An ethanologenic yeast exhibiting unusual metabolism in the fermentation of lignocellulosic hexose sugars

Three lignocellulosic substrate mixtures [liquid fraction of acid-catalyzed steam-exploded softwood, softwood spent sulfite liquor (SSL) and hardwood SSL] were separately fermented by the industrially employed SSL-adapted strain Tembec T1 and a natural galactose-assimilating isolate (Y-1528) of Saccharomyces cerevisiae to compare fermentative efficacy. Both strains were confirmed as S. cerevisiae via molecular genotyping. The performance of strain Y-1528 exceeded that of Tembec T1 on all three substrate mixtures, with complete hexose sugar consumption ranging from 10 to 18 h for Y-1528, vs 24 to 28 h for T1. Furthermore, Y-1528 consumed galactose prior to glucose and mannose, in contrast to Tembec T1, which exhibited catabolite repression of galactose metabolism. Ethanol yields were comparable regardless of the substrate utilized. Strains T1 and Y-1528 were also combined in mixed culture to determine the effects of integrating their distinct metabolic capabilities during defined hexose sugar and SSL fermentations. Sugar consumption in the defined mixture was accelerated, with complete exhaustion of hexose sugars occurring in just over 6 h. Galactose was consumed first, followed by glucose and mannose. Ethanol yields were slightly reduced relative to pure cultures of Y-1528, but normal growth kinetics was not impeded. Sugar consumption in the SSLs was also accelerated, with complete utilization of softwood- and hardwood-derived hexose sugars occurring in 6 and 8 h, respectively. Catabolite repression was absent in both SSL fermentations.     

Sequence Analyses of a Broad Host-Range Plasmid Containing ermT from a Tylosin-Resistant Lactobacillus sp. Isolated from Swine Feces

Anaerobic bacteria resistant to the macrolide antibiotics tylosin and erythromycin were isolated from the feces of swine. One of the strains, 121B, was initially identified by 16S rDNA sequence analysis as an unknown Lactobacillus sp. The strain was found to contain at least two plasmids, one of which was capable of replicating and providing erythromycin and tylosin resistance to Bacillus subtilis, Streptococcus gordonii, and Escherichia coli. DNA sequence analyses of the 4,232-bp plasmid, p121BS, identified one open reading frame encoding a methylase gene highly similar (>98% amino acid identity, >99% DNA sequence identity) to the ermT gene from the Lactobacillus reuteri plasmid pGT633. This is only the second ermT gene to be reported. p121BS also contains two additional open reading frames with significant amino acid similarities to replication proteins from Lactobacillus and other Gram-positive bacteria. Received: 13 October 2000 / Accepted: 4 December 2000     

Genetic analysis of a locus on the Bacteroides ovatus chromosome which contains xylan utilization genes.

Bacteroides ovatus, a gram-negative obligate anaerobe found in the human colon, can utilize xylan as a sole source of carbohydrate. Previously, a 3.8-kbp segment of B. ovatus chromosomal DNA, which contained genes encoding a xylanase (xylI) and a bifunctional xylosidase-arabinosidase (xsa), was cloned, and expression of the two genes was studied in Escherichia coli (T. Whitehead and R. Hespell, J. Bacteriol. 172:2408-2412, 1990). In the present study, we have used segments of the cloned region to construct insertional disruptions in the B. ovatus chromosomal locus containing these two genes. Analysis of these insertional mutants demonstrated that (i) xylI and xsa are probably part of the same operon, with xylI upstream of xsa, (ii) the true B. ovatus promoter was not cloned on the 3.5-kbp DNA fragment which expressed xylanase and xylosidase in E. coli, (iii) there is at least one gene upstream of xylI which could encode an arabinosidase, and (iv) xylosidase rather than xylanase may be a rate-limiting step in xylan utilization. Insertional mutations in the xylI-xsa locus reduced the rate of growth on xylan, but the concentration of residual sugars at the end of growth was the same as that with the wild type. Thus, a slower rate of growth on xylan was not accompanied by less extensive digestion of xylan. Mutants in which xylI had been disrupted still expressed some xylanase activity. This second activity was associated with membranes and produced xylose from xylan, whereas the xylI gene product partitioned primarily with the soluble fraction and produced xylobiose from xylan.     

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)     

Biological pretreatment of corn stover with Phlebia brevispora NRRL‐13108 for enhanced enzymatic hydrolysis and efficient ethanol production

Biological pretreatment of lignocellulosic biomass by white‐rot fungus can represent a low‐cost and eco‐friendly alternative to harsh physical, chemical, or physico‐chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid‐state cultivation of corn stover with Phlebia brevispora NRRL‐13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed‐batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed‐batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL‐13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365–374, 2017     

Survival of the recombinant Bacteroides thetaiotaomicron strain BTX in in vitro rumen incubations

The survival of Bacteroides thetaiotaomicron strain BTX under rumen-simulating conditions was studied. Strain BTX is a recombinant variant of strain 5482 engineered for the production of high levels of xylanase, an enzyme important in the degradation of hemicellulose. Strain BTX was not inhibited by compounds present in rumen fluid and it grew well in media containing rumen fluid (up to 75%) or high concentrations of volatile fatty acids (total concentration, 100 mmol l⁻¹). The ability of strain BTX to compete with other micro-organisms under rumen-like conditions was studied in in vitro incubations of rumen contents. These experiments employed a consecutive batch culture (CBC) system consisting of alfalfa suspended in a rumen fluid buffer inoculated with blended rumen contents and maintained by transfers (10%, v/v) at 48 h intervals. CBC cultures contained a diversity of microbial morphotypes and accumulated fermentation products in rumen-like proportions. When added alone, the numbers of BTX cells were maintained for only a few hours, and then declined precipitously until undetectable after 48 h. If CBC cultures were also supplemented with chondroitin sulphate (a mucopolysaccharide used by Bact. thetaiotaomicron ), strain BTX grew and the pattern of its population generally followed that of the total population of ruminal bacteria in these cultures. When transferred into fresh CBC cultures containing chondroitin sulphate, BTX was again able to grow and increase in numbers, but to a diminished degree. Although BTX was able to survive and maintain itself in chondroitin sulphate supplemented cultures, this was at a very low level (10¹⁰ ml⁻¹). The potential for manipulation of rumen function by inoculation with recombinant bacteria is discussed.     

Genetic structure and variability of the endemic and vulnerable <Emphasis Type="Italic">Vellozia gigantea</Emphasis> (Velloziaceae) associated with the landscape in the Espinhaço Range,in southeastern Brazil: implications for conservation

The Espinhaço Range, in eastern Brazil, has a peculiar landscape that has influenced the vegetation pattern of the region because of its valleys, canyons, ranges and disjunct rock outcrops found at high elevations. In this region, the vegetation type known as campos rupestres (rupestrian fields), which occurs in the disjunct outcrops, has high levels of species richness and endemism. Vellozia gigantea, a 6-m tall dracenoid monocot, is a vulnerable species endemic to this vegetation and has a narrow distribution that extends approximately 27 km. This region is located in a disturbed area, where populations are divided into three geographical groups, separated by a canyon and a valley. For this study, we used ISSR markers to measure the genetic diversity of the species and test the hypothesis that the canyon and the valley constitute geographical barriers to gene flow in V. gigantea. Nine populations and 173 individuals were analyzed using nine ISSR primers, which produced 89 fragments. In spite of being a vulnerable species with a narrow distribution, the populations of V. gigantea have high genetic diversity (mean percentage of polymorphic loci = 56.6%; mean Shannon’s index of diversity = 0.278; mean expected heterozygosity = 0.183). Genetic divergence among populations was high (Φ_ST = 0.28), and principal coordinate, neighborjoining and Bayesian analyses showed that only the canyon may constitute a partial barrier to gene flow in this species. Groups of populations separated by the canyon should be managed separately because they contain different gene pools.     

Al-hyperaccumulator Vochysiaceae from the Brazilian Cerrado store aluminum in their chloroplasts without apparent damage

We investigated the pattern of aluminum (Al) accumulation in leaf tissues of native hyperaccumulator Vochysiaceae species Qualea grandiflora,Callisthene major, and Vochysia pyramidalis, from the Brazilian Cerrado. Non-accumulator Sclerolobium paniculatum was used as a control species. We expected a strong compartmentalization of Al in non-active leaf cell compartments such as cell walls and vacuoles in Al-accumulating species and the absence of Al in critical metabolic sites such as the chloroplasts. Plant leaves were harvested in the field and cut in small segments for histological analysis; hematoxylin dye was used for Al localization in tissues. Results of soil analysis of the three sites and the concentration of Al in leaves indicated that there is no direct relationship between Al availability in soils and Al hyperaccumulation among the Vochysiaceae species evaluated. The cross-sections of leaf tissues showed hematoxylin color in the palisade and spongy parenchyma cells (chloroplast) of Q. grandiflora and C. major. The vascular system of Q. grandiflora was not colored, but some cells from the xylem region of C. major were stained. In contrast, the adaxial and abaxial epidermal cells of V. pyramidalis were colored by hematoxylin, as were some cells from the vascular bundle, but color formation was not observed in the cells of palisade parenchyma. Al was not detected in leaves of S. paniculatum. We concluded that, although hyperaccumulation of Al is a common trait in the Vochysiaceae family, the processes of storage and detoxification in leaf tissues differ among the species. Two of the three hyperaccumulator species use chloroplasts as a sink for Al, with no apparent signs of toxicity. Therefore, the physiological role of Al in plant tissues remains to be elucidated.     

Individual differences in the elevated plus-maze and the forced swim test

The elevated plus-maze is an apparatus composed of enclosed and open (elevated) arms and time spent in the open arms by a rat can be increased/decreased by anxiolytic/anxiogenic agents. In the forced swim test, floating behavior is used as an index of behavioral despair and can be decreased by antidepressant agents. As the comorbidity between anxiety and depression is a remarkable issue in human behavioral disorders, a possible relationship between the behaviors seen in the cited tests is of great relevance. In the present study, fifty-four male rats (Rattus norvegicus) were submitted to a plus-maze session and to a 2-day forced swim protocol. According to their time in the open arms, they were divided into three groups: Low Open, Medium Open and High Open. Some plus-maze measures were found to be coherent with time in the open arms and are suggested to also be reliable anxiety indexes. In the forced swim test, the Low Open group showed decreases in floating duration from forced swim Session 1 to Session 2, an alteration opposite to that observed in the other groups (particularly, the Medium Open group). The Low Open group also showed increases in floating latency, again in sharp contrast with the alteration found in the other groups. Accordingly, positive and negative correlation were found between time in the open arms and floating duration and latency, respectively. Results are compared to previous studies and mediation of the effect by reactivity to aversive stimulation or alterations induced by open arm exposure is discussed.