Efficient production of polyhydroxybutyrate using lignocellulosic biomass derived from oil palm trunks by the inhibitor-tolerant strain Burkholderia ambifaria E5-3.

Lignocellulosic biomass is a valuable, renewable substrate for the synthesis of polyhydroxybutyrate (PHB), an ecofriendly biopolymer. In this study, bacterial strain E5-3 was isolated from soil in Japan; it was identified as Burkholderia ambifaria strain E5-3 by 16 S rRNA gene sequencing. The strain showed optimal growth at 37 °C with an initial pH of 9. It demonstrated diverse metabolic ability, processing a broad range of carbon substrates, including xylose, glucose, sucrose, glycerol, cellobiose, and, notably, palm oil. Palm oil induced the highest cellular growth, with a PHB content of 65% wt. The strain exhibited inherent tolerance to potential fermentation inhibitors derived from lignocellulosic hydrolysate, withstanding 3 g/L 5-hydroxymethylfurfural and 1.25 g/L acetic acid. Employing a fed-batch fermentation strategy with a combination of glucose, xylose, and cellobiose resulted in PHB production 2.7-times that in traditional batch fermentation. The use of oil palm trunk hydrolysate, without inhibitor pretreatment, in a fed-batch fermentation setup led to significant cell growth with a PHB content of 45% wt, equivalent to 10 g/L. The physicochemical attributes of xylose-derived PHB produced by strain E5-3 included a molecular weight of 722 kDa, a number-average molecular weight of 191 kDa, and a polydispersity index of 3.78. The amorphous structure of this PHB displayed a glass transition temperature of 4.59 °C, while its crystalline counterpart had a melting point of 171.03 °C. This research highlights the potential of lignocellulosic feedstocks, especially oil palm trunk hydrolysate, for PHB production through fed-batch fermentation by B. ambifaria strain E5-3, which has high inhibitor tolerance.

Electrotransformation of thermophilic bacterium Caldimonas manganoxidans.

Caldimonas manganoxidans is a Gram-negative, thermophilic, bioplastic-producing bacterium that is a promising strain to overcome the drawbacks of existing bioplastic manufacturing methods. However, genetic manipulation of this species has not previously been studied. Here, we developed an optimized electrotransformation protocol for C. manganoxidans by screening conditions, including the bacterial growth phase, electroporation buffer, pulse strength, and recovery time. The optimized transformation protocol obtained (3.1 ± 0.78) × 108 colony-forming units/µg DNA of plasmid pBBR1MCS-2. High transformation efficiency was observed when using plasmid DNA isolated from C. manganoxidans. The DNA methylases of Escherichia coli did not affect the transformation efficiency of C. manganoxidans. The electrotransformation technique proposed here will be beneficial for the genetic manipulation of thermophilic Caldimonas species.

Enhanced polyhydroxybutyrate (PHB) production by newly isolated rare actinomycetes Rhodococcus sp. strain BSRT1-1 using response surface methodology.

Poly-ß-hydroxybutyrate (PHB) is a biodegradable polymer, synthesized as carbon and energy reserve by bacteria and archaea. To the best of our knowledge, this is the first report on PHB production by a rare actinomycete species, Rhodococcus pyridinivorans BSRT1-1. Response surface methodology (RSM) employing central composite design, was applied to enhance PHB production in a flask scale. A maximum yield of 3.6 ± 0.5 g/L in biomass and 43.1 ± 0.5 wt% of dry cell weight (DCW) of PHB were obtained when using RSM optimized medium, which was improved the production of biomass and PHB content by 2.5 and 2.3-fold, respectively. The optimized medium was applied to upscale PHB production in a 10 L stirred-tank bioreactor, maximum biomass of 5.2 ± 0.5 g/L, and PHB content of 46.8 ± 2 wt% DCW were achieved. Furthermore, the FTIR and 1H NMR results confirmed the polymer as PHB. DSC and TGA analysis results revealed the melting, glass transition, and thermal decomposition temperature of 171.8, 4.03, and 288 °C, respectively. In conclusion, RSM can be a promising technique to improve PHB production by a newly isolated strain of R. pyridinivorans BSRT1-1 and the properties of produced PHB possessed similar properties compared to commercial PHB.

Small temperature variations are a key regulator of reproductive growth and assimilate storage in oil palm (Elaeis guineensis).

Oil palm is an important crop for global vegetable oil production, and is widely grown in the humid tropical regions of Southeast Asia. Projected future climate change may well threaten palm oil production. However, oil palm plantations currently produce large amounts of unutilised biological waste. Oil palm stems - which comprise two-thirds of the waste - are especially relevant because they can contain high levels of non-structural carbohydrates (NSC) that can serve as feedstock for biorefineries. The NSC in stem are also considered a potent buffer to source-sink imbalances. In the present study, we monitored stem NSC levels and female reproductive growth. We then applied convergent cross mapping (CCM) to assess the causal relationship between the time-series. Mutual causal relationships between female reproductive growth and the stem NSC were detected, with the exception of a relationship between female reproductive organ growth and starch levels. The NSC levels were also influenced by long-term cumulative temperature, with the relationship showing a seven-month time lag. The dynamic between NSC levels and long-term cumulative rainfall showed a shorter time lag. The lower temperatures and higher cumulative rainfall observed from October to December identify this as a period with maximum stem NSC stocks.

Direct production of polyhydroxybutyrate from waste starch by newly-isolated Bacillus aryabhattai T34-N4.

Polyhydroxybutyrate (PHB) is a natural microbial polyester produced by a variety of bacteria and archaea from renewable resources. PHB resembles some petrochemical plastics but is completely biodegradable. It is desirable to identify suitable microbial strains and develop processes that can directly use starch from agricultural wastes without commercial amylase treatment. Here, PHB production using starch from agricultural waste was developed using a newly isolated strain, Bacillus aryabhattai T34-N4. This strain hydrolyzed cassava pulp and oil palm trunk starch and accumulated up to 17 wt% PHB of the cell dry weight. The α-amylase of this strain, AmyA, showed high activity in the presence of cassava pulp starch (69.72 U) and oil palm trunk starch (70.53 U). High expression of amyA was recorded in the presence of cassava pulp starch, whereas low expression was detected in the presence of glucose. These data suggest that starch saccharification by amyA allows strain T34-N4 to grow and directly produce PHB from waste starch materials such as cassava pulp and oil palm trunk starch, which may be used as low-cost substrates.

Detoxification of Sap from Felled Oil Palm Trunks for the Efficient Production of Lactic Acid.

The availability of fermentable sugars in high concentrations in the sap of felled oil palm trunks and the thermophilic nature of the recently isolated Bacillus coagulans strain 191 were exploited for lactic acid production under non-sterile conditions. Screening indicated that strain 191 was active toward most sugars including sucrose, which is a major component of sap. Strain 191 catalyzed a moderate conversion of sap sugars to lactic acid (53%) with a productivity of 1.56 g/L/h. Pretreatment of oil palm sap (OPS) using alkaline precipitation improved the sugar fermentability, providing a lactic acid yield of 92% and productivity of 2.64 g/L/h. To better characterize potential inhibitors in the sap, phenolic, organic, and mineral compounds were analyzed using non-treated sap and saps treated with activated charcoal and alkaline precipitation. Phthalic acid, 3,4-dimethoxybenzoic acid, aconitic acid, syringic acid, and ferulic acid were reduced in the sap after treatment. High concentrations of Mg, P, K, and Ca were also precipitated by the alkaline treatment. These results suggest that elimination of excess phenolic and mineral compounds in OPS can improve the fermentation yield. OPS, a non-food resource that is readily available in bulk quantities from plantation sites, is a promising source for lactic acid production.

Evaluation of Enzymatic Deinking of Non-impact Ink Laser-Printed Paper Using Crude Enzyme from Penicillium rolfsii c3-2(1) IBRL.

Application of microbial enzymes for paper deinking is getting tremendous attention due to the rapidly increasing of waste paper every year. This study reports the deinking efficiency of laser-printed paper by the lignocellulolytic enzyme from Penicillium rolfsii c3-2(1) IBRL strain compared to other enzyme sources as well as commercial available enzymes. High enzymatic deinking efficiency of approximately 82 % on laser-printed paper was obtained by pulp treatment with crude enzyme from P. rolfsii c3-2(1) IBRL. However, this crude enzyme was found to reduce the paper strength properties of the pulp based on the results of tensile, tear and burst indices, most probably due to the cellulose degradation. This was further proven by the low viscosity of paper pulp obtained after enzymatic treatment and increasing of sugar production during the treatment. Balancing to this detrimental effect on paper pulp, high deinking efficiency was achieved within a short period of time, in which the enzymatic treatment was conducted for 30 min that enabled contribution to higher brightness index obtained, thus promoting savings of time and energy consumption, therefore environmental sustainability. Extensive research should be conducted to understand the nature and mechanism of enzymatic deinking process by the crude enzyme from P. rolfsii c3-2(1) IBRL in order to improve paper strength properties.

Characterization of oil-palm trunk residue degradation enzymes derived from the isolated fungus, Penicillium rolfsii c3-2(1) IBRL.

This study characterizes crude enzymes derived from Penicillium rolfsii c3-2(1) IBRL, a mesophilic fungus isolated from the local soil of Malaysia. Prior to enzyme activity evaluation, P. rolfsii c3-2(1) IBRL was inoculated into a broth medium containing oil-palm trunk residues for the preparation of crude enzymes. Oil-palm trunk residues were optimally hydrolysed at pH5.0 and 50°C. P. rolfsii c3-2(1) IBRL-derived crude enzymes displayed higher thermal stability compared with the commercial enzymes, Celluclast 1.5 L and Acellerase 1500. Moreover, the hydrolysing activities of the P. rolfsii c3-2(1) IBRL-derived crude enzymes (xylan, arabinan, and laminarin) were superior compared to that of Celluclast 1.5 L and Acellerase 1500, and exhibit 2- to 3-fold and 3- to 4-fold higher oil-palm trunk residues-hydrolysing specific activity, respectively. This higher hydrolysis efficiency may be attributed to the weak 'lignin-binding' ability of the P. rolfsii c3-2(1) IBRL-derived enzymes compared to the commercial enzymes.

Growth inhibition of thermotolerant yeast, Kluyveromyces marxianus, in hydrolysates from cassava pulp.

In this study, we report the inhibition of Kluyveromyces marxianus TISTR5925 growth and ethanol fermentation in the presence of furan derivatives and weak acids (acetic acid and lactic acid) at high temperatures. Cassava pulp, obtained as the waste from starch processing, was collected from 14 starch factories located in several provinces of Thailand. At a high temperature (42 °C), the cassava pulp hydrolysate from some starch factories strongly inhibited growth and ethanol production of both K. marxianus (strain TISTR5925) and Saccharomyces cerevisiae (strain K3). HPLC detected high levels of lactic acid and acetic acid in the hydrolysates, suggesting that these weak acids impaired the growth of K. marxianus at high temperature. We isolated Trp-requiring mutants that had reduced tolerance to acetic acid compared to the wild-type. This sensitivity to acetic acid was suppressed by supplementation of the medium with tryptophan.

Efficient ethanol production from separated parenchyma and vascular bundle of oil palm trunk.

For efficient utilization of both starchy and cellulosic materials, oil palm trunk was separated into parenchyma (PA) and vascular bundle (VB). High solid-state simultaneous saccharification and fermentation (HSS-SSF) using 30% (w/v) PA, containing 46.7% (w/w) starch, supplemented with amylases and Saccharomyces cerevisiae K3, produced 6.1% (w/v) ethanol. Subsequent alkali-pretreatment using sodium hydroxide was carried out with starch-free PA (sfPA) and VB. Enzymatic digestibility of 5% (w/v) pretreated sfPA and VB was 92% and 97%, respectively, using 18 FPU of commercial cellulase supplemented with 10 U of Novozyme-188 per gram of substrate. Likewise, HSS-SSF using 30% (w/v) alkali-pretreated sfPA and VB, with cellulases and yeast, resulted in high ethanol production (8.2% and 8.5% (w/v), respectively). These results show that HSS-SSF using separated PA and VB is a useful fermentation strategy, without loss of starchy and cellulosic materials, for oil palm trunk.

Isolation and characterization of a new cellulosome-producing Clostridium thermocellum strain.

The anaerobic thermophilic bacterium, Clostridium thermocellum, is a potent cellulolytic microorganism that produces large extracellular multienzyme complexes called cellulosomes. To isolate C. thermocellum organisms that possess effective cellulose-degrading ability, new thermophilic cellulolytic strains were screened from more than 800 samples obtained mainly from agriculture residues in Thailand using microcrystalline cellulose as a carbon source. A new strain, C. thermocellum S14, having high cellulose-degrading ability was isolated from bagasse paper sludge. Cellulosomes prepared from S14 demonstrated faster degradation of microcrystalline cellulose, and 3.4- and 5.6-fold greater Avicelase activity than those from C. thermocellum ATCC27405 and JW20 (ATCC31449), respectively. Scanning electron microscopic analysis showed that S14 had unique cell surface features with few protuberances in contrast to the type strains. In addition, the cellulosome of S14 was resistant to inhibition by cellobiose that is a major end product of cellulose hydrolysis. Saccharification tests conducted using rice straw soaked with sodium hydroxide indicated the cellulosome of S14 released approximately 1.5-fold more total sugars compared to that of ATCC27405. This newly isolated S14 strain has the potential as an enzyme resource for effective lignocellulose degradation.

Direct ethanol production from cassava pulp using a surface-engineered yeast strain co-displaying two amylases, two cellulases, and ß-glucosidase.

In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying α-amylase (α-AM), glucoamylase, endoglucanase, cellobiohydrase, and ß-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley ß-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes.

Ethanol and lactic acid production using sap squeezed from old oil palm trunks felled for replanting.

Old oil palm trunks that had been felled for replanting were found to contain large quantities of high glucose content sap. Notably, the sap in the inner part of the trunk accounted for more than 80% of the whole trunk weight. The glucose concentration of the sap from the inner part was 85.2g/L and decreased towards the outer part. Other sugars found in relatively low concentrations were sucrose, fructose, galactose, xylose, and rhamnose. In addition, oil palm sap was found to be rich in various kinds of amino acids, organic acids, minerals and vitamins. Based on these findings, we fermented the sap to produce ethanol using the sake brewing yeast strain, Saccharomyces cerevisiae Kyokai no.7. Ethanol was produced from the sap without the addition of nutrients, at a comparable rate and yield to the reference fermentation on YPD medium with glucose as a carbon source. Likewise, we produced lactic acid, a promising material for bio-plastics, poly-lactate, from the sap using the homolactic acid bacterium Lactobacillus lactis ATCC19435. We confirmed that sugars contained in the sap were readily converted to lactic acid with almost the same efficiency as the reference fermentation on MSR medium with glucose as a substrate. These results indicate that oil palm trunks felled for replanting are a significant resource for the production of fuel ethanol and lactic acid in palm oil-producing countries such as Malaysia and Indonesia.

Purification and characterization of a multienzyme complex produced by Paenibacillus curdlanolyticus B-6.

Paenibacillus curdlanolyticus B-6 showed effective degradation activities for xylan and cellulose and produced an extracellular multienzyme complex (approximately 1,450 kDa) containing several xylanases and cellulases. To characterize the multienzyme complex, we purified the complex from culture supernatants by four kind of chromatography. The purified multienzyme complex was composed of a 280-kDa protein with xylanase activity, a 260-kDa protein that was a truncated form on the C-terminal side of the 280-kDa protein, two xylanases of 40 and 48 kDa, and 60 and 65 kDa proteins having both xylanase and carboxymethyl cellulase activities. The 280-kDa protein resembled the scaffolding proteins of cellulosomes based on its migratory behavior in polyacrylamide gels and as a glycoprotein. Cloning of the 40-kDa major xylanase subunit named Xyn11A revealed that Xyn11A contained two functional domains which belonged to glycosyl hydrolase family-11 and to carbohydrate-binding module family-36, respectively, and a glycine- and asparagine-rich linker. However, an amino acid sequence similar to a dockerin domain, which is crucial to cellulosome assembly, was not found in Xyn11A. These results suggest that the multienzyme complex produced by P. curdlanolyticus B-6 should assemble by a mechanism distinct from the cohesin-dockerin interactions known in cellulosomes.

Synthesis of Clostridium cellulovorans minicellulosomes by intercellular complementation.

The ability of two strains of bacteria to cooperate in the synthesis of an enzyme complex (a minicellulosome) was examined. Three strains of Bacillus subtilis were constructed to express Clostridium cellulovorans genes engB, xynB, and minicbpA. MiniCbpA, EngB, and XynB were synthesized and secreted into the medium by B. subtilis. When the strains with the minicbpA and engB genes or with xynB were cocultured, minicellulosomes were synthesized, consisting in one case of miniCbpA and EngB and in the second case of miniCbpA and XynB. Both minicellulosomes showed their respective enzymatic activities. We call this phenomenon "intercellular complementation." Interesting implications concerning bacterial cooperation are suggested from these results.

Degradation of cellulosome-produced cello-oligosaccharides by an extracellular non-cellulosomal beta-glucan glucohydrolase, BglA, from Clostridium cellulovorans.

Clostridium cellulovorans degrades cellulose efficiently to small oligosaccharides, which are used as an energy source. To characterize enzymes related to degrading small oligosaccharides, a gene was cloned for an extracellular non-cellulosomal beta-glucan glucohydrolase (BglA) classified as a family-1 glycosyl hydrolase in C. cellulovorans. Recombinant BglA (rBglA) had higher activity on long glucooligomers than on cellobiose. When cellulosomes and rBglA were incubated with cellulose, the oligosaccharides produced were degraded more effectively to cellobiose and glucose, than with cellulosomes alone, indicating that BglA facilitated the degradation of accessible cello-oligosaccharides produced from cellulose by C. cellulovorans cellulosomes. Thus, this is an example of an extracellular non-cellulosomal enzyme working in a cooperative manner with cellulosomes to degrade cellulose to sugars.

Properties of cellulosomal family 9 cellulases from Clostridium cellulovorans.

The cellulosomal family 9 cellulase genes engH, engK, engL, engM, and engY of Clostridium cellulovorans have been cloned and sequenced. We compared the enzyme activity of family 9 cellulosomal cellulases from C. cellulovorans and their derivatives. EngH has the highest activity toward soluble cellulose derivatives such as carboxymethylcellulose (CMC) as well as insoluble cellulose such as acid-swollen cellulose (ASC). EngK has high activity toward insoluble cellulose such as ASC and Avicel. The results of thin-layer chromatography showed that the cleavage products of family 9 cellulases were varied. These results indicated that family 9 endoglucanases possess different modes of attacking substrates and produce varied products. To investigate the functions of the carbohydrate-binding module (CBM) and the catalytic module, truncated derivatives of EngK, EngH, and EngY were constructed and characterized. EngHDeltaCBM and EngYDeltaCBM devoid of the CBM lost activity toward all substrates including CMC. EngKDeltaCBM and EngMDeltaCBM did not lose activity toward CMC but lost activity toward Avicel. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.

Isothermal titration calorimetric studies on the binding of a family 6 carbohydrate-binding module of Clostridium thermocellum xynA with xlylooligosaccharides.

The family 6 carbohydrate-binding module (CBM) of Clostridium thermocellum XynA was expressed, and the binding equilibria of the CBM with xylooligosaccharides (degree of polymerization DP = 2-8) were observed by isothermal titration calorimetry (ITC) at pH 8. The association constant, Ka, increased with increasing DP from 5 x 10(3) M(-1) (DP = 2) to approximately 5 x 10(5) M(-1) (DP = 5-8) at 20 degrees C. The Ka values at 60 degrees C were about 1/10 of those at 20 degrees C. The binding was found to be an enthalpy-driven reaction. The DP dependence of the thermodynamic parameters of the binding reaction suggested the size of the ligand-binding site to be 5 xylose units long.

Characterization of a cellulase containing a family 30 carbohydrate-binding module (CBM) derived from Clostridium thermocellum CelJ: importance of the CBM to cellulose hydrolysis.

Clostridium thermocellum CelJ is a modular enzyme containing a family 30 carbohydrate-binding module (CBM) and a family 9 catalytic module at its N-terminal moiety. To investigate the functions of the CBM and the catalytic module, truncated derivatives of CelJ were constructed and characterized. Isothermal titration calorimetric studies showed that the association constants (K(a)) of the CBM polypeptide (CBM30) for the binding of cellopentaose and cellohexaose were 1.2 x 10(4) and 6.4 x 10(4) M(-1), respectively, and that the binding of CBM30 to these ligands is enthalpically driven. Qualitative analyses showed that CBM30 had strong affinity for cellulose and beta-1,3-1,4-mixed glucan such as barley beta-glucan and lichenan. Analyses of the hydrolytic action of the enzyme comprising the CBM and the catalytic module showed that the enzyme is a processive endoglucanse with strong activity towards carboxymethylcellulose, barley beta-glucan and lichenan. By contrast, the catalytic module polypeptide devoid of the CBM showed negligible activity toward these substrates. These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity.

Linear alignment of four sulfur atoms in bis[(8-phenylthio)naphthyl] disulfide: contribution of linear S4 hypervalent four-centre six-electron bond to the structure.

The four sulfur atoms in bis[8-(phenylthio)naphthyl]-1,1'-disulfide are demonstrated to align linearly by the X-ray crystallographic analysis, where the linear S4 alignment is stabilized by the four-centre six-electron interaction.