Enhanced Xylitol Production by Precultivation of Candida guilliermondii Cells in Sugarcane Bagasse Hemicellulosic Hydrolysate

The present work evaluated the key enzymes involved in xylitol production (xylose reductase [XR] and xylitol dehydrogenase [XDH]) and their correlation with xylose, arabinose, and acetic acid assimilation during cultivation of Candida guilliermondii FTI 20037 cells in sugarcane bagasse hemicellulosic hydrolysate. For this purpose, inocula previously grown either in sugarcane bagasse hemicellulosic hydrolysate (SBHH) or in semidefined medium (xylose as a substrate) were used. The highest xylose/acetic acid consumption ratio (1.78) and the lowest arabinose consumption (13%) were attained in the fermentation using inoculum previously grown in semidefined medium (without acetic acid and arabinose). In this case, the highest values of XR (1.37 U mg prot⁻¹) and XDH (0.91 U mg prot⁻¹) activities were observed. The highest xylitol yield (∼0.55 g g⁻¹) and byproducts (ethanol and glycerol) formation were not influenced by inoculum procedure. However, the cell previously grown in the hydrolysate was effective in enhancing xylitol production by keeping the XR enzyme activity at high levels (around 0.99 U·mg_prot⁻¹), reducing the XDH activity (34.0%) and increasing xylitol volumetric productivity (26.5%) with respect to the inoculum cultivated in semidefined medium. Therefore, inoculum adaptation to SBHH was shown to be an important strategy to improve xylitol productivity.     

Efficient Production of Lumichrome by Microbacterium sp. Strain TPU 3598

Lumichrome is a photodegradation product of riboflavin and is available as a photosensitizer and fluorescent dye. To develop new efficient methods of lumichrome production, we isolated bacterial strains with high lumichrome productivity from soil. The strain with highest productivity was identified as Microbacterium sp. strain TPU 3598. Since this strain inductively produced lumichrome when cultivated with riboflavin, we developed two different methods, a cultivation method and a resting cell method, for the production of large amounts of lumichrome using the strain. In the cultivation method, 2.4 g (9.9 mmol) of lumichrome was produced from 3.8 g (10.1 mmol) of riboflavin at the 500-ml scale (98% yield). The strain also produced 4.7 g (19.4 mmol) of lumichrome from 7.6 g (20.2 mmol) of riboflavin (96% yield) by addition of riboflavin during cultivation at the 500-ml scale. In the resting cell method, 20 g of cells (wet weight) in 100 ml of potassium phosphate buffer, pH 7.0, produced 2.4 g of lumichrome from 3.8 g of riboflavin (98% yield). Since the lumichrome production by these methods was carried out in suspension, the resulting lumichrome was easily purified from the cultivation medium or reaction mixture by centrifugation and crystallization. Thus, the biochemical methods we describe here are a significant improvement in terms of simplicity and yield over the existing chemical, photolytic, and other biochemical methods of lumichrome production.     

假丝酵母发酵玉米芯半纤维素水解液生产木糖醇

采用一株驯化过的假丝酵母(Candida sp.)直接发酵经过简单脱毒处理的玉米芯半纤维素水解液生产木糖醇。确定了水解液的最适浓缩倍数在3.0～3.72的范围内。利用正交实验,确定了摇瓶分批发酵工艺条件的最适组合为：摇床转速180r/min,起始C/N为50,起始pH 5.5,接种量5% (体积比)。在此基础上,重点研究了在发酵罐中通气量对酵母发酵玉米芯水解液生产木糖醇的影响。结果表明采用先高后低的分段通气发酵在木糖醇得率方面明显优于恒定通气发酵;其中,在0～24h,3.75 L/min;24～108h,1.25 L/min的分段通气条件下(装液量为2.5L),木糖醇得率(木糖醇/木糖,g/g) 达到0.75 g/g。该结果将有助于建立一种高效的、大规模的利用玉米芯半纤维素水解液发酵生产木糖醇的工艺。     

Production of valine by a Bacillus sp.

A bacterium isolated from Burdwan (India) soil was found to accumulate L-valine in the growth medium and identified to be a strain of Bacillus subtilis. The strain is able to grow and accumulate valine in a purely synthetic medium, but supplementation of the synthetic medium with either Casamino acids or yeast extract or with both, significantly improves the yield. The entire fermentation period can be divided into a growth phase and a production phase, which can be prolonged by adjustment of pH to the neutral range. Among the different carbon and nitrogen sources tested glucose at 8.5% and L-glutamic acid at 0.8%, respectively, were found most suitable. Cane sugar molasses tested as a substitute for glucose significantly stimulated growth but valine production was less. Different vitamins tested stimulated growth and valine yield and an inoculum level of 10% (v/v) of the medium was found to be optimal. The yield of valine under optimal conditions was found to be 4.53 g per litre of the medium. Valine has been isolated in crystalline form from the fermented broth by ion exchange resin chromatography and found to be a pure sample of the L-isomer.     

A New Alkali-Thermostable Azoreductase from Bacillus sp. Strain SF

A screening for dye-decolorizing alkali-thermophilic microorganisms resulted in a Bacillus sp. strain isolated out of the wastewater drain of a textile finishing company. An NADH-dependent azoreductase of this strain, Bacillus sp. strain SF, was found to be responsible for the decolorization of azo dyes. This enzyme was purified by a combination of ammonium sulfate precipitation and anion-exchange and affinity chromatography and had a molecular mass of 61.6 kDa and an isoelectric point at pH 5.3. The pH optimum of the azoreductase depended on the substrate and was within the range of pHs 8 to 9, while the temperature maximum was reached at 80°C. Decolorization only took place in the absence of oxygen and was enhanced by FAD, which was not consumed during the reaction. A 26% similarity of this azoreductase to chaperonin Cpn60 from a Bacillus sp. was found by peptide mass mapping experiments. Substrate specificities of the azoreductase were studied by using synthesized model substrates based on di-sodium-(R)-benzyl-azo-2,7-dihydroxy-3,6-disulfonyl-naphthaline. Those dyes with NO₂ substituents, especially in the ortho position, were degraded fastest, while analogues with a methyl substitution showed the lowest degradation rates.     

Biodegradation of p-Cresol by Bacillus sp. Strain PHN 1

A bacterium capable of utilizing p-cresol as sole source of carbon and energy was isolated from soil and identified as a Bacillus species. The organism also utilized phenol, o-cresol, m-cresol, 4-hydroxybenzoic acid, and gentisic acid as growth substrates. The organism degraded p-cresol to 4-hydroxybenzoic acid, which was further metabolized by a gentisate pathway, as evidenced by isolation and identification of metabolites and enzyme activities in the cell-free extract. Such a bacterial strain can be used for bioremediation of environments contaminated with phenolic compounds.     

木糖发酵产氢菌的筛选及其生长产氢特性研究

利用改进的Hungate厌氧技术, 从牛粪堆肥中分离出一株能有效利用木糖发酵产氢的中温菌HR-1。通过16S rRNA系统发育树分析表明, 菌株 HR-1 与丙酮丁醇梭菌Clostridium acetobutylicum ATCC 824 相似性最高为96%, 结合生理生化和生长特性分析表明, HR-1是梭菌属Clostridium的一个新种, 命名为Clostridium sp. HR-1。菌株HR-1为单胞生长的规则杆状菌(0.3 mm ~0.6 mm)×(1.4 mm~2.3 mm), 革兰氏染色为阴性, 无荚膜、无鞭毛、表面光滑、无明显凸起, 专性厌氧菌。HR-1可在10°C~45°C, pH 4.0~10.0条件下生长; 37°C和pH 8.0分别为其最适生长条件。发酵PYG的主要发酵产物有氢气、二氧化碳、乙酸、丁酸及少量乙醇。HR-1可以利用有机氮源和无机氮源生长并产氢, 酵母提取物是其最佳产氢氮源。HR-1在木糖浓度为3 g/L和初始pH 6.5条件下, 其比产氢量为1.84 mol-H2/mol-木糖, 最大比产氢速率为10.52 mmol H2/h·g-细胞干重。HR-1可以亦利用葡萄糖、半乳糖、纤维二糖、甘露糖和果糖等碳源生长并发酵产氢, 发酵葡萄糖时比产氢量为2.36 mol-H2/mol-葡萄糖。     

Characterization of Thermotolerant Chitinase from the Strain Cohnella sp. IB P-192 and Its Application for the Production of Bioactive Chitosan Oligomers

Applied Biochemistry and Microbiology - Thermostable exochitinase was purified from a culture medium of a moderately thermophilic strain Cohnella sp. IB-P192 via ultrafiltration, affinity sorption,...     

Fermentative Production of Guanosine by 8-Azaguanine Resistant of Bacillus subtilis

Two forms of the restriction enzyme HindIII were alternated with each other under some physiological or biochemical conditions. Addition of a low amount of phase T7 to the culture of HindIII-producing Haemophilus influenzae Rd, resulted in appearance of some amounts of the P2 fraction of HindIII, which was eluted with a high concentration of KCI from a phosphocellulose column. Higher amounts of T7 caused a decrease of the P2 fraction; finally the alternative PI fraction of HindIII, which was eluted with a lower concentration of KCI, remained exclusively.

Addition of disaccharides such as maltose and trehalose to the bacterial extract, yielded more P2, although the disaccharides inhibited to this enzyme. Urea showed an interesting distribution of these two forms of HindIII. Phosphocellulose chromatography in the presence of 2 m urea generated a broad peak of HindIII Activity. Addition of 4 m urea, on the contrary, showed only one active peak of this enzyme. The HindIII could be purified by the following DEAE-cellulose chromatography.

These results indicate the presence of only one kind of HindIII molecule, which was alternated between free and bound forms, and a certain kind of factor that would equilibrate these two forms.     

Mechanism of Algal Aggregation by Bacillus sp. Strain RP1137

Alga-derived biofuels are one of the best alternatives for economically replacing liquid fossil fuels with a fungible renewable energy source. Production of fuel from algae is technically feasible but not yet economically viable. Harvest of dilute algal biomass from the surrounding water remains one of the largest barriers to economic production of algal biofuel. We identified Bacillus sp. strain RP1137 in a previous study and showed that this strain can rapidly aggregate several biofuel-producing algae in a pH- and divalent-cation-dependent manner. In this study, we further characterized the mechanism of algal aggregation by RP1137. We show that aggregation of both algae and bacteria is optimal in the exponential phase of growth and that the density of ionizable residues on the RP1137 cell surface changes with growth stage. Aggregation likely occurs via charge neutralization with calcium ions at the cell surface of both algae and bacteria. We show that charge neutralization occurs at least in part through binding of calcium to negatively charged teichoic acid residues. The addition of calcium also renders both algae and bacteria more able to bind to hydrophobic beads, suggesting that aggregation may occur through hydrophobic interactions. Knowledge of the aggregation mechanism may enable engineering of RP1137 to obtain more efficient algal harvesting.     

Fermentative Production of Thymidine by a Metabolically Engineered Escherichia coli Strain

Thymidine is an important precursor in the production of various antiviral drugs, including azidothymidine for the treatment of AIDS. Since thymidine-containing nucleotides are synthesized only by the de novo pathway during DNA synthesis, it is not easy to produce a large amount of thymidine biologically. In order to develop a host strain to produce thymidine, thymidine phosphorylase, thymidine kinase, and uridine phosphorylase genes were deleted from an Escherichia coli BL21 strain to develop BLdtu. Since the genes coding for the enzymes related to the nucleotide salvage pathway were disrupted, BLdtu was unable to utilize thymidine or thymine, and thymidine degradation activity was completely abrogated. We additionally expressed T4 thymidylate synthase, T4 nucleotide diphosphate reductase, bacteriophage PBS2 TMP phosphohydrolase, E. coli dCTP deaminase, and E. coli uridine kinase in the BLdtu strain to develop a thymidine-producing strain (BLdtu24). BLdtu24 produced 649.3 mg liter⁻¹ of thymidine in a 7-liter batch fermenter for 24 h, and neither thymine nor uridine was detected. However, the dUTP/dTTP ratio was increased in BLdtu24, which could lead to increased double-strand breakages and eventually to cell deaths during fermentation. To enhance thymidine production and to prevent cell deaths during fermentation, we disrupted a gene (encoding uracil-DNA N-glycosylase) involved in DNA excision repair to suppress the consumption of dTTP and developed BLdtug24. Compared with the thymidine production in BLdtu24, the thymidine production in BLdtug24 was increased by ∼1.2-fold (740.3 mg liter⁻¹). Here, we show that a thymidine-producing strain with a relatively high yield can be developed using a metabolic engineering approach.Thymidine, which is composed of 2-deoxyribose and a thymine base, is a commercially useful precursor in the chemical synthesis of various antiviral drugs, including stavudine and zidovudine (azidothymidine), the active ingredient in a formulation for the treatment of AIDS (18, 19). Because thymidine is required only in DNA synthesis, intracellular thymidine levels are very low and are tightly controlled (40). For the production of precursors for antiviral drugs, thymidine is either biologically produced in a low yield by a few modified microorganisms or chemically synthesized through a very costly process (17, 33, 48, 49). Thus, there is a need for developing a more efficient strain for thymidine production on a large scale.In nature, there are two distinct pathways for dTTP synthesis, the salvage and de novo pathways. The salvage pathway enables the cells to utilize preformed nucleobases and nucleosides for nucleotide synthesis, using thymidine phosphorylase (deoA), uridine phosphorylase (udp), and thymidine kinase (tdk) (Fig. ​(Fig.1)1) (40).Open in a separate windowFIG. 1.Thymidine biosynthetic pathway. The steps engineered in this study are indicated by the bold arrows and lines. Components of the catabolism are as follows: pyrA, carbamoylphosphate synthase; pyrBI, aspartate-carbamoyl transferase; pyrC, dihydroorotase; pyrD, dihydroorotate oxidase; pyrE, orotate phosphoribosyltransferase; pyrF, OMP decarboxylase; pyrG, CTP synthetase; pyrH, UMP kinase; TMPase, TMP phosphohydrolase; nrd, nucleotide diphosphate reductase; tdΔI, T4 thymidylate synthase (intron deleted); thyA, thymidylate synthase; dcd, dCTP deaminase; udk, uridine kinase; deoA, thymidine phosphorylase; tdk, thymidine kinase; udp, uridine phosphorylase; dut, deoxyribonucleotide triphosphatase; ndk, nucleotide diphosphate kinase; tmk, TMP kinase; ung, uracil-DNA N-glycosylase; upp, uracil phosphoribosyl-transferase; cdd, cytidine deaminase; codA, cytosine deaminase.As the name indicates, the de novo pathway enables the cells to synthesize nucleobases de novo. The de novo pathway leading to thymidine biosynthesis starts with the condensation of aspartate and carbamoylphosphate, synthesized by carbamoylphosphate synthase (pyrA) (41). This condensation reaction is catalyzed by aspartate-carbamoyl transferase (pyrBI) to produce carbamoyl aspartate, which undergoes several reactions to produce UMP, the common precursor for the synthesis of the pyrimidine ribonucleoside and deoxynucleosides (Fig. ​(Fig.1)1) (39-41). For thymidine biosynthesis, UMP is converted to UDP in a reaction catalyzed by UMP kinase (pyrH), and UDP is converted to dUDP by ribonucleoside diphosphate reductase (nrdAB), which is regulated by NTP effectors through binding to specific allosteric sites on ribonucleotide diphosphate reductase (nrdA). Escherichia coli can synthesize dUMP from both dCDP and dUDP. The major pathway involves phosphorylation of dCDP to dCTP, deamination of dCTP to dUTP, and hydrolysis of dUTP to dUMP. Only 20 to 30% of the cellular dUMP is supplied by hydrolysis of dUTP (29, 37). The deamination of dCTP (dcd) is located at a branch point in the pyrimidine metabolic pathway. Because of its importance, dcd is regulated by a positive homotropic cooperativity toward dCTP and by a feedback inhibition by dTTP (29, 31, 40).Deoxyuridine triphosphatase (dUTPase [dut]) is a pyrophosphatase that contains zinc ions (42). dUTPase catalyzes the hydrolysis of dUTP to PP_i and dUMP, a substrate for thymidylate synthase (thyA). Generally, the intracellular concentration of dUTP is <10 nmol per 1 g dry cell weight (DCW), and that of dTTP exceeds 500 nmol per 1 g DCW (5, 39, 52). The intracellular dUTP-to-dTTP ratio is increased in dut-deficient mutants, leading to an increased frequency of misincorporation of uracil for thymine in DNA (34). This incorporation is transient only because uracil is removed from DNA via a subsequent excision repair initiated by uracil-DNA N-glycosylase, which is encoded by ung (15, 50). Attempted repair of deoxyuridine residues from DNA without adequate dTTP available to complete the repair reaction can result in multiple single-strand breaks, eventually leading to double-strand breaks (15). Indeed, single- and double-strand breaks accumulate in thymidine-deprived cells (16). In such cells, the loss of uracil glycosylase activity should decrease DNA breaks arising from attempted repair and thereby decrease the toxicity of thymidine depletion.The synthesis of dTMP from dUMP involves the transfer of a methylene group and two reducing equivalents from 5,10-methylenetetrahydrofolate to dUMP, catalyzed by the dimeric enzyme thymidylate synthase (thyA). Even though ThyA catalyzes the committed step for de novo synthesis of dTTP, neither the activity of the enzyme nor the expression of the thyA gene seems to be regulated (2, 3).The general strategy used for the development of a thymidine-overproducing strain involves the alleviation of control mechanisms in key pathways. Several different microorganisms have been modified for thymidine production, including E. coli, Brevibacterium helvolum, and Corynebacterium ammoniagenes, by classical mutagenesis methods, and they were selected based on their capacity to grow on toxic thymidine analogues (30, 33, 48, 49). In these studies, feedback inhibition-resistant variants of thymidine biosynthetic enzymes were obtained by random mutation, and high-producing variants were selected. The most optimum B. helvolum strain obtained by this procedure produced 500 mg liter⁻¹ of thymidine by batch fermentation (33). However, engineered B. helvolum and E. coli mutants also produced thymine, deoxyuridine, and uracil, which are unfavorable for thymidine production since it increases costs during the purification process (30, 33, 48, 49). Furthermore, these thymidine-producing strains have residual thymidine degradation activities, resulting in decreased productivities.Thus, we tried to develop a more efficient thymidine-producing strain by enhancing the de novo pathway leading to thymidine biosynthesis and by disrupting the thymidine salvage pathway. The strategy reported here is based on disrupting genes which encode enzymes involved in thymidine degradation and on expressing foreign genes in the de novo pathway leading to thymidine biosynthesis which encode enzymes that are expected to be less sensitive to feedback inhibition by thymidine than the original enzymes in the host strain. The T4 ribonucleotide diphosphate reductase (nrdAB) operon, T4 thioredoxin (nrdC), T4 thymidylate synthase (td), and PBS2 TMP phosphohydrolase (TMPase) were expressed in an E. coli mutant strain which was modified to block the salvage pathway (deoA, tdk, and udp). In order to increase the influx of dUMP, E. coli dCTP deaminase (dcd), deoxyuridine triphosphatase (dut), and uridine kinase (udk) were expressed with phage-derived genes. We found that the dUTP/dTTP ratio was increased by increasing the level of dUTP in our mutant, leading to the frequent misincorporation of dUTP in DNA. In order to prevent frequent temporary DNA breaks and gaps by excision repair caused by the increased intracellular dUTP/dTTP ratio, uracil-DNA N-glycosylase (ung) was additionally disrupted.     

Optimization of Alkaline Protease Production from Bacillus sp. by Response Surface Methodology

High yields (1939 U/ml) of an alkaline protease were obtained in batch fermentation of a Bacillus sp. using a response surface methodology. The interaction of four variables, viz., starch, peptone, incubation time, and inoculum density, suggested inoculum density to be an insignificant variable. However, incubation time had a profound effect on protease yields at all the concentrations of carbon and nitrogen used. The response surface raised and flattened with increase in time of incubation, and maximum protease production up to 1939 U/ml was obtained after 96 h of incubation. The model equation obtained was validated experimentally at maximum starch (15 mg/ml) and peptone (7.5 mg/ml) concentration with increased incubation time up to 144 h in the presence of minimum inoculum density (1%). An overall 2.6-fold increase in protease production was obtained as compared with mean observed response (750 U/ml) at zero level of all variables. Received: 19 July 2001 / Accepted: 15 August 2001     

克雷伯氏菌生产胞外多糖的研究进展

从克雷伯氏菌胞外多糖的组成、功能及发酵工艺等方面概述了克雷伯氏菌发酵生产胞外多糖研究进展,并讨论了该发酵过程中存在的问题;同时指出,在现有基础上应用数学工具模拟优化发酵工艺并改良分离提取方法,并进一步对其进行开发应用是今后研究的重点。     

Isolation and Characterization of Chitosanase from the Strain Bacillus sp. 739

The specific nature of the chitosanase activity of the strain Bacillus sp. 739 was determined. Maximum enzyme activity was observed in a medium containing biomass of the fruiting bodies of the fungus Macrolepiota procera. The chitosanase was purified to homogeneity by chromatography on DEAE-Sephadex A-50 and Toyopearl HW-50. The molecular weight of the enzyme assessed by electrophoresis (the Laemmli procedure) approximated 46 kDa. The temperature and pH optima of the purified chitosanase were in the ranges 45–55°C and 6.0–6.5, respectively. Time to half-maximum inactivation of the enzyme at 50°C was equal to 1 h. With colloidal chitosan as the substrate, the value of K of the purified chitosanase was equal to 25 mg/ml. The enzyme also exhibited a weak ability to hydrolyze colloidal chitin.     

芽孢杆菌木聚糖酶的发酵条件研究

本文研究了芽孢杆菌Ｌ２３产木聚糖酶的时间曲线,碳源种类和浓度,添加物,发酵起始ｐｈ以及接种量对产酶的影响。该菌经３７℃培养５０小时,酶活力为３０ＩＵ／ｍｌ,酶最适反应温度为５７℃,最适ｐＨ值为７．０。     

Feasibility of Manufacturing Cellulose Nanocrystals from the Solid Residues of Second-Generation Ethanol Production from Sugarcane Bagasse

The reuse of the solid residues generated in the production of second-generation (2G) ethanol to obtain high-value products is a potential strategy for improving the economic and environmental viability of the overall process. This study evaluated the feasibility of using the residual solids remaining after the enzymatic hydrolysis of sugarcane bagasse for the production of cellulose nanocrystals (CNC), a valuable bionanomaterial. To this end, sugarcane bagasse subjected to steam explosion (SEB) or liquid hot water (LHWB) pretreatment was hydrolysed using different loadings of a commercial cellulase cocktail. Samples of SEB and LHWB were hydrolysed enzymatically, resulting in glucose releases close to 40 g/L, which would be suitable for producing 2G ethanol by microbial fermentation. The solid residues after the enzymatic hydrolysis step presented cellulose contents of up to 54 %, indicating that a significant amount of recalcitrant crystalline cellulose remained available for subsequent use. These solid residues were purified and submitted to acid hydrolysis, resulting in the successful formation of CNC with crystallinity close to 80 %, lengths of 193–246 nm and diameters of 14–18 nm. The CNC produced presented morphology, dimensions, physical-chemical characteristics, thermal stability and crystallinity within the ranges reported for this type of material. Moreover, the enzyme loading or the type of hydrothermal pretreatment process employed here showed no significant effects on the CNC obtained, indicating that these variables could be flexibly adjusted according to specific interests.     

Characterization of Lignocellulolytic Activities from a Moderate Halophile Strain of Aspergillus caesiellus Isolated from a Sugarcane Bagasse Fermentation

A moderate halophile and thermotolerant fungal strain was isolated from a sugarcane bagasse fermentation in the presence of 2 M NaCl that was set in the laboratory. This strain was identified by polyphasic criteria as Aspergillus caesiellus. The fungus showed an optimal growth rate in media containing 1 M NaCl at 28°C and could grow in media added with up to 2 M NaCl. This strain was able to grow at 37 and 42°C, with or without NaCl. A. caesiellus H1 produced cellulases, xylanases, manganese peroxidase (MnP) and esterases. No laccase activity was detected in the conditions we tested. The cellulase activity was thermostable, halostable, and no differential expression of cellulases was observed in media with different salt concentrations. However, differential band patterns for cellulase and xylanase activities were detected in zymograms when the fungus was grown in different lignocellulosic substrates such as wheat straw, maize stover, agave fibres, sugarcane bagasse and sawdust. Optimal temperature and pH were similar to other cellulases previously described. These results support the potential of this fungus to degrade lignocellulosic materials and its possible use in biotechnological applications.     

Endophytic Colonization of Balloon Flower by Antifungal Strain Bacillus sp. CY22

Endophytic Bacillus sp. CY22 was previously isolated from the root interior of the balloon flower (Platycodon grandiflorum) (Cho et al., Biosci. Biotechnol. Biochem., 66, 1270-1275 (2002)). Three-month-old balloon flower seedlings were inoculated with 10⁷ cfu/ml of strain CY22R3, a rifampicin-resistant strain of CY22, and external and internal root colonization was assessed 2 and 4 weeks later. After inoculation, large numbers of bacteria were observed on the root surface by scanning electron microscopy. More detailed studies using optical and transmission electron microscopy confirmed that Bacillus sp. CY22 was endophytically established within intercellular spaces, cortical cells, and aerenchymas of root. Also, Bacillus sp. CY22 showed antibiotic activities against several phytopathogens by producing the antibiotic iturin A. In the pot test, root rot of balloon flower seedlings caused by Rhizoctonia solani was suppressed when the Bacillus sp. CY22R3 was inoculated into the soil.     

Production of alkaline xylanase by an alkaliphilic Bacillus sp. isolated from an alkalinesoda lake

An alkaline xylanase-producing alkaliphilic Bacillus sp. AR-009 was isolated from analkaline soda lake in Ethiopia. The enzyme was optimally active at pH 9 and was stable over abroad pH range. The optimum temperature for xylanase activity, assayed at pH 9, was60°–65°C. Measured at pH 8 and 9, the enzyme had good stability at 55° and60°C. At both pH values, over 80% of its original activity was retained after heating for2·5 h at 55°C. At 60°C, the enzyme maintained 63% of its original activity after2·5 h incubation while at pH 9 it retained 54% of its original activity after 1 h heating. Theseproperties qualify the enzyme to be novel and potentially important for application in someindustrial processes.     

Production of xylanases from a newly isolated alkalophilic thermophilic Bacillus sp.

A new thermophilic strain of Bacillus SPS-0 which produces thermostable xylanases was isolated from a hot spring in Portugal. Xylanase production was 50 nkat/ml in the presence of wheat bran arabinoxylan. The temperature and pH for optimum activity were 75°C and 6–9, respectively. The hydrolysis patterns demonstrated that crude xylanases yield mainly xylose and xylobiose from xylan, whereas xylose and arabinose were produced from destarched wheat bran. An increase in xylose release was observed when SPS-0 xylanase was supplemented by a ferulic acid esterase. © Rapid Science Ltd. 1998