Fermentation of Xylan,Corn Fiber,or Sugars to Acetoin and Butanediol by Bacillus polymyxa Strains

Bacillus polymyxa can produce levo-butanediol, a potential biogradable anti-freeze, and ethanol, a fuel additive, using starch-based fermentations. To explore use of less expensive biomass fermentation substrates, we screened B. polymyxa strains for good growth on xylans. During aerobic growth on glucose, six selected xylanolytic strains produced mainly acetoin and butanediol plus lesser amounts of acetaldehyde and ethanol. Undesirable acetoin formation was eliminated by anaerobic growth on glucose, but substrate usage, butanediol, and other fermentation products were greatly reduced. High xylanase activity occurred with growth on xylans or corn fiber, and about 50–65% of oatspelt xylan and 25–35% of the corn fiber were used during aerobic growth, but unexpectedly no butanediol and only small levels of acetoin were produced. Aerobic growth on arabinose, arabinose plus glucose, or xylose plus glucose resulted in both acetoin and butanediol formation. Little or no butanediol was made from xylose alone. Growth on an acid hydrolysate of corn fiber that contained a mixture of these sugars resulted in the formation of acetoin, acetaldehyde, and ethanol, but very little butanediol. The data suggest B. polymyxa is limited in conversion of xylan-rich biomass sources or their hydrolysates to butanediol. This limitation might be overcome by using better cultivation conditions and/or genetically engineered strains.     

Degradation and utilization by Butyrivibrio fibrisolvens H17c of xylans with different chemical and physical properties.

Hemicelluloses, mainly xylans, can be a major component of diets consumed by ruminants and undergo various degrees of microbial digestion in the rumen. The ability of Butyrivibrio fibrisolvens, a major xylanolytic ruminal species, to degrade and utilize nine chemically and physically different xylans for growth was examined. The arabinoxylans used included two isolated from corncobs (CCX-A and CCX-B), a native xylan excreted by corn cell tissue cultures (CX), an oxalic acid-treated, arabinose-depleted CX, and oat spelt xylan. Except for CCX-A, these xylans were extensively converted within 3 h of growth to acid-alcohol-soluble forms that remained at high levels for the duration of culture growth. These xylans contain mainly xylose and arabinose with small amounts of uronic acids. For a given xylan, all three components were used at about the same rate and extent. During the early stages of growth B. fibrisolvens also rapidly solubilized glucuronoxylans from birchwood, larchwood, 4-O-methylglucuronoxylan, and the xylose homopolymer xylan isolated from beechwood (BEWX). In contrast to the findings for the arabinoxylans, little acid-alcohol-soluble carbohydrate remained in these cultures after 9 h of growth, except for BEWX. Initially, with birchwood, larchwood, and 4-O-methylglucuronoxylan the uronic acid components were preferentially used over the xylose. Final xylan utilization measured at 72 h for all xylans varied from 57% for CCX-A to 92% for BEWX and was correlated with the initial 12-h utilization rate for a given xylan. Since CCX-A and BEWX are both highly water insoluble, this aspect did not appear to influence overall utilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation and Utilization of Xylans by the Rumen AnaerobePrevotella bryantii(formerlyP. ruminicolasubsp.brevis) B14

Freshly harvested whole cells from cultures ofP. bryantiiB₁4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantiiB₁4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation withP. bryantiishowed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobeRuminococcus flavefaciensgave little increase in overall pentose utilization suggesting that externalP. bryantiixylanases are as effective as the clonedR. flavefaciensenzymes in releasing products that can be utilised byP. bryantiicells. The xylanase system ofP. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.     

Degradation and utilization of xylans by the rumen anaerobe Prevotella bryantii (formerly P. ruminicola subsp. brevis) B(1)4

Freshly harvested whole cells from cultures of P. bryantii B(1)4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantii B(1)4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation with P. bryantii showed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens gave little increase in overall pentose utilization suggesting that external P. bryantii xylanases are as effective as the cloned R. flavefaciens enzymes in releasing products that can be utilised by P. bryantii cells. The xylanase system of P. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.     

Xylanolytic activities ofSpirochaeta thermophila

Spirochetes capable of degrading xylan or cellulose have not been commonly isolated, nor have their polysaccharolytic activities been characterized.Spirochaeta thermophila strain RI 19.B1 is xylanolytic and grows well at 65°C with oatspelt (OX), birchwood (BX), corncob (CCX-A) xylans, or glucuronoxylan (MGX) as the energy source. All xylans were extensively degraded and utilized during growth. About 72–82% of the initial hexuronic acids and 57–79% of initial pentoses disappeared during growth.S. thermophila possessed xylanase, xylosidase, and arabinofuranosidase enzyme activities. Low levels of these activities were detected with growth on glucose, but high expression of these activities occurred during growth on OX. All three activities were cell-associated and were more stable in cells than cell extracts. Xylan-degrading activities were measured with cells or cell extracts exposed (60 min) to a variety of temperatures (65°–85°C) and pHs (5.0–8.0). More than 50% loss of activities occurred at temperatures above 75°C. Although pH stability was affected by buffer, the optimal range was pH 6.5–7.5. These temperature and pH profiles for xylan-degrading activities coincide with those found for the growth ofS. thermophila.     

Enhanced production of polyhydroxyalkanoates (PHAs) from beechwood xylan by recombinant Escherichia coli

Microbial conversion of plant biomass to value-added products is an attractive option to address the impacts of petroleum dependency. In this study, a bacterial system was developed that can hydrolyze xylan and utilize xylan-derived xylose for growth and production of polyhydroxyalkanoates (PHAs). A β-xylosidase and an endoxylanase were engineered into a P(LA-co-3HB)-producing Escherichia coli strain to obtain a xylanolytic strain. Although PHA production yields using xylan as sole carbon source were minimal, when the xylan-based media was supplemented with a single sugar (xylose or arabinose) to permit the accumulation of xylan-derived xylose in the media, PHA production yields increased up to 18-fold when compared to xylan-based production, and increased by 37 % when compared to production from single sugar sources alone. ¹H-Nuclear magnetic resonance (NMR) analysis shows higher accumulation of xylan-derived xylose in the media when xylan was supplemented with arabinose to prevent xylose uptake by catabolite repression. ¹H-NMR, gel permeation chromatography, and differential scanning calorimetry analyses corroborate that the polymers maintain physical properties regardless of the carbon source. This study demonstrates that accumulation of biomass-derived sugars in the media prior to their uptake by microbes is an important aspect to enhance PHA production when using plant biomass as feedstock.     

Xylose and arabinose utilization by the rumen bacterium Butyrivibrio fibrisolvens

Abstract The rumen bacterium Butyrivibrio fibrisolvens strain D1 co-utilized xylose and glucose in batch culture, but there was a marked preference for glucose over arabinose. When both pentoses were provided, xylose was preferred over arabinose. Strain D1 co-utilized a combination of either pentose and cellobiose, but preferred pentoses over maltose. Pentose sugars were depleted less rapidly in the presence of sucrose than controls containing only pentose. In contrast, B. fibrisolvens strain A38 exhibited a strong preference for disaccharides, including maltose, over either xylose or arabinose. Theoretical maximum growth yields for strain D1v in single-substrate continuous culture were highest for sucrose and cellobiose and the maintenance energy coefficient for arabinose was at least 3.8-fold greater than for other substrates. We suggest that B. fibrisolvens may have evolved a mechanism to utilize certain sugars before arabinose in order to avoid this high maintenance energy expenditure.     

Heterologous expression,purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

Numerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6–7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20–30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxylanase described so far in filamentous fungi. This is one of the few reports describing the heterologous expression and characterization of a xylanase from a fungus isolated from Antarctica.     

Characterization of Thermoanaerobacter Glucose Isomerase in Relation to Saccharidase Synthesis and Development of Single-Step Processes for Sweetener Production

Regulation of glucose isomerase synthesis was studied in Thermoanaerobacter strain B6A, which fermented a wide variety of carbohydrates including glucose, xylose, lactose, starch, and xylan. Glucogenic amylase activities and β-galactosidase were produced constitutively, whereas the synthesis of glucose isomerase was induced by either xylose or xylan. Production of these saccharidase activities was not significantly repressed by the presence of glucose or 2-deoxyglucose in the growth media. Glucose isomerase production was optimized by controlling the culture pH at 5.5 during xylose fermentation. The apparent temperature and pH optima for these cell-bound saccharidase activities were as follows: glucose isomerase, 80°C, pH 7.0 to 7.5; glucogenic amylase, 70°C, pH 5.0 to 5.5; and β-galactosidase, 60°C, pH 6.0 to 6.5 Glucose isomerase, glucogenic amylase, and β-galactosidase were produced in xylose-grown cells that were active and stable at 60 to 70°C and pH 6.0 to 6.5. Under single-step process conditions, these saccharidase activities in whole cells or cell extracts converted starch or lactose directly into fructose mixtures. A total of 96% of initial liquefied starch was converted into a 49:51 mixture of glucose and fructose, whereas 85% of initial lactose was converted into a 40:31:29 mixture of galactose, glucose, and fructose.     

Optimization of cellulase-free xylanase production by a novel yeast strain

Summary A novel yeast strain, NCIM 3574, isolated from a decaying wood produced up to 570 IU ml^–1 of xylanolytic enzymes when grown on medium containing 4% xylan. The yeast strain also produced xylanase activity (40–50 IU ml^–1) in the presence of soluble carbon sources like xylose or arabinose. No xylanase activity was detected when the organism was grown on glucose. The crude xylanase preparation showed no activity towards cellulolytic substrates but low levels of -xylosidase (0.1 IU ml^–1) and -l-arabinofuranosidase (0.05 IU ml^–1) were detected. The temperature and pH optima for the crude xylanase preparation were 55°C and 4.5 respectively. The crude xylanase produced mainly xylose from xylan within 5 min. Prolonged hydrolysis of xylan produced xylobiose and arabinose, in addition to xylose, as the end products. The presence of arabinose as one of the end products in xylan hydrolysate could be due to the low levels of arabinofuranosidase enzyme present in the crude fermentation broth.     

Relationships between activities of xylanases and xylan structures

Structures of five water-soluble xylans have been determined. Four purified xylanase enzymes have been studied for the hydrolysis of the xylans. Different xylanases have different activities against various xylan structures. The key factors that influence the rate of xylan hydrolysis are chain length and degree of substitution. Two family 11 xylanases, Orpinomyces pc2 xylanase and Trichoderma longibrachiatum xylanase, can rapidly hydrolyze xylans that have a chain length greater than 8 xylose residues, and their hydrolytic rates are not sensitive to substituents on the xylan backbone. A family 11 xylanase from Aureobasidium pullulans is most effective on xylans that have a long chain (greater than 19 xylose residues), and also is effective against substituent groups. Although Thermatoga maritima xylanase is also more active on a long xylan chain (greater than 19 xylose residues), its hydrolytic rate is greatly reduced by substituents on xylan backbones.     

Purification and Characterization of an alpha-l-Arabinofuranosidase from Butyrivibrio fibrisolvens GS113

An alpha-l-arabinofuranosidase (EC 3.2.1.55) was purified from the cytoplasm of Butyrivibrio fibrisolvens GS113. The native enzyme had an apparent molecular mass of 240 kDa and was composed of eight polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.0, a pH optimum of 6.0 to 6.5, a pH stability of 4.0 to 8.0, and a temperature optimum of 45 degrees C and was stable to 55 degrees C. The K(m) and V(max) for p-nitrophenyl-alpha-l-arabinofuranoside were 0.7 mM and 109 mumol/min/mg of protein, respectively. The enzyme was specific for the furanoside configuration and also readily cleaved methylumbelliferyl-alpha-l-arabinofuranoside but had no activity on a variety of other nitrophenyl- or methylumbelliferyl glycosides. When the enzyme was incubated with cellulose, carboxymethyl cellulose, or arabinogalactan, no release of sugars was found. Arabinose was found as the hydrolysis product of oatspelt xylan, corn endosperm xylan, or beet arabinan. No activity was detected when either coumaric or ferulic acid ester linked to arabinoxylobiose was used as substrates, but arabinoxylobiose was degraded to arabinose and xylobiose. Since B. fibrisolvens GS113 possesses essentially no extracellular arabinofuranosidase activity, the major role of the purified enzyme is apparently in the assimilation of arabinose-containing xylooligosaccharides generated from xylosidase, phenolic esterase, xylanase, and other enzymatic activities on xylans.     

Organisation and Variable Incidence of Genes Concerned with the Utilization of Xylans in the Rumen Cellulolytic Bacterium Ruminococcus flavefaciens

Strains of the rumen cellulolytic bacterium Ruminococcus flavefaciens vary in their ability to utilise isolated plant xylans for growth. Here an 11.5 kb fragment of genomic DNA from the xylan-utilizing R. flavefaciens strain 17 that contains the xynD gene, which encodes an extracellular xylanase/β -(1,3-1,4)-glucanase, was analysed. Sequencing revealed five consecutive open reading frames downstream from xynD on the same strand, preceded by the divergently transcribed ORF3. These include the following genes likely to be involved in utilisation of xylan breakdown products: xylA, encoding a β -(1,4)-xylosidase, xsi, encoding a xylose isomerase and ORF8 encoding part of an ABC-type sugar transporter. The products of ORF3 and of a partial ORF1 found upstream of xynD, show significant sequence similarity to AraC-type regulatory proteins while ORF4 and ORF7 show no close relationship to other known proteins. Homologues of the xylA and xsi genes, and inducible β -xylosidase activity, were readily detectable in three xylan-utilizing R. flavefaciens strains 17, B1a and C94 but not in two xylan non-utilizing strains, C1a and B34b, suggesting that this cluster may be absent from xylan non-utilizing strains.     

Xylan-degrading activity in yeasts: Growth on xylose,xylan and hemicelluloses

The ability to grow in liquid media withD-xylose, xylan from deciduous trees, and hemicelluloses from conifers was tested in 95 strains of 35 genera of yeasts and yeast-like organisms. Of 54 strains thriving on xylose, only 13 (generaAureobasidium, Cryptococcus andTrichosporon) utilized xylan and hemicelluloses as growth substrates. The árowth media of these strains were found to contain xylandegrading enzymes splitting the substrate to xylose and a mixture of xylose oligosaccharides. The ability of these yeasts to utilize the wood components (hitherto unknown in the genusCryptococeus) makes them potential producers of microbial proteins from industrial wood wastes containing xylose oligosaccharides, xylan, and hemicelluloses as the major saccharide components without previous saccharification.     

Xylanolytic Activity of Clostridium acetobutylicum

Of 20 strains of Clostridium spp. screened, 17 hydrolyzed larch wood xylan. Two strains of Clostridium acetobutylicum, NRRL B527 and ATCC 824, hydrolyzed xylan but failed to grow on solid media with larch xylan as the sole carbon source; however, strain ATCC 824 was subsequently found to grow on xylan under specified conditions in a chemostat. These two strains possessed cellulolytic activity and were therefore selected for further studies. In cellobiose-limited continuous cultures, strain NRRL B527 produced maximum xylanase activity at pH 5.2. Strain ATCC 824 produced higher xylanase, xylopyranosidase, and arabinofuranosidase activities in chemostat culture with xylose than with any other soluble carbon source as the limiting nutrient. The activities of these enzymes were markedly reduced when the cells were grown in the presence of excess glucose. The xylanase showed maximum activity at pH 5.8 to 6.0 and 65°C. The enzyme was stable on the alkaline side of pH 5.2 but was unstable below this pH value. The extracellular xylanolytic activity from strain ATCC 824 hydrolyzed 12% of the larch wood xylan during a 24-h incubation period, yielding xylose, xylobiose, and xylotriose as the major hydrolysis products. Strain ATCC 824, after being induced to grow in batch culture in xylan medium supplemented with a low concentration of xylose, failed to grow reproducibly in unsupplemented xylan medium. A mutant obtained by mutagenesis with ethyl methanesulfonate was able to grow reproducibly in batch culture on xylan. Both the parent strain and the mutant were able to grow with xylan as the sole source of carbohydrate in continuous culture with the pH maintained at either 5.2 or 6.0. Under these conditions, the cells utilized approximately 50% of the xylan.     

Fermentation of xylans by Butyrivibrio fibrisolvens and other ruminal bacteria

The ability of Butyrivibrio fibrisolvens and other ruminal bacteria (6 species, 18 strains) to ferment a crude xylan from wheat straw or to ferment xylans from larchwood or oat spelts was studied. Liquid cultures were monitored for carbohydrate utilization, cell growth (protein), and fermentation acid production. B. fibrisolvens 49, H17c, AcTF2, and D1 grew almost as well on one or more of the xylans as they did on cellobiose-maltose. B. fibrisolvens 12, R28, A38, X10C34, ARD22a, and X6C61 exhibited moderate growth on xylans. Partial fermentation of xylans was observed with Bacteroides ruminicola B14, Bacteroides succinogenes S85, Ruminococcus albus 7, Ruminococcus flavefaciens C94 and FD1, and Succinivibrio dextrinosolvens 22B. All xylans tested appeared to have a small fraction of carbohydrate that supported low levels of growth of nonxylanolytic strains such as Selenomonas ruminantium HD4. Compared to growth on hexoses, the same array of fermentation acids was produced upon growth on xylans for most strains; however, reduced lactate levels were observed for B. fibrisolvens 49 and Selenomonas ruminantium HD4. Measurements of enzyme activities of B. fibrisolvens AcTF2, 49, H17c, and D1 indicated that the xylobiase activities were cell associated and that the xylanase activities were predominantly associated with the culture fluid. The pattern of expression of these enzymes varied both between strains and between the carbon sources on which the strains were grown.     

Effect of Methanobrevibacter smithii on Xylanolytic Activity of Anaerobic Ruminal Fungi

Three different ruminal anaerobic fungi, Neocallimastix frontalis PNK2, Sphaeromonas communis B7, and Piromonas communis B19, were grown axenically or in coculture with Methanobrevibacter smithii on xylan. N. frontalis and S. communis in monoculture and coculture accumulated xylobiose, xylose, and arabinose in the growth medium; arabinose was not metabolized, but xylobiose and xylose were subsequently used. The transient accumulation of xylose was much less evident in cocultures. Both the rate and extent of xylan utilization were increased by coculturing, and metabolite profiles became acetogenic as a result of interspecies hydrogen transfer; more acetate and less lactate were formed, while formate and hydrogen did not accumulate. For each of the three fungi, there were marked increases in the specific activities of extracellular xylanase (up to fivefold), alpha-l-arabinofuranosidase (up to fivefold), and beta-d-xylosidase (up to sevenfold) upon coculturing. The stimulating effect on fungal enzymes from coculturing with M. smithii was independent of the growth substrate, and the magnitude of the stimulation varied according to the enzymes and the incubation time. For an N. frontalis-M. smithii coculture, the positive stimulation was maintained during an extended (18-day) incubation period, and this affected not only hemicellulolytic enzymes but also polysaccharidase and glycoside hydrolase enzymes that were not involved in xylan breakdown. The specific activity of cell-bound endopeptidase was not increased under the coculture conditions used in this study. The higher enzyme activities in cocultures are discussed in relation to catabolite repression.     

Fermentation of xylans by Butyrivibrio fibrisolvens and other ruminal bacteria.

The ability of Butyrivibrio fibrisolvens and other ruminal bacteria (6 species, 18 strains) to ferment a crude xylan from wheat straw or to ferment xylans from larchwood or oat spelts was studied. Liquid cultures were monitored for carbohydrate utilization, cell growth (protein), and fermentation acid production. B. fibrisolvens 49, H17c, AcTF2, and D1 grew almost as well on one or more of the xylans as they did on cellobiose-maltose. B. fibrisolvens 12, R28, A38, X10C34, ARD22a, and X6C61 exhibited moderate growth on xylans. Partial fermentation of xylans was observed with Bacteroides ruminicola B14, Bacteroides succinogenes S85, Ruminococcus albus 7, Ruminococcus flavefaciens C94 and FD1, and Succinivibrio dextrinosolvens 22B. All xylans tested appeared to have a small fraction of carbohydrate that supported low levels of growth of nonxylanolytic strains such as Selenomonas ruminantium HD4. Compared to growth on hexoses, the same array of fermentation acids was produced upon growth on xylans for most strains; however, reduced lactate levels were observed for B. fibrisolvens 49 and Selenomonas ruminantium HD4. Measurements of enzyme activities of B. fibrisolvens AcTF2, 49, H17c, and D1 indicated that the xylobiase activities were cell associated and that the xylanase activities were predominantly associated with the culture fluid. The pattern of expression of these enzymes varied both between strains and between the carbon sources on which the strains were grown.