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.

A review on microbes mediated resource recovery and bioplastic (polyhydroxyalkanoates) production from wastewater.

BACKGROUND: Plastic is widely utilized in packaging, frameworks, and as coverings material. Its overconsumption and slow degradation, pose threats to ecosystems due to its toxic effects. While polyhydroxyalkanoates (PHA) offer a sustainable alternative to petroleum-based plastics, their production costs present significant obstacles to global adoption. On the other side, a multitude of household and industrial activities generate substantial volumes of wastewater containing both organic and inorganic contaminants. This not only poses a threat to ecosystems but also presents opportunities to get benefits from the circular economy. Production of bioplastics may be improved by using the nutrients and minerals in wastewater as a feedstock for microbial fermentation. Strategies like feast-famine culture, mixed-consortia culture, and integrated processes have been developed for PHA production from highly polluted wastewater with high organic loads. Various process parameters like organic loading rate, organic content (volatile fatty acids), dissolved oxygen, operating pH, and temperature also have critical roles in PHA accumulation in microbial biomass. Research advances are also going on in downstream and recovery of PHA utilizing a combination of physical and chemical (halogenated solvents, surfactants, green solvents) methods. This review highlights recent developments in upcycling wastewater resources into PHA, encompassing various production strategies, downstream processing methodologies, and techno-economic analyses. SHORT CONCLUSION: Organic carbon and nitrogen present in wastewater offer a promising, cost-effective source for producing bioplastic. Previous attempts have focused on enhancing productivity through optimizing culture systems and growth conditions. However, despite technological progress, significant challenges persist, such as low productivity, intricate downstream processing, scalability issues, and the properties of resulting PHA.

Characteristics and functional outcomes of varus displaced proximal humerus fractures.

BACKGROUND: The purpose of this study was to compare fracture characteristics and functional outcomes between patients with proximal humerus fractures with and without initial varus displacement. METHODS: A retrospective review of 325 patients with proximal humerus fractures was performed. Patients with initial varus displacement were placed in Varus cohort and were age- and sex-matched 1:1 with a second cohort presenting proximal humerus fractures without varus displacement, referred to as Fracture cohort. Varus fracture displacement was defined when the most proximal aspect of humeral head was below the most proximal aspect of greater tuberosity on initial radiographs, and the head shaft angle was <130°. RESULTS: There were 60 patients in V cohort and 60 patients in F cohort. Statistical analysis revealed that there were significant differences in initial horizontal offset (38.8 vs. 45.9 mm), initial anterior angulation angle (36.5° vs. 16.4°), postoperative head shaft angle (132.2° vs. 141.3°), last head shaft angle (122.2° vs. 138.5°), difference for head shaft angles (10.0° vs. 2.7°), postoperative horizontal offset (43.4 vs. 45.3 mm), last horizontal offset (38.4 vs. 42.8 mm), difference for offsets (4.9 vs. 2.5 mm), complications (15 vs. 7 cases), and revision surgery (7 vs. 1 case) between two cohorts. Overall satisfactory results were achieved in most patients regardless of varus displacement, pain-VAS and Constant scores in V cohort were inferior to the scores in F cohort. The cut-off value of postoperative head shaft angle for good/excellent outcomes was 135.5° using receiver operating characteristic curve analyses. CONCLUSION: Varus displaced proximal humerus fractures were accompanied by decreased horizontal offset and increased anterior angulation angle, and had a course of more varization and horizontal shortening compared with those without initial varus displacement. Patients with varus displaced fractures were associated with worse functional outcomes, and these factors might affect functional outcomes. LEVEL OF EVIDENCE: Prognostic, cohort study, Level III.

Optimization of polyhydroxyalkanoate production in Halomonas sp. YLGW01 using mixed volatile fatty acids: a study on mixture analysis and fed-batch strategy.

Polyhydroxyalkanoate (PHA) is one of the most promising materials for replacing petroleum-based plastics, and it can be produced from various renewable biomass sources. In this study, PHA production was conducted using Halomonas sp. YLGW01 utilizing mixed volatile fatty acids (VFAs) as carbon sources. The ratio and concentration of carbon and nitrogen sources were optimized through mixture analysis and organic nitrogen source screening, respectively. It was found that the highest cell dry weight (CDW) of 3.15 g/L and PHA production of 1.63 g/L were achieved when the ratio of acetate to lactate in the mixed VFAs was 0.45:0.55. Furthermore, supplementation of organic nitrogen sources such as soytone resulted in a ninefold increase in CDW (reaching 2.32 g/L) and a 22-fold increase in PHA production (reaching 1.60 g/L) compared to using inorganic nitrogen sources. Subsequently, DO-stat, VFAs consumption rate stat, and pH-stat fed-batch methods were applied to investigate and evaluate PHA productivity. The results showed that when pH-stat-based VFAs feeding was employed, a CDW of 7 g/L and PHA production of 5.1 g/L were achieved within 68 h, with a PHA content of 73%. Overall, the pH-stat fed-batch strategy proved to be effective in enhancing PHA production by Halomonas sp. YLGW01 utilizing VFAs.

Surgical outcomes of segmental diaphyseal forearm fractures in adults: a small case series on plate osteosynthesis, intramedullary nailing, and other surgical methods.

BACKGROUND: Segmental fractures often result from high-energy or indirect trauma that causes bending or torsional forces with axial loading. We evaluated surgical outcomes of patients with forearm segmental diaphyseal fractures. METHODS: We retrospectively analyzed data from patients with forearm segmental fractures for which they underwent surgery at the Pusan National University Trauma Center from March 2013 to March 2022. We also analyzed accompanying injuries, injury severity score (ISS), injury mechanism, occurrence of open fracture, surgical technique, and treatment results. RESULTS: Fifteen patients were identified, one with bilateral segmental diaphyseal forearm bone fracture, for a total of 16 cases. Nine of the patients were male. The overall mean age was 50 years, and the mean follow-up period was 16.2 months. Six cases who underwent surgery using plate osteosynthesis achieved bone union without length deformity at final follow-up. Three of seven patients who underwent intramedullary nailing alone underwent reoperation due to nonunion. Six cases achieved bone union at final follow-up, three of which showed length deformity. Three patients underwent surgery using a hybrid method of IM nailing, plates, and mini cables. One patient who underwent surgery with a plate and one patient who underwent surgery with IM nailing alone showed nonunion and were lost to follow-up. CONCLUSION: Plate osteosynthesis is considered the gold standard for treatment of adult forearm diaphyseal segmental fractures. In this study, IM nailing was associated with high rates of non-union and length deformity. However, the combination of IM nailing and a plate-cable system may be an acceptable alternative in segmental diaphyseal forearm fracture, achieving a union rate similar to that provided by plate fixation.

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.

Humeral head coverage in arthroscopic partial repair of massive rotator cuff tears improves functional outcomes: an analysis of influential factors.

BACKGROUND: Although partial rotator cuff repair has shown good outcomes, differences in clinical outcomes remain concerns. This study was performed to determine whether patients with humeral head coverage would show better functional outcomes than patients without humeral head coverage and to identify the factors for humeral head coverage after arthroscopic partial repair of massive tears. METHODS: We performed a retrospective study of 63 patients with massive rotator cuff tears who underwent arthroscopic partial repair between 2012 and 2018. Two to four margin convergences were first performed; then, the Mason-Allen technique was performed. The patients were divided into 2 groups: those with humeral head coverage (38 cases) and those without humeral head coverage (25 cases). The following factors were evaluated: age; sex; hypertension; diabetes; osteoporosis; preoperative and postoperative pseudoparalysis, visual analog scale (VAS) pain score, Constant score, acromiohumeral distance, and subacromial bony spur; and subscapularis tear and repair. Muscle atrophy and fatty degeneration were evaluated by magnetic resonance imaging preoperatively, and the integrity of the repaired cuff was evaluated by ultrasonography at a minimum of 2 years after surgery. RESULTS: Compared with preoperative values, significant improvements in VAS pain scores (from 6.27 to 2.32 in patients with humeral head coverage and from 7.00 to 2.81 in those without humeral head coverage) and Constant scores (from 51.35 to 75.95 and from 44.62 to 69.81, respectively) were observed in both groups (P < .001). Statistical analysis revealed that postoperative VAS pain scores (2.32 vs. 2.81) and Constant scores (75.95 vs. 69.81) in patients with humeral head coverage were superior to those in patients without humeral head coverage (P = .044 and P = .003, respectively). The integrity of the repaired cuff was evaluated by ultrasonography, and partial tears were found in 4 of 37 patients with humeral head coverage and 2 of 26 patients without humeral head coverage (P = .816). Univariable logistic regression analysis revealed that age (P < .001), comorbidity (P = .005), symptom duration (P = .023), preoperative shoulder mobility (P < .001), maintained acromiohumeral distance (P = .006), subscapularis tear (P = .026), and less preoperative supraspinatus and infraspinatus muscle atrophy (P = .001 and P = .010, respectively) had significant correlations with humeral head coverage. CONCLUSIONS: Overall satisfactory results were achieved in most patients regardless of high retear rates, but patients with partial repair covering the humeral head were associated with better outcomes than patients without humeral head coverage. Multivariable regression analysis revealed that age (<70 years, P = .003), capability of shoulder mobility (P = .005), maintenance of the acromiohumeral space (>7 mm, P = .016), and less atrophy of the rotator cuff muscles (P = .021) were favorable factors to achieve humeral head coverage during surgical partial repair of massive rotator cuff tears.

Risk factors for nonunion in oblique lateral interbody fusion.

BACKGROUND: Compared with posterior interbody fusion techniques, oblique lateral interbody fusion (OLIF) offers a larger fusion bed with greater intervertebral space access, use of larger cages, more sufficient discectomy, and better end-plate preparation. However, the fusion rate of OLIF is similar to that of other interbody fusions. This study aimed to examine the factors associated with nonunion in OLIF. METHODS: This study examined 201 disc levels from 124 consecutive patients who underwent OLIF for lumbar degenerative diseases with 1-year regular follow-up. Demographic and surgical factors were reviewed from the medical records. Radiological factors measured were sagittal parameters, intervertebral disc angle (DA) before surgery and at the final follow-up, presence of vertebral end-plate lesions, and cage subsidence. Multivariable logistic regression analysis was performed to identify the factors associated with nonunion. RESULTS: Among the 201 discs, 185 (92.0%) achieved union at 1-year followed up. Smoking, surgery at the L5-S1 level, not performing laminectomy, and a large intervertebral DA were factors associated with nonunion in OLIF (all P < 0.05). Multivariable logistic regression analysis showed two independent variables (surgery at L5-S1 level and not performing laminectomy) as risk factors for nonunion in OLIF. CONCLUSIONS: Not performing laminectomy and surgery at the L5-S1 level were risk factors for nonunion in OLIF. To reduce the nonunion rate, surgeons should consider additional stabilization strategies for the L5-S1 OLIF and perform laminectomy.

Enhanced isobutanol production from acetate by combinatorial overexpression of acetyl-CoA synthetase and anaplerotic enzymes in engineered Escherichia coli.

Acetic acid is an abundant material that can be used as a carbon source by microorganisms. Despite its abundance, its toxicity and low energy content make it hard to utilize as a sole carbon source for biochemical production. To increase acetate utilization and isobutanol production with engineered Escherichia coli, the feasibility of utilizing acetate and metabolic engineering was investigated. The expression of acs, pckA, and maeB increased isobutanol production by up to 26%, and the addition of TCA cycle intermediates indicated that the intermediates can enhance isobutanol production. For isobutanol production from acetate, acetate uptake rates and the NADPH pool were not limiting factors compared to glucose as a carbon source. This work represents the first approach to produce isobutanol from acetate with pyruvate flux optimization to extend the applicability of acetate. This technique suggests a strategy for biochemical production utilizing acetate as the sole carbon source.

Butyrate-based n-butanol production from an engineered Shewanella oneidensis MR-1.

n-Butanol is considered as the next-generation biofuel, because its physiochemical properties are very similar to fossil fuels and it could be produced by Clostridia under anaerobic culture. Due to the difficulties of strict anaerobic culture, a host which can be used with facultative environment was being searched for n-butanol production. As an alternative, Shewanella oneidensis MR-1, which is known as facultative bacteria, was selected as a host and studied. A plasmid containing adhE2 encoding alcohol dehydrogenase, various CoA transferases (ctfAB, atoAD, pct, and ACT), and acs encoding acetyl-CoA synthetase were introduced and examined to S. oneidensis MR-1 to produce n-butanol. As a result, ctfAB, acs, and adhE2 overexpression in S. oneidensis-pJM102 showed the highest n-butanol production in the presence of 2% of N-acetylglucosamine (NAG), 0.3% of butyrate, and 0.1 mM of IPTG for 96 h under microaerobic condition. When more NAG and butyrate were fed, n-butanol production was enhanced, producing up to 160 mg/L of n-butanol. When metal ions or extra electrons were added to S. oneidensis-pJM102 for n-butanol production, metal ion as electron acceptor or supply of extra electron showed no significant effect on n-butanol production. Overall, we made a newly engineered S. oneidensis that could utilize NAG and butyrate to produce n-butanol. It could be used in further microaerobic condition and electricity supply studies.

Production of (3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer from coffee waste oil using engineered Ralstonia eutropha.

Polyhydroxyalkonate (PHA) is a type of polymer that has the potential to replace petro-based plastics. To make PHA production more economically feasible, there is a need to find a new carbon source and engineer microbes to produce a commercially valuable polymer. Coffee waste is an inexpensive raw material that contains fatty acids. It can act as a sustainable carbon source and seems quite promising with PHA production in Ralstonia eutropha, which is a well-known microbe for PHA accumulation, and has the potential to utilize fatty acids. In this study, to make poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)), which has superior properties in terms of biodegradability, biocompatibility, and mechanical strength, engineered strain Ralstonia eutropha Re2133 overexpressing (R)-specific enoyl coenzyme-A hydratase (phaJ) and PHA synthetase (phaC2) with deletion of acetoacetyl Co-A reductases (phaB1, phaB2, and phaB3) was used to produce PHA from coffee waste oil. At a coffee oil concentration of 1.5%, and C/N ratio of 20, the R. eutropha Re2133 fermentation process results in 69% w/w of DCW PHA accumulation and consists of HB (78 mol%) and HHx (22 mol%). This shows the feasibility of using coffee waste oil for P(HB-co-HHx) production, as it is a low-cost fatty acid enriched waste material.

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.

Combinatorial application of two aldehyde oxidoreductases on isobutanol production in the presence of furfural.

Furfural is a toxic by-product formulated from pretreatment processes of lignocellulosic biomass. In order to utilize the lignocellulosic biomass on isobutanol production, inhibitory effect of the furfural on isobutanol production was investigated and combinatorial application of two oxidoreductases, FucO and YqhD, was suggested as an alternative strategy. Furfural decreased cell growth and isobutanol production when only YqhD or FucO was employed as an isobutyraldehyde oxidoreductase. However, combinatorial overexpression of FucO and YqhD could overcome the inhibitory effect of furfural giving higher isobutanol production by 110% compared with overexpression of YqhD. The combinatorial oxidoreductases increased furfural detoxification rate 2.1-fold and also accelerated glucose consumption 1.4-fold. When it compares to another known system increasing furfural tolerance, membrane-bound transhydrogenase (pntAB), the combinatorial aldehyde oxidoreductases were better on cell growth and production. Thus, to control oxidoreductases is important to produce isobutanol using furfural-containing biomass and the combinatorial overexpression of FucO and YqhD can be an alternative strategy.

Isobutanol production from an engineered Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is one of the most well-known metal-reducing bacteria and it has been extensively studied for microbial fuel cell and bioremediation aspects. In this study, we have examined S. oneidensis MR-1 as an isobutanol-producing host by assessing three key factors such as isobutanol synthetic genes, carbon sources, and electron supply systems. Heterologous Ehrlich pathway genes, kivD encoding ketoisovalerate decarboxylase and adh encoding alcohol dehydrogenase, were constructed in S. oneidensis MR-1. Among the composition of carbon sources examined, 2% of N-acetylglucosamine, 1.5% of pyruvate and 2% of lactate were found to be the most optimal nutrients and resulted in 10.3 mg/L of isobutanol production with 48 h of microaerobic incubation. Finally, the effects of metal ions (electron acceptor) and direct electron transfer systems on isobutanol production were investigated, and Fe(2+) ions increased the isobutanol production up to 35%. Interestingly, deletion of mtrA and mtrB, genes responsible for membrane transport systems, did not have significant impact on isobutanol production. Finally, we applied engineered S. oneidensis to a bioelectrical reactor system to investigate the effect of a direct electron supply system on isobutanol production, and it resulted in an increased growth and isobutanol production (up to 19.3 mg/L). This report showed the feasibility of S. oneidensis MR-1 as a genetic host to produce valuable biochemicals and combine an electron-supplying system with biotechnological applications.

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.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) from butyrate using engineered Ralstonia eutropha.

Polyhydroxyalkanoates (PHAs), a promising family of bio-based polymers, are considered to be alternatives to traditional petroleum-based plastics. Copolymers like poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) have been shown to exhibit favorable physical and mechanical properties, due to decreased crystallinity resulting from the presence of medium-chain-length 3-hydroxyhexanoate (3HHx) monomers. In this study, we produced P(HB-co-HHx) using engineered Ralstonia eutropha strains containing deletions of the acetoacetyl-CoA reductase (phaB) genes and replacing the native PHA synthase with phaC2 from Rhodococcus aetherivorans I24 and by using butyrate, a short-chain organic acid, as the carbon source. Although the wild-type R. eutropha did not produce P(HB-co-HHx) when grown on mixed acids or on butyrate as the sole carbon source, we are able to produce polymer containing up to 40 wt% 3HHx monomer with the aforementioned engineered R. eutropha strains using various concentrations of just butyrate as the sole carbon source. This is the first report for the production of P(HB-co-HHx) copolymer in R. eutropha using butyrate.

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.

Expanding the utilization of alkane mixtures: Enhancing medium chain length polyhydroxyalkanoate production in Pseudomonas resinovorans through alkane monooxygenase overexpression.

Medium chain length Polyhydroxyalkanoate (mcl-PHA) is a biodegradable bioplastic material with promising applications in various fields, including the medical, packaging, and agricultural industries. This mcl-PHA can be biosynthesized by microorganisms from various carbon sources, and notably, it can also be produced from alkane mixtures contained in pyrolysis oil derived from low-grade waste plastics. In this study, Pseudomonas resinovorans was engineered to overexpress alkane monooxygenase from Lysinibaillus fusiformis JJY0216, enhancing its ability to utilize alkanes as carbon sources and thereby increasing mcl-PHA production. The engineered strain, P. resinovorans JJY01, demonstrated a notable increase in cell dry weight (CDW) to 0.97 g/L and mcl-PHA production to 0.33 g/L from an optimized alkane mixture, achieving a 1.7-fold enhancement compared to the wild type. The PHA content reached 39.5 %, which is 3.1 times higher than the wild type. Further optimization through fed-batch cultivation resulted in P. resinovorans JJY01 achieving 5.65 g/L of CDW, 3.07 g/L of PHA, and a PHA content of 57.5 % within 96 h. In addition, produced mcl-PHA were characterized through various analytical techniques to assess their physical properties and monomer compositions, highlighting the potential of mcl-PHA produced by P. resinovorans JJY01 as a candidate for medical-grade biopolymers.

Production of Polyhydroxybutyrate by halotolerant Halomonas cerina YK44 using sugarcane molasses and soybean flour in tap water.

As environmental pollution intensifies, the interest in bioplastics is growing. The bioplastic polyhydroxyalkanoates (PHAs), which are produced and degraded by microorganisms, have received considerable attention. However, the production cost of PHA is still high, and several ways to increase economy of PHA production have been studied. Therefore, as one way of solution, Halomonas species were screened and evaluated with cheap substrates such as molasses and soybean flour. Among tested strains, Halomonas cerina YK44 was selected and used for polyhydroxybutyrate (PHB) production with molasses and soybean flour together, whose combination was not evaluated well before, in tap water. The medium composition optimization showed maximum PHB production at 4 % sugarcane molasses, 2 % NaCl, 0.05 % soybean flour, and pH 8 in tap water (9.2 g/L DCW, 7.3 g/L PHB, and 79.7 % PHB contents). However, cell growth of halotolerant H. cerina YK44 was disturbed by 0.2 % furfural, which existed in biomass based sugars as inhibitors. Physical and thermal analyses revealed that PHB film started from sugarcane molasses and soybean flour was no different from that initiated from simple sugars (Tm was 175.8 °C and 176.2 °C, PDI was 1.29, and 1.31, respectively).

Strategic Use of Vegetable Oil for Mass Production of 5-Hydroxyvalerate-Containing Polyhydroxyalkanoate from δ-Valerolactone by Engineered Cupriavidus necator.

Although efforts have been undertaken to produce polyhydroxyalkanoates (PHA) with various monomers, the low yield of PHAs because of complex metabolic pathways and inhibitory substrates remains a major hurdle in their analyses and applications. Therefore, we investigated the feasibility of mass production of PHAs containing 5-hydroxyvalerate (5HV) using δ-valerolactone (DVL) without any pretreatment along with the addition of plant oil to achieve enough biomass. We identified that PhaCBP-M-CPF4, a PHA synthase, was capable of incorporating 5HV monomers and that C. necator PHB-4 harboring phaCBP-M-CPF4 synthesized poly(3HB-co-3HHx-co-5HV) in the presence of bean oil and DVL. In fed-batch fermentation, the supply of bean oil resulted in the synthesis of 49 g/L of poly(3HB-co-3.7 mol% 3HHx-co-5.3 mol%5HV) from 66 g/L of biomass. Thermophysical studies showed that 3HHx was effective in increasing the elongation, whereas 5HV was effective in decreasing the melting point. The contact angles of poly(3HB-co-3HHx-co-5HV) and poly(3HB-co-3HHx) were 109 and 98°, respectively. In addition, the analysis of microbial degradation confirmed that poly(3HB-co-3HHx-co-5HV) degraded more slowly (82% over 7 days) compared to poly(3HB-co-3HHx) (100% over 5 days). Overall, the oil-based fermentation strategy helped produce more PHA, and the mass production of novel PHAs could provide more opportunities to study polymer properties.