerance to inhibitors, cell growth, PHB production and sugar consumption rate. In addition, the engineered strain harboring pncB showed higher PHB production using lignocellulosic hydrolysates than the wild-type strain. Therefore, the application of NAD salvage pathway genes improves the tolerance of Cupriavidus necator to lignocellulosic-derived inhibitors and should be used to optimize PHB production.

Application of acetyl-CoA acetyltransferase (AtoAD) in Escherichia coli to increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Polyhydroxyalkanoate (PHA) is a family of biodegradable polymers, and incorporation of different monomers can alter its physical properties. To produce the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) containing a high level of 3-hydroxyvalerate (3HV) by altering acetyl-CoA pool levels, we overexpressed an acetyl-CoA acetyltransferase (atoAD) in an engineered E. coli strain, YH090, carrying PHA synthetic genes bktB, phaB, and phaC. It was found that, with introduction of atoAD and with propionate as a co-substrate, 3HV fraction in PHA was increased up to 7.3-fold higher than a strain without atoAD expressed in trans (67.9 mol%). By the analysis of CoA pool concentrations in vivo and in vitro using HPLC and LC-MS, overexpression of AtoAD was shown to decrease the amount of acetyl-CoA and increase the propionyl-CoA/acetyl-CoA ratio, ultimately resulting in an increased 3HV fraction in PHA. Finally, synthesis of P(3HB-co-3HV) containing 57.9 mol% of 3HV was achieved by fed-batch fermentation of YJ101 with propionate.

Starch based polyhydroxybutyrate production in engineered Escherichia coli.

Every year, the amount of chemosynthetic plastic accumulating in the environment is increasing, and significant time is required for decomposition. Bio-based, biodegradable plastic is a promising alternative, but its production is not yet a cost effective process. Decreasing the production cost of polyhydroxyalkanoate by utilizing renewable carbon sources for biosynthesis is an important aspect of commercializing this biodegradable polymer. An Escherichia coli strain that expresses a functional amylase and accumulate polyhydroxybutyrate (PHB), was constructed using different plasmids containing the amylase gene of Panibacillus sp. and PHB synthesis genes from Ralstonia eutropha. This engineered strain can utilize starch as the sole carbon source. The maximum PHB production (1.24 g/L) was obtained with 2% (w/v) starch in M9 media containing 0.15% (w/v) yeast extract and 10 mM glycine betaine. The engineered E. coli SKB99 strain can accumulate intracellular PHB up to 57.4% of cell dry mass.

Efficient polyhydroxybutyrate production using acetate by engineered Halomonas sp. JJY01 harboring acetyl-CoA acetyltransferase.

Polyhydroxybutyrate (PHB) is a well-known biodegradable bioplastic synthesized by microorganisms and can be produced from volatile fatty acids (VFAs). Among VFAs acetate can be utilized by Halomonas sp. YLGW01 for growth and PHB production. In this study, Halomonas sp. JJY01 was developed through introducing acetyl-CoA acetyltransferase (atoAD) with LacIq-Ptrc promoter into Halomonas sp. YLGW01. The effect of expression of atoAD on acetate was investigated by comparison with acetate consumption and PHB production. Shake-flask study showed that Halomonas sp. JJY01 increased acetate consumption rate, PHB yield and PHB production (0.27 g/L/h, 0.075 g/g, 0.72 g/L) compared to the wild type strain (0.17 g/L/h, 0.016 g/g, 0.11 g/L). In 10 L fermenter scale fed-batch fermentation, the growth of Halomonas sp. JJY01 resulted in higher acetate consumption rate, PHB yield and PHB titer (0.55 g/L/h, 0.091 g/g, 4.6 g/L) than wild type strain (0.35 g/L/h, 0.067 h/h, 2.9 g/L). These findings demonstrate enhanced acetate utilization and PHB production through the introduction of atoAD in Halomonas strains.

Polyhydroxybutyrate production from crude glycerol using a highly robust bacterial strain Halomonas sp. YLGW01.

Petrochemical-based plastics are hardly biodegradable and a major cause of environmental pollution, and polyhydroxybutyrate (PHB) is attracting attention as an alternative due to its similar properties. However, the cost of PHB production is high and is considered the greatest challenge for its industrialization. Here, crude glycerol was used as a carbon source for more efficient PHB production. Among the 18 strains investigated, Halomonas taeanenisis YLGW01 was selected for PHB production due to its salt tolerance and high glycerol consumption rate. Furthermore, this strain can produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3 HV)) with 17 % 3 HV mol fraction when a precursor is added. PHB production was maximized through medium optimization and activated carbon treatment of crude glycerol, resulting in 10.5 g/L of PHB with 60 % PHB content in fed-batch fermentation. Physical properties of the produced PHB were analyzed, i.e., weight average molecular weight (6.8 × 105), number average molecular weight (4.4 × 105), and the polydispersity index (1.53). In the universal testing machine analysis, the extracted intracellular PHB showed a decrease in Young's modulus, an increase in Elongation at break, greater flexibility than authentic film, and decreased brittleness. This study confirmed that YLGW01 is a promising strain for industrial PHB production using crude glycerol.

Lignocellulosic hydrolysate based biorefinery for marine exopolysaccharide production and application of the produced biopolymer in environmental clean-up.

The present study deals with the utilization of lignocellulosic hydrolysate-based carbon source for exopolysaccharide (EPS) production using newly reported marine Echinicola sediminis BBL-M-12. This bacterium produced 7.56 g L-1 and 5.32 g L-1 of EPS on supplementing 30 g L-1 glucose and 10 g L-1 xylose as the sole carbon source, respectively. Whereas on feeding Miscanthus hydrolysate (MCH) with glucose content adjusting to 20 g L-1, E. sediminis BBL-M-12 produced 6.18 g L-1 of EPS. The inhibitors study showed bacterium could tolerate higher concentrations of fermentation inhibitors include furfural (0.05%), 5-hydroxymethylfurfural (0.1%), vanillin (0.1%) and acetate (0.5%). Moreover, the EPS composition was greatly altered with the type and concentration of carbon source supplied, although ß-D-Glucopyranose, ß-D-Galactopyranose, and ß-D-Xylopyranose were the dominant monomers detected. Interestingly, E. sediminis BBL-M-12 EPS revealed excellent environmental applications like clay flocculation, oil emulsification, and removal of humic acid, textile dye, and heavy metal from the aqueous phase.

Finding of novel polyhydroxybutyrate producer Loktanella sp. SM43 capable of balanced utilization of glucose and xylose from lignocellulosic biomass.

Polyhydroxybutyrate (PHB) is a potential substitute for plastics derived from fossil fuels, owing to its biodegradable and biocompatible properties. Lignocellulosic biomass could be used to reduce PHB production costs; however, the co-utilization of sugars, such as glucose and xylose, without catabolite repression is a difficult problem to be solved. Here, we selected a novel Loktanella sp. SM43 from a marine environment and optimized the conditions for PHB production. Loktanella sp. SM43 showed high PHB production (66.5% content) from glucose. When glucose and xylose were used together, this strain showed high utilization of both substrates compared to other high PHB-producers such as Halomonas sp. and Cupriavidus necator, which showed glucose preference. Loktanella sp. SM43 showed high growth and PHB production with lignocellulosic hydrolysates. When pine tree hydrolysates were used, PHB production was the highest at 3.66 ± 0.01 g/L, followed by Miscanthus (3.46 ± 0.09 g/L) and barley straw hydrolysate (3.36 ± 0.36 g/L). Overall, these results reveal the potential of Loktanella sp. SM43 to produce PHB using various lignocellulosic hydrolysates as feedstock and the first systematic study for PHB production with Loktanella sp. The approach of screening novel strains is a strategy to overcome co-utilization of sugars without genetic engineering.

Finding of novel lactate utilizing Bacillus sp. YHY22 and its evaluation for polyhydroxybutyrate (PHB) production.

Polyhydroxyalkanoates (PHAs) and their derivatives are biopolymers that have the potential of replacing petroleum-based plastics and can be produced and degraded via bacterial metabolism. However, there are only a few studies on polyhydroxybutyrate (PHB) production using lactate, one of the major waste organic acids that could be implemented in the production of polylactic acid (PLA). Herein, we screened and characterized the PHA-producing microbial strains isolated from saltern soil from Docho Island (South Korea). Among the 24 identified microorganisms that can use lactate as a carbon source, Bacillus sp. YHY22, a newly reported strain, produced the highest amount of PHB: 4.05 g/L with 6.25 g/L dry cell weight, which is 64.7% PHB content under optimal production conditions. Bacillus sp. YHY22 could form the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer with propionate addition. Moreover, Bacillus sp. YHY22 produced PHB in non-sterilized 2% lactate and 8% NaCl marine broth culture medium, suggesting that its production can occur in high salinity media without additional sterilization steps, rendering fermentation cost- and time-efficient.

Lipase mediated functionalization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with ascorbic acid into an antioxidant active biomaterial.

Naturally produced polyhydroxyalkanoates (PHAs) biopolymers have limited medical applications due to their brittle and hydrophobic nature. In this study poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) copolymer was produced using engineered Escherichia coli YJ101, and further functionalized with ascorbic acid using Candida antarctica lipase B mediated esterification. Copolymer P(3HB-co-3HV)-ascorbic acid showed lower degree of crystallinity (9.96%), higher thermal degradation temperature (294.97 °C) and hydrophilicity (68°) as compared to P(3HB-co-3HV). Further, P(3HB-co-3HV)-ascorbic acid biomaterial showed 14% scavenging effect on 1,1-diphenyl-2-picryl-hydrazyl (DPPH), and 1.6 fold increase in biodegradability as compared to P(3HB-co-3HV). Improvement of PHAs polymer properties by adding functional groups could be a good approach to increase their biodegradability, economic value and important applications in the medical field.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) terpolymer production from volatile fatty acids using engineered Ralstonia eutropha.

One of the advantages of microbial synthesis of polyhydroxyalkanoates (PHAs) is the production of diverse polymers with different properties by the addition of different monomers, such as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx). Considering the number of possible variables, terpolymers can have more variations than copolymers. In this study, we aimed to synthesize the terpolymer P(3HB-co-3HV-co-3HHx) from volatile fatty acids such as propionate and butyrate using the recombinant Ralstonia eutropha strain (Re2133/pCB81), containing deletions in the phaB1, phaB2, and phaB3 genes, and overexpression of synthetic PHA operon (phaC2, phaA, phaJ). This strain produced terpolymers depending on the ratio of two different carbon sources, namely, propionic and butyric acids; however, wild type R. eutropha could not produce the same type of polymer. The incorporation of 3-hydroxyvalerate and 3-hydroxyhexanoate monomers was confirmed by gas chromatography and H-nuclear magnetic resonance spectroscopy, and the parameters affecting the terpolymer composition were obtained based on regression. In addition, the thermal analysis showed that this terpolymer has properties different from those of the copolymer, obtained from the same composition of volatile acids. Depending on the ratio of two volatile acids, the composition of the terpolymer can be regulated resulting in different properties.

Development of novel on-line capillary gas chromatography-based analysis method for volatile organic compounds produced by aerobic fermentation.

Many volatile compounds, such as isoprene, a precursor used in the synthesis of natural rubber, have been produced through fermentation using genetically engineered microorganisms. Despite this biotechnological success, measuring the concentrations of volatile compounds during fermentation is difficult because of their high volatility. In current systems, off-line analytical methods usually lead to product loss, whereas on-line methods raise the production cost due to the requirement of complex devices. Here, we developed a novel on-line gas chromatography (GC)-based system for analyzing the concentration of isoprene with the aim to minimize the cost and requirement for devices as compared to current strategies. In this system, a programmable logic controller is used to combine conventional GC with a syringe pump module (SPM) directly connected to the exhaust pipe of the fermentor, and isoprene-containing samples are continuously pumped from the SPM into the GC using an air cylinder recycle stream. We showed that this novel system enables isoprene analysis during fermentation with convenient equipment and without the requirement of an expensive desorption tube. Furthermore, this system may be extended to the detection of other volatile organic compounds in fermentation or chemical processes.

Engineering of artificial microbial consortia of Ralstonia eutropha and Bacillus subtilis for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production from sugarcane sugar without precursor feeding.

Ralstonia eutropha is a well-known microbe reported for polyhydroxyalkonate (PHA) production, and unable to utilize sucrose as carbon source. Two strains, Ralstonia eutropha H16 and Ralstonia eutropha 5119 were co-cultured with sucrose hydrolyzing microbes (Bacillus subtilis and Bacillus amyloliquefaciens) for PHA production. Co-culture of B. subtilis:R. eutropha 5119 (BS:RE5) resulted in best PHA production (45% w/w dcw). Optimization of the PHA production process components through response surface resulted in sucrose: NH4Cl:B. subtilis: R. eutropha (3.0:0.17:0.10:0.190). Along with the hydrolysis of sucrose, B. subtilis also ferments sugars into organic acid (propionic acid), which acts as a precursor for HV monomer unit. Microbial consortia of BS:RE5 when cultured in optimized media led to the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV) with 66% w/w of dcw having 16 mol% HV fraction. This co-culture strategy overcomes the need for metabolic engineering of R. eutropha for sucrose utilization, and addition of precursor for copolymer production.

L-Glycine Alleviates Furfural-Induced Growth Inhibition during Isobutanol Production in Escherichia coli.

Lignocellulose is now a promising raw material for biofuel production. However, the lignin complex and crystalline cellulose require pretreatment steps for breakdown of the crystalline structure of cellulose for the generation of fermentable sugars. Moreover, several fermentation inhibitors are generated with sugar compounds, majorly furfural. The mitigation of these inhibitors is required for the further fermentation steps to proceed. Amino acids were investigated on furfural-induced growth inhibition in E. coli producing isobutanol. Glycine and serine were the most effective compounds against furfural. In minimal media, glycine conferred tolerance against furfural. From the IC50 value for inhibitors in the production media, only glycine could alleviate growth arrest for furfural, where 6 mM glycine addition led to a slight increase in growth rate and isobutanol production from 2.6 to 2.8 g/l under furfural stress. Overexpression of glycine pathway genes did not lead to alleviation. However, addition of glycine to engineered strains blocked the growth arrest and increased the isobutanol production about 2.3-fold.

Production of itaconate by whole-cell bioconversion of citrate mediated by expression of multiple cis-aconitate decarboxylase (cadA) genes in Escherichia coli.

Itaconate, a C5 unsaturated dicarboxylic acid, is an important chemical building block that is used in manufacturing high-value products, such as latex and superabsorbent polymers. Itaconate is produced by fermentation of sugars by the filamentous fungus Aspergillus terreus. However, fermentation by A. terreus involves a long fermentation period and the formation of various byproducts, resulting in high production costs. E. coli has been developed as an alternative for producing itaconate. However, fermentation of glucose gives low conversion yields and low productivity. Here, we report the whole-cell bioconversion of citrate to itaconate with enhanced aconitase and cis-aconitate decarboxylase activities by controlling the expression of multiple cadA genes. In addition, this bioconversion system does not require the use of buffers, which reduces the production cost and the byproducts released during purification. Using this whole-cell bioconversion system, we were able to catalyze the conversion of 319.8 mM of itaconate (41.6 g/L) from 500 mM citrate without any buffer system or additional cofactors, with 64.0% conversion in 19 h and a productivity of 2.19 g/L/h. Our bioconversion system suggests very high productivity for itaconate production.

Functional Study of Lysine Decarboxylases from Klebsiella pneumoniae in Escherichia coli and Application of Whole Cell Bioconversion for Cadaverine Production.

Klebsiella pneumoniae is a gram-negative, non-motile, rod-shaped, and encapsulated bacterium in the normal flora of the intestines, mouth, skin, and food, and has decarboxylation activity, which results in generation of diamines (cadaverine, agmatine, and putrescine). However, there is no specific information on the exact mechanism of decarboxylation in K. pnuemoniae. Specifically lysine decarboxylases that generate cadaverine with a wide range of applications has not been shown. Therefore, we performed a functional study of lysine decarboxylases. Enzymatic characteristics such as optimal pH, temperature, and substrates were examined by overexpressing and purifying CadA and LdcC. CadA and LdcC from K. pneumoniae had a preference for L-lysine, and an optimal reaction temperature of 37°C and an optimal pH of 7. Although the activity of purified CadA from K. pneumoniae was lower than that of CadA from E. coli, the activity of K. pneumoniae CadA in whole cell bioconversion was comparable to that of E. coli CadA, resulting in 90% lysine conversion to cadaverine with pyridoxal 5'-phosphate L-lysine.