Enhancing polyethylene degradation: a novel bioprocess approach using Acinetobacter nosocomialis pseudo-resting cells.

Despite the discovery of several bacteria capable of interacting with polymers, the activity of the natural bacterial isolates is limited. Furthermore, there is a lack of knowledge regarding the development of bioprocesses for polyethylene (PE) degradation. Here, we report a bioprocess using pseudo-resting cells for efficient degradation of PE. The bacterial strain Acinetobacter nosocomialis was isolated from PE-containing landfills and characterized using low-density PE (LDPE) surface oxidation when incubated with LDPE. We optimized culture conditions to generate catalytic pseudo-resting cells of A. nosocomialis that are capable of degrading LDPE films in a bioreactor. After 28 days of bioreactor operation using pseudo-resting cells of A. nosocomialis, we observed the formation of holes on the PE film (39 holes per 217 cm2, a maximum diameter of 1440 µm). This study highlights the potential of bacteria as biocatalysts for the development of PE degradation processes. KEY POINTS: • New bioprocess has been proposed to degrade polyethylene (PE). • Process with pseudo-resting cells results in the formation of holes in PE film. • We demonstrated PE degradation using A. nosocomialis as a biocatalyst.

Efforts to install a heterologous Wood-Ljungdahl pathway in Clostridium acetobutylicum enable the identification of the native tetrahydrofolate (THF) cycle and result in early induction of solvents.

Here, we report the construction of a Clostridium acetobutylicum strain ATCC 824 (pCD07239) by heterologous expression of carbonyl branch genes (CD630_0723∼CD630_0729) from Clostridium difficile, aimed at installing a heterologous Wood-Ljungdahl pathway (WLP). As part of this effort, in order to validate the methyl branch of the WLP in the C. acetobutylicum, we performed 13C-tracing analysis on knockdown mutants of four genes responsible for the formation of 5-methyl-tetrahydrofolate (5-methyl-THF) from formate: CA_C3201, CA_C2310, CA_C2083, and CA_C0291. While C. acetobutylicum 824 (pCD07239) could not grow autotrophically, in heterotrophic fermentation, it began producing butanol at the early growth phase (OD600 of 0.80; 0.162 g/L butanol). In contrast, solvent production in the parent strain did not begin until the early stationary phase (OD600 of 7.40). This study offers valuable insights for future research on biobutanol production during the early growth phase.

Environmental toxicity and decomposition of polyethylene.

In the more than 100 years since the invention of plastics, various plastic polymers have been developed that exhibit different characteristics and have been widely used in production and life. In 2020 alone, nearly 400 million tons of plastics were produced globally. However, while plastic such as polyethylene brings us convenience, it also threatens environmental sustainability and human health. Due to insufficient recycling efficiency, millions of tons of polyethylene pollutants accumulate in terrestrial or marine environments each year. Polyethylene is elastic, chemically stable, and non-biodegradable, and the traditional disposal methods include landfilling and incineration. These methods are costly, unsustainable, and further increase the burden on the environment. Therefore, recent research has increasingly focused on the biodegradation of polyethylene. In this work, we briefly summarized polyethylene's properties and environmental toxicity. We also reviewed the recent advances in the biodegradation of polyethylene with a summary of traditional abiotic methods. Finally, we proposed a brief research direction in polyethylene study with the aspect of environmental toxicology and industrial applications of decomposition technology.

Metabolic engineering of microorganisms for the production of ethanol and butanol from oxides of carbon.

The utilized biomass is an important consideration for sustainable biofuel production. To avoid competing with food needs, researchers have turned their attention to non-food lignocellulosic biomasses as potential feedstocks for biofuel production. However, the saccharification of a lignocellulosic biomass produces a large amount of lignin as waste. To overcome this hurdle, biomass gasification has been suggested as an alternative to saccharification. During biomass gasification, oxides of carbon (CO, CO2) and hydrogen are produced as a major product. Accordingly, microorganisms capable of utilizing these oxides of carbon have gained attention as hosts for the production of biofuels, such as ethanol and butanol. In this work, we reviewed the Calvin cycle and Wood-Ljungdahl pathway for utilizing oxides of carbon in cyanobacteria and acetogens, respectively, and discussed the metabolic engineering strategies that may be used to produce ethanol and butanol from oxides of carbon through these routes.

Microbial production of butyl butyrate, a flavor and fragrance compound.

Butyl butyrate (BB) has been widely used as a flavor and fragrance compound in the beverage, food, perfume, and cosmetic industries. Currently, BB is produced through two-step processes; butanol and butyrate are first produced and are used as precursors for the esterification reactions to yield BB in the next step. Recently, an alternative process to the current process has been developed by using microorganisms for the one-pot BB production. In the one-pot BB process, alcohol acyl transferases (AATs) and lipases play roles in the esterification of butanol together with their co-substrates butyryl-CoA and butyrate, respectively. In this paper, we review the characteristics of two enzymes including AAT and lipase in the esterification reaction. Also, we review the one-pot processes for BB production by employing the wild-type and engineered Clostridium species and the engineered Escherichia coli strains, with the combination of AATs and lipases.

Metabolic engineering of Clostridium acetobutylicum for the production of butyl butyrate.

Butyl butyrate is widely used as a fragrance additive for foods and beverages. The first step in the currently used process is the production of precursors, including butanol and butyrate, from petroleum using chemical catalysts, followed by the conversion of precursors to butyl butyrate by immobilized lipase. In this work, we engineered Clostridium acetobutylicum for the selective, one-step production of butyl butyrate from glucose. C. acetobutylicum ATCC 824, possessing a strong carbon flux that yields butanol and butyryl-CoA, was selected as a host and was engineered by introducing alcohol acyltransferases (AATs) from Fragaria x ananassa (strawberry) or Malus sp. (apple). Batch culture of the engineered C. acetobutylicum strain CaSAAT expressing the strawberry SAAT gene produced 50.07 mg/L of butyl butyrate with a selectivity of 84.8% of total esters produced. Also, the engineered C. acetobutylicum strain CaAAAT expressing the apple AAAT gene produced 40.60 mg/L of butyl butyrate with a selectivity of 87.4%. This study demonstrated the feasibility of the one-step fermentation of butyl butyrate from glucose in the engineered C. acetobutylicum, as a proof of concept.

Metabolic engineering of Clostridium acetobutylicum for butyric acid production with high butyric acid selectivity.

A typical characteristic of the butyric acid-producing Clostridium is coproduction of both butyric and acetic acids. Increasing the butyric acid selectivity important for economical butyric acid production has been rather difficult in clostridia due to their complex metabolic pathways. In this work, Clostridium acetobutylicum was metabolically engineered for highly selective butyric acid production. For this purpose, the second butyrate kinase of C. acetobutylicum encoded by the bukII gene instead of butyrate kinase I encoded by the buk gene was employed. Furthermore, metabolic pathways were engineered to further enhance the NADH-driving force. Batch fermentation of the metabolically engineered C. acetobutylicum strain HCBEKW (pta(-), buk(-), ctfB(-) and adhE1(-)) at pH 6.0 resulted in the production of 32.5g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AA ratio) of 31.3g/g from 83.3g/L of glucose. By further knocking out the hydA gene (encoding hydrogenase) in the HCBEKW strain, the butyric acid titer was not further improved in batch fermentation. However, the BA/AA ratio (28.5g/g) obtained with the HYCBEKW strain (pta(-), buk(-), ctfB(-), adhE1(-) and hydA(-)) was 1.6 times higher than that (18.2g/g) obtained with the HCBEKW strain at pH 5.0, while no improvement was observed at pH 6.0. These results suggested that the buk gene knockout was essential to get a high butyric acid selectivity to acetic acid in C. acetobutylicum.

Proteomic analyses of the phase transition from acidogenesis to solventogenesis using solventogenic and non-solventogenic Clostridium acetobutylicum strains.

The fermentation carried out by the solvent-producing bacterium, Clostridium acetobutylicum, is characterized by two distinct phases: acidogenic and solventogenic phases. Understanding the cellular physiological changes occurring during the phase transition in clostridial fermentation is important for the enhanced production of solvents. To identify protein changes upon entry to stationary phase where solvents are typically produced, we herein analyzed the proteomic profiles of the parental wild type C. acetobutylicum strains, ATCC 824, the non-solventogenic strain, M5 that has lost the solventogenic megaplasmid pSOL1, and the synthetic simplified alcohol forming strain, M5 (pIMP1E1AB) expressing plasmid-based CoA-transferase (CtfAB) and aldehyde/alcohol dehydrogenase (AdhE1). A total of 68 protein spots, corresponding to 56 unique proteins, were unambiguously identified as being differentially present after the phase transitions in the three C. acetobutylicum strains. In addition to changes in proteins known to be involved in solventogenesis (AdhE1 and CtfB), we identified significant alterations in enzymes involved in sugar transport and metabolism, fermentative pathway, heat shock proteins, translation, and amino acid biosynthesis upon entry into the stationary phase. Of these, four increased proteins (AdhE1, CAC0233, CtfB and phosphocarrier protein HPr) and six decreased proteins (butyrate kinase, ferredoxin:pyruvate oxidoreductase, phenylalanyl-tRNA synthetase, adenylosuccinate synthase, pyruvate kinase and valyl-tRNA synthetase) showed similar patterns in the two strains capable of butanol formation. Interestingly, significant changes of several proteins by post-translational modifications were observed in the solventogenic phase. The proteomic data from this study will improve our understanding on how cell physiology is affected through protein levels patterns in clostridia.

Effect of Abiotic Signals on the Accumulation of Saponarin in Barley Leaves in Hydroponics Under Artificial Lights.

Functional flavonoid production is a new agenda in the agricultural industry, and young barley leaves (YBL) are one of the highlighted crops due to their health-beneficial flavonoid, saponarin. For the year-round cultivation of a high saponarin content of YBL, abiotic signal effects on the biosynthesis and metabolism in YBL need to be understood clearly. In this research, the effects of reactive oxygen species (ROS)-related abiotic signals, such as light, potassium, and sodium, were investigated on the biosynthetic metabolism in YBL cultivation under artificial lights. A higher quantity of blue-rich white light (6500 K of light temperature) irradiation enhanced ROS levels and the related enzyme activities (APX and CAT), as well as photosynthesis and saponarin amount, while red-rich white light (3000 K of light temperature) increased the photosynthesis only. In addition, 1.0 g L-1 K+ treatment in water slightly reduced ROS levels and increased saponarin accumulation in YBL. These blue-rich light and K+ supplemental conditions relatively increased OGT expression and reduced 4-coumaric acid and isovitexin as saponarin precursors. Furthermore, the relative ratio of lutonarin as an oxidized product of saponarin increased in increments of light quantity. Finally, the abiotic conditions for saponarin production were optimized with the mixture solution treatment of 1.0 g L-1 Na+ and 1.0 g L-1 K+ under 500 PPFD of 6500 K light, and the saponarin amount per leaf was 219.5 µg plant-1; it was comparable amount with that under sunlight condition.

Acetone-butanol-ethanol production with high productivity using Clostridium acetobutylicum BKM19.

Conventional acetone-butanol-ethanol (ABE) fermentation is severely limited by low solvent titer and productivities. Thus, this study aims at developing an improved Clostridium acetobutylicum strain possessing enhanced ABE production capability followed by process optimization for high ABE productivity. Random mutagenesis of C. acetobutylicum PJC4BK was performed by screening cells on fluoroacetate plates to isolate a mutant strain, BKM19, which exhibited the total solvent production capability 30.5% higher than the parent strain. The BKM19 produced 32.5 g L(-1) of ABE (17.6 g L(-1) butanol, 10.5 g L(-1) ethanol, and 4.4 g L(-1) acetone) from 85.2 g L(-1) glucose in batch fermentation. A high cell density continuous ABE fermentation of the BKM19 in membrane cell-recycle bioreactor was studied and optimized for improved solvent volumetric productivity. Different dilution rates were examined to find the optimal condition giving highest butanol and ABE productivities. The maximum butanol and ABE productivities of 9.6 and 20.0 g L(-1) h(-1) , respectively, could be achieved at the dilution rate of 0.85 h(-1) . Further cell recycling experiments were carried out with controlled cell-bleeding at two different bleeding rates. The maximum solvent productivities were obtained when the fermenter was operated at a dilution rate of 0.86 h(-1) with the bleeding rate of 0.04 h(-1) . Under the optimal operational condition, butanol and ABE could be produced with the volumetric productivities of 10.7 and 21.1 g L(-1) h(-1) , and the yields of 0.17 and 0.34 g g(-1) , respectively. The obtained butanol and ABE volumetric productivities are the highest reported productivities obtained from all known-processes.

Metabolic engineering of Clostridium acetobutylicum for enhanced production of butyric acid.

Clostridium acetobutylicum has been considered as an attractive platform host for biorefinery due to its metabolic diversity. Considering its capability to overproduce butanol through butyrate, it was thought that butyric acid can also be efficiently produced by this bacterium through metabolic engineering. The pta-ctfB-deficient C. acetobutylicum CEKW, in which genes encoding phosphotransacetylase and CoA-transferase were knocked out, was assessed for its potential as a butyric acid producer in fermentations with four controlled pH values at 5.0, 5.5, 6.0, and 6.4. Butyric acid could be best produced by fermentation of the CEKW at pH 6.0, resulting in the highest titer of 26.6 g/l, which is 6.4 times higher than that obtained with the wild type. However, due to the remaining solventogenic ability of the CEKW, 3.6 g/l solvents were also produced. Thus, the CEKW was further engineered by knocking out the adhE1-encoding aldehyde/alcohol dehydrogenase to prevent solvent production. Batch fermentation of the resulting C. acetobutylicum HCEKW at pH 6.0 showed increased butyric acid production to 30.8 g/l with a ratio of butyric-to-acetic acid (BA/AA) of 6.6 g/g and a productivity of 0.72 g/l/h from 86.9 g/l glucose, while negligible solvent (0.8 g/l ethanol only) was produced. The butyric acid titer, BA/AA ratio, and productivity obtained in this study were the highest values reported for C. acetobutylicum, and the BA/AA ratio and productivity were also comparable to those of native butyric acid producer Clostridium tyrobutyricum. These results suggested that the simultaneous deletion of the pta-ctfB-adhE1 in C. acetobutylicum resulted in metabolic switch from biphasic to acidogenic fermentation, which enhanced butyric acid production.

Complete Genome Sequence of Acinetobacter nosocomialis GNU001, Isolated from a Plastic-Containing Landfill.

Due to the hazard of plastic waste exposed to the environment, microorganisms capable of degrading different polymeric pollutants have gained attention. Here, we report the complete genome sequence of Acinetobacter nosocomialis GNU001, which was isolated from a landfill. The genome was composed of a circular chromosome of 3,850,149 bp and a plasmid.

Harnessing lignocellulosic biomass for butanol production through clostridia for sustainable waste management: recent advances and perspectives.

The escalating waste generation rates, driven by population growth, urbanization, and consumption patterns, have made waste management a critical global concern with significant environmental, social, and economic repercussions. Among the various waste sources, lignocellulosic biomass represents a significant proportion of agricultural, agro-industrial, and municipal wastes. Biofuels are gaining attention as a promising substitute to fossil fuels, and butanol is one such biofuel that has been identified as a potential candidate due to its compatibility with existing fuel infrastructure, lower volatility, and higher energy density. Sustainable management of lignocellulosic biomass waste and its utilization in fermentation are viable alternatives to produce butanol via the promising microbial catalyst clostridia. This review provides an overview of lignocellulosic biomass waste management, focusing on recent advances in strain development for butanol production from renewable biomass with an emphasis on future perspectives.

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for isopropanol-butanol-ethanol fermentation.

Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh(B-593)) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.

Bio-based production of C2-C6 platform chemicals.

Platform chemicals composed of 2-6 carbons derived from fossil resources are used as important precursors for making a variety of chemicals and materials, including solvents, fuels, polymers, pharmaceuticals, perfumes, and foods. Due to concerns regarding our environment and the limited nature of fossil resources, however, increasing interest has focused on the development of sustainable technologies for producing these platform chemicals from renewable resources. The techniques and strategies for developing microbial strains for chemicals production have advanced rapidly, and it is becoming feasible to develop microbes for producing additional types of chemicals, including non-natural molecules. In this study, we review the current status of the bio-based production of major C2-C6 platform chemicals, focusing on the microbial production of platform chemicals that have been used for the production of chemical intermediates, building block compounds, and polymers.

Continuous butanol production with reduced byproducts formation from glycerol by a hyper producing mutant of Clostridium pasteurianum.

Butanol, a four-carbon primary alcohol (C(4)H(10)O), is an important industrial chemical and has a good potential to be used as a superior biofuel. Bio-based production of butanol from renewable feedstock is a promising and sustainable alternative to substitute petroleum-based fuels. Here, we report the development of a process for butanol production from glycerol, which is abundantly available as a byproduct of biodiesel production. First, a hyper butanol producing strain of Clostridium pasteurianum was isolated by chemical mutagenesis. The best mutant strain, C. pasteurianum MBEL_GLY2, was able to produce 10.8 g l(-1) butanol from 80 g l(-1) glycerol as compared to 7.6 g l(-1) butanol produced by the parent strain. Next, the process parameters were optimized to maximize butanol production from glycerol. Under the optimized batch condition, the butanol concentration, yield, and productivity of 17.8 g l(-1), 0.30 g g(-1), and 0.43 g l(-1) h(-1) could be achieved. Finally, continuous fermentation of C. pasteurianum MBEL_GLY2 with cell recycling was carried out using glycerol as a major carbon source at several different dilution rates. The continuous fermentation was run for 710 h without strain degeneration. The acetone-butanol-ethanol productivity and the butanol productivity of 8.3 and 7.8 g l(-1) h(-1), respectively, could be achieved at the dilution rate of 0.9 h(-1). This study reports continuous production of butanol with reduced byproducts formation from glycerol using C. pasteurianum, and thus could help design a bioprocess for the improved production of butanol.

Escherichia coli W as a new platform strain for the enhanced production of L-valine by systems metabolic engineering.

A less frequently employed Escherichia coli strain W, yet possessing useful metabolic characteristics such as less acetic acid production and high L-valine tolerance, was metabolically engineered for the production of L-valine. The ilvA gene was deleted to make more pyruvate, a key precursor for L-valine, available for enhanced L-valine biosynthesis. The lacI gene was deleted to allow constitutive expression of genes under the tac or trc promoter. The ilvBN(mut) genes encoding feedback-resistant acetohydroxy acid synthase (AHAS) I and the L-valine biosynthetic ilvCED genes encoding acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and branched chain amino acid aminotransferase, respectively, were amplified by plasmid-based overexpression. The global regulator Lrp and L-valine exporter YgaZH were also amplified by plasmid-based overexpression. The engineered E. coli W (ΔlacI ΔilvA) strain overexpressing the ilvBN(mut) , ilvCED, ygaZH, and lrp genes was able to produce an impressively high concentration of 60.7 g/L L-valine by fed-batch culture in 29.5 h, resulting in a high volumetric productivity of 2.06 g/L/h. The most notable finding is that there was no other byproduct produced during L-valine production. The results obtained in this study suggest that E. coli W can be a good alternative to Corynebacterium glutamicum and E. coli K-12, which have so far been the most efficient L-valine producer. Furthermore, it is expected that various bioproducts including other amino acids might be more efficiently produced by this revisited platform strain of E. coli.

Characterization of an organic solvent-tolerant polysaccharide lyase from Microbulbifer thermotolerans DAU221.

Alginate and its derivatives are annually produced approximately 30,000 tons or more and are applied to various industries as they are natural polymers. The global market for alginate and its derivatives has been growing steadily. There is little research compared to other enzymes produced through biomass degradation or modification. An alginate lyase, MtAl138, from Microbulbifer thermotolerans DAU221 was cloned and identified in Escherichia coli BL21 (DE3). MtAl138 contains a highly conserved motif (R538TELR, Q607IH609, and YFKAGVY716NQ), which indicates that it belongs to the polysaccharide lyase family 7 (PL7). MtAl138, with a molecular weight of 77 kDa worked optimally at 45 °C and pH 7.4. MtAl138 showed twice as much activity as when there was no NaCl when there was between 100 and 600 mM NaCl. Moreover, its activity increased in organic solvents such as benzene, hexane, methanol, and toluene. Based on the thin layer chromatography analyses, MtAl38 is an endo-type enzyme that produces di-, tri-, or tetrasaccharides from polyG and polyM. This study provided that MtAl138 is an endoenzyme that showed outstanding enzymatic activity at concentrated salt solutions and organic solvents, which makes it a reasonably attractive enzyme for use in various industries.

Clostridium acetobutylicum atpG-Knockdown Mutants Increase Extracellular pH in Batch Cultures.

ATPase, a key enzyme involved in energy metabolism, has not yet been well studied in Clostridium acetobutylicum. Here, we knocked down the atpG gene encoding the ATPase gamma subunit in C. acetobutylicum ATCC 824 using a mobile group II intron system and analyzed the physiological characteristics of the atpG gene knockdown mutant, 824-2866KD. Properties investigated included cell growth, glucose consumption, production of major metabolites, and extracellular pH. Interestingly, in 2-L batch fermentations, 824-2866KD showed no significant difference in metabolite biosynthesis or cell growth compared with the parent ATCC 824. However, the pH value in 824-2866KD cultures at the late stage of the solventogenic phase was abnormally high (pH 6.12), compared with that obtained routinely in the culture of ATCC 824 (pH 5.74). This phenomenon was also observed in batch cultures of another C. acetobutylicum, BEKW-2866KD, an atpG-knockdown and pta-buk double-knockout mutant. The findings reported in this study suggested that ATPase is relatively minor than acid-forming pathway in ATP metabolism in C. acetobutylicum.

Control of the galactose-to-glucose consumption ratio in co-fermentation using engineered Escherichia coli strains.

Marine biomasses capable of fixing carbon dioxide have attracted attention as an alternative to fossil resources for fuel and chemical production. Although a simple co-fermentation of fermentable sugars, such as glucose and galactose, has been reported from marine biomass, no previous report has discussed the fine-control of the galactose-to-glucose consumption ratio in this context. Here, we sought to finely control the galactose-to-glucose consumption ratio in the co-fermentation of these sugars using engineered Escherichia coli strains. Toward this end, we constructed E. coli strains GR2, GR2P, and GR2PZ by knocking out galRS, galRS-pfkA, and galRS-pfkA-zwf, respectively, in parent strain W3110. We found that strains W3110, GR2, GR2P, and GR2PZ achieved 0.03, 0.09, 0.12, and 0.17 galactose-to-glucose consumption ratio (specific galactose consumption rate per specific glucose consumption rate), respectively, during co-fermentation. The ratio was further extended to 0.67 by integration of a brief process optimization for initial sugar ratio using GR2P strain. The strategy reported in this study will be helpful to expand our knowledge on the galactose utilization under glucose conditions.