[Show abstract][Hide abstract] ABSTRACT: Biochemical production processes require water and nutrient resources for culture media preparation, but aqueous waste is generated after the target products are extracted. In this study, culture waste (including cells) produced from a lab-scale fermenter was fed into a microbial fuel cell-membrane bioreactor (MFC-MBR) system. Electrical energy was generated via the interaction between the microbial consortia and the solid electrode in the MFC. The treated wastewater was reclaimed in this process which was reused as a solvent and a nutrient source in subsequent fermentation. Polarization testing showed that the MFC produced a maximum current density of 37.53 A m(-3) with a maximum power density of 5.49 W m(-3). The MFC was able to generate 0.04 kWh of energy per cubic meter of culture waste treated. The lab-scale fermenters containing pure cultures of an engineered Pseudomonas spp. were used to generate 2-pyrone-4,6-dicarboxylic acid (PDC), a high value platform chemical. When the MFC-MBR-treated wastewater was used for the fermenter culture medium, a specific bacterial growth rate of 1.00 ± 0.05 h(-1) was obtained with a PDC production rate of 708.11 ± 64.70 mg PDC L(-1) h(-1). Comparable values for controls using pure water were 0.95 ± 0.06 h(-1) and 621.01 ± 22.09 mg PDC L(-1) h(-1) (P > 0.05), respectively. The results provide insight on a new approach for more sustainable bio-material production while at the same time generating energy, and suggest that the treated wastewater can be used as a solvent and a nutrient source for the fermentation production of high value platform chemicals.
Bioprocess and Biosystems Engineering 05/2015; DOI:10.1007/s00449-015-1420-9 · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The decarboxylation reaction of protocatechuate has been described as a bottleneck and a rate-limiting step in cis,cis-muconate (ccMA) bioproduction from renewable feedstocks such as sugar. Because sugars are already in high demand in the development of many bio-based products, our work focuses on improving protocatechuate decarboxylase (Pdc) activity and ccMA production in particular, from lignin-related aromatic compounds. We previously had transformed an Escherichia coli strain using aroY, which had been used as a protocatechuate decarboxylase encoding gene from Klebsiella pneumoniae subsp. pneumoniae A170-40, and inserted other required genes from Pseudomonas putida KT2440, to allow the production of ccMA from vanillin. This recombinant strain produced ccMA from vanillin, however the Pdc reaction step remained a bottleneck during incubation. In the current study, we identify a way to increase protocatechuate decarboxylase activity in E. coli through enzyme production involving both aroY and kpdB; the latter which encodes for the B subunit of 4-hydroxybenzoate decarboxylase. This permits expression of Pdc activity at a level approximately 14-fold greater than the strain with aroY only. The expression level of AroY increased, apparently as a function of the co-expression of AroY and KpdB. Our results also imply that ccMA may inhibit vanillate demethylation, a reaction step that is rate limiting for efficient ccMA production from lignin-related aromatic compounds, so even though ccMA production may be enhanced, other challenges to overcome vanilate demethylation inhibition still remain.
Journal of Biotechnology 10/2014; 192. DOI:10.1016/j.jbiotec.2014.10.027 · 2.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sphingobium sp. strain SYK-6 is able to assimilate lignin-derived biaryls, including a biphenyl compound, 5,5'-dehydrodivanillate (DDVA). Previously, ligXa (SLG_07770), which is similar to oxygenase components of Rieske-type non-heme iron aromatic ring-hydroxylating oxygenases, was identified to be essential for the conversion of DDVA; however, the genes encoding electron transfer components remained unknown. Disruption of putative electron transfer component genes scattered through the SYK-6 genome indicated that SLG_08500 and SLG_21200, which showed approximately 60% amino acid sequence identities with ferredoxin and ferredoxin reductase of dicamba O-demethylase, were essential for the normal growth of SYK-6 on DDVA. LigXa and the gene products of SLG_08500 (LigXc) and SLG_21200 (LigXd) were purified and were estimated to be a trimer, a monomer, and a monomer. LigXd contains FAD as the prosthetic group, and showed much higher reductase activity toward 2,6-dichlorophenolindophenol with NADH than NADPH. A mixture of purified LigXa, LigXc, and LigXd converted DDVA into 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl in the presence of NADH, indicating that DDVA O-demethylase is a three-component monooxygenase. This enzyme requires Fe(II) for its activity and is highly specific for DDVA with the Km value of 63.5 μM and kcat of 6.1 sec(-1). Genome searches in six other sphingomonads revealed genes similar to ligXc and ligXd (>58% amino acid sequence identities) with a limited number of electron transfer component genes, yet a diverse number of oxygenase component genes were found. This fact implies that these few electron transfer components are able to interact with numerous oxygenase components, and the conserved LigXc and LigXd orthologs are important in sphingomonads.
[Show abstract][Hide abstract] ABSTRACT: Cycloaddition reactions of a lignin-derived pyrone derivative, 2-pyrone-4,6-dicarboxylic acid and its dimethyl ester with alkenyl acetates and alkynes under thermal conditions proceed with decarboxylation to give isophthalic acids and various mono- and disubstituted isophthalates. A ruthenium complex catalyzes the reaction with alkynes under milder conditions and affords products with different regioselectivity from that observed under thermal conditions.
[Show abstract][Hide abstract] ABSTRACT: Subcritical water extraction of low-molecular-weight phenolic compounds from oil palm biomass (trunk, bark, petiole, rachis, leaves, empty fruit bunch fiber, midrib spine leaflets, stalk of fruit bunches, flesh, kernel shells, and albumen) was conducted. It was elucidated that gallic acid, protocatechuicaldehyde, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillic acid, syringic acid, vanillin, syringaldehyde p-coumaric acid and ferulic acid, all of which could be used as 2-pyrone-4,6-dicarboxylic acid (PDC) precursors, were contained in all parts of oil palm, although their composition differed. The peak yield of p-hydroxybenzoic acid was obtained among the PDC precursors. With regard to extraction conditions, temperature: 200°C, time: 20-60 min, and liquor ratio: 50-125 were the most efficient. The kernel shell exhibited the highest yield of PDC precursors, followed by the trunk, empty fruit bunch fiber, and bark. The results of our study indicate the oil palm is a potentially valuable source of PDC precursors.
Japan Agricultural Research Quarterly 01/2014; 48(3):355-362. DOI:10.6090/jarq.48.355 · 0.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The degradation of 2-chloro-4,5-O-(4'-methyl-7', 8'-diphenyl)ether (CMDPE), an analog of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), mediated by Geobacillus sp. UZO 3 cell-free extract was monitored. Ethyl acetate extracts of a complete reaction mixture incubated at 65°C for 18 h were analyzed either by thin layer chromatography (TLC) fractionation coupled with spectrometric detection or by gas chromatography-mass spectrometry (GC-MS). The reaction product 4-methylumbelliferone (4MU) was successfully isolated by TLC and visualized by a transilluminator at 450 nm. The 4MU, 4-chlorophenol, and reaction intermediate 6-chlorophenoxy-4-methylumbelliferone were all successfully detected by GC-MS. The presence of these compounds suggest that Geobacillus sp. UZO 3 cell-free extract also catalyzes the reductive cleavage of the diaryl ether bonds of CMDPE in a similar mechanism previously reported in 2,7-DCDD. In the present study, the authors describe a simple and highly sensitive fluorescent assay for a new dioxin degrading enzyme(s).
[Show abstract][Hide abstract] ABSTRACT: The gene encoding manganese peroxidase of a white-rot fungus Phanerochaete crassa WD1694 was cloned and sequenced. Four genomic clones were sequenced in which 3 clones were existed as alleles. The analysis of intron–exon structures divided the 4 clones into three subfamilies that corresponded to mnp2 and mnp3 of Phanerochaete chrysosporium, and a new subfamily possessing only five introns. The purified P. crassa WD1694 MnP consisted of 4 isozymes with same molecular weight, same N-terminal sequence, and different pI. N-terminal sequence of deduced protein of P. crassa mnpB3 gene was identical to those of 4 MnP isozymes; however, the other 3 mnp genes had different N-terminal sequence. The molecular weight of encoded mature protein of mnpB3 gene and purified MnP had a gap that could be difference between MnP proteins encoded by single gene. The results suggested that 4 MnP isozymes of P. crassa WD1694 arose from single gene.
[Show abstract][Hide abstract] ABSTRACT: Guaiacylglycerol-beta-guaiacyl ether (GGE) is one of the most important phenolic compound for studying the chemistry and biochemistry of lignin. GGE contains two asymmetric carbons at the alpha and beta positions of its side chain: therefore, theoretically it can exist as four different stereoisomers. It has been proposed that a Gram-negative bacterium, Sphingobium sp. SYK-6 (formerly referred as Sphingomonas paucimobilis SYK-6), degrades GGE enantiospecifically via cleavage of the ether bond. The cleavage was thought to proceed in two steps, each catalyzed by a different enantiospecific enzyme. In the first step, the alcohol residue at the alpha position of the side chain in GGE was thought to be oxidized enantiospecifically by four distinct C alpha-dehydrogenases (LigD, LigN, LigL and LigO), to produce two enantiomers of 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-propan-1-one (erone). To study this enantiospecific degradation step by the four dehydrogenases, we synthesized all four stereoisomers of GGE and both enantiomers of erone and separated them into enantiopure samples. The stereoisomers and our synthetic methods to prepare them are useful both for microbial and chemical investigations of lignin degradation.
[Show abstract][Hide abstract] ABSTRACT: The glutathione S-transferases, LigF and LigE, of Sphingobium sp. strain SYK-6 respectively play a role in cleavage of the beta-aryl ether of (+)-(beta S)-alpha-(2-methoxyphenoxy)-beta-hydroxypropiovanillone (MPHPV) and (-)(-beta R)-MPHPV. The ligP gene, which showed 59% similarity to ligE at the amino acid level, was isolated from SYK-6. LigP produced in Escherichia coli revealed enantioselectivity for (-)-(beta R)-MPHPV, and ligE and ligP alone contributed to the degradation of (-)-(beta R)-MPHPV in SYK-6.
[Show abstract][Hide abstract] ABSTRACT: Two cholesterol groups were for the first time attached to a lignin-derived metabolic intermediate, 2-pyrone-4,6-dicarboxylic acid (PDC). This PDC molecule showed a liquid crystalline mesophase and interesting organogelation behavior. The relationship between molecular structure and assembly properties was investigated by modifying the PDC core and a spacer group between the PDC and cholesterol moieties. Although all derivatives displayed similar phase transition behavior with a SmA phase, only a few of them could form organogels. Detailed morphology investigations of the xerogels by X-ray diffraction (XRD) and scanning electron microscopy (SEM) suggested that both the PDC moiety and a carbamate spacer are essential for the occurrence of organogelation. Hydrogen bonds of the carbamate spacers and van der Waals interactions of the cholesterol moieties lead to a dipolar-induced interaction of the pyrone rings. The physical gels were probably stabilized by these cooperative interactions. The IR and H-1 NMR spectroscopies clearly revealed the presence of weak intermolecular interactions, such as hydrogen bonds and dipolar-type interactions, for efficient gelator molecules.
Bulletin of the Chemical Society of Japan 06/2011; 84(6-6):667-674. DOI:10.1246/bcsj.20110055 · 2.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We characterized the ability of the cell free extract from polychlorinated dibenzo-p-dioxins degrading bacterium Geobacillus sp. UZO 3 to reduce even highly chlorinated dibenzo-p-dioxins such as octachlorodibenzo-p-dioxins in incineration fly ash. The degradation of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) as a model dioxin catalyzed by the cell free extract from this strain implicates that the ether bonds of 2,7-DCDD molecule undergo reductive cleavage, since 4',5-dichloro-2-hydroxydiphenyl ether and 4-chlorophenol were detected as intermediate products of 2,7-DCDD degradation.
[Show abstract][Hide abstract] ABSTRACT: Highly monodispersed nanometric particles, with a polyfunctional nature due to the presence of phenolic and aliphatic hydroxyl groups, were obtained by the simultaneous enzymatic saccharification and physical comminution of lignin, which is a woody biomass resource.
Green Chemistry 11/2010; 12(11):1914-1916. DOI:10.1039/C0GC00140F · 6.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peroxidase activity staining localized at hyphal tips of white-rot fungus Phanerochaete crassa WD1694 that was cultivated in a shaken liquid culture containing unbleached kraft pulp was investigated. Manganese peroxidase
was detected in culture solution, washing solution of mycelium, and mycelial extract. Glyoxal oxidase was detected only in
mycelial extract and was not detected in culture solution. Addition of hydrogen peroxide generated peroxidase activity staining
in the culture solution. Addition of catalase resulted in no staining in the culture of P. crassa WD1694, and the addition of methylglyoxal resulted in marked peroxidase activity staining at hyphal tips and on hyphal wall.
In an optimized culture, glyoxal oxidase was produced in culture solution. Although the production of glyoxal oxidase and
manganese peroxidase had a positive correlation, the secretion and the peak of glyoxal oxidase was observed 3 and 2 days later
than those of manganese peroxidase. The N-terminal sequence of purified glyoxal oxidase had very high homology with that of
P. chrysosporium. These results elucidated the hydrogen peroxide supply system in lignin biodegradation by white-rot fungi, i.e., while remaining
on the hyphal cell wall, glyoxal oxidase provides hydrogen peroxide to manganese peroxidase that had diffused into the culture
Key wordsGlyoxal oxidase-Lignin-
-Manganese peroxidase-Hyphal tip
[Show abstract][Hide abstract] ABSTRACT: Copolymers of L-lactic acid (LA) and 2-pyrone-4,6-dicarboxylic acid (PDC), a chemically stable metabolic intermediate of lignin, were prepared by dehydrated polycondensation based on stepwise oligomerization followed by post-polymerization in vacuo. When the polymerization was performed in the presence of methanesulfonic acid as a catalyst, the molecular weights of the resulting copolymers were sufficiently high. Furthermore, expansion of the polymeric surface area was found to be an important factor in facilitating dehydration and thereby producing high molecular weight polymers. PDC feed ratio significantly affected the molecular weight because of the different polymerization capability from LA. Relationship between the PDC feed ratio and the molecular weight of the resulting polyesters was for the first time demonstrated quantitatively. The obtained copolymers were characterized by 1H- and 13C-NMR, IR, and thermal analysis. The high molecular weight copolymers possessed the higher decomposition temperatures than PLLA and their fusible temperature ranges were reasonably expanded.
Journal of Macromolecular Science Part A 06/2010; Part A: Pure and Applied Chemistry(6-Vol. 47):564-570. DOI:10.1080/10601321003742121 · 0.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The organogelation behavior of a lignin-derived stable metabolic intermediate bearing two cholesteryl groups was examined for the first time, and it was found that the carbamate spacers possessing a hydrogen-bonding ability are essential for gelation of aliphatic or aromatic organic solvents.
[Show abstract][Hide abstract] ABSTRACT: A novel bacterium, designated strain OX-01(T), was isolated from acidic soil, taxonomically investigated and identified as an agent that catabolizes (+)-catechin into taxifolin. Strain OX-01(T) is a Gram-reaction-negative, aerobic, non-sporulating, non-motile and rod-shaped bacterium. 16S rRNA gene sequence analysis identified this strain as a member of the genus Burkholderia and occupying a phylogenetic position closest to, but clearly distinct from, Burkholderia sacchari. Strain OX-01(T) does not have any nif genes, which are required for N(2)-fixation, in its genome, a feature that is similar to B. sacchari, which lacks nifH, but is distinct from the N(2)-fixing features of many other phylogenetically related taxa, such as Burkholderia ferrariae, B. heleia, B. mimosarum, B. nodosa, B. silvatlantica, B. tropica and B. unamae. Strain OX-01(T) has the following chemotaxonomic characteristics: the major ubiquinone is Q-8, the DNA G+C content is 64 mol% and the major fatty acids are C(16 : 0), C(17 : 0) cyclo and C(18 : 1)ω7c. It also has a unique profile of carbohydrate utilization among other species of the genus Burkholderia. The strain cannot assimilate many pentoses, hexoses and oligosaccharides, whereas it can catabolize (+)-catechin and its putative aromatic derivatives, such as 4-hydroxy-3-methoxycinnamic acid, protocatechuic acid, p-hydroxybenzoic acid, trans-p-coumaric acid and vanillic acid. Based on its morphological, physiological and chemotaxonomic characteristics, together with DNA-DNA relatedness values and 16S rRNA gene sequence comparison data, we show that strain OX-O1(T) represents a novel species of the genus Burkholderia, for which the name Burkholderia oxyphila sp. nov. is proposed. The type strain is OX-01(T) (=NBRC 105797(T) =DSM 22550(T)).
International Journal of Systematic and Evolutionary Microbiology 03/2010; 61(Pt 2):249-54. DOI:10.1099/ijs.0.017368-0 · 2.80 Impact Factor