Identification of three proteins up-regulated by raw starch in Cytophaga sp.

Raw starch-digesting amylases (RSDAs) in many microorganisms convert starch granules into maltodextrins and simple sugars. We cloned and sequenced from Cytophaga sp. an RSDA with an excellent raw starch digestion activity. This RSDA was highly inducible by raw starch, but not by other sugars, suggesting that an unknown signal transduction mechanism is involved in the degradation of raw starch. We used a proteomic approach to investigate the effect of raw starch on protein expression in Cytophaga sp. Using MALDI–TOF MS protein analysis, we have identified three proteins up-regulated by raw starch, i.e., a 60-kDa chaperonin (cpn60), glutaminase, and pyruvate phosphate dikinase (PPDK). Subsequent time-course studies detected an increased expression of RSDA as well as the highest expression of PPDK occurring 6 h post-incubation with raw corn starch, implying that the latter enzyme may work along with RSDA on the digestion of raw starch. Finding these proteins up-regulated by raw starch may provide an insight into how Cytophaga sp. cells respond to raw starch stimulation.     

Degradation of staphylococcal enterotoxin A by a Pseudomonas aeruginosa isolate from raw milk

Recently, we found that staphylococcal enterotoxin A (SEA)-producing Staphylococcus aureus strains produced SEA in raw milk with microbial contaminants at high temperatures like 40 °C only. Moreover, the concentration of SEA produced in raw milk gradually decreased after the peak. The reason(s) for SEA degradation in raw milk was studied in this study. Degradation of SEA spiked in raw milk was observed at 40 °C, but not at 25 °C. A Pseudomonas aeruginosa isolate from raw milk degraded SEA spiked in broth at 40 °C. A sample partially purified with a chromatographic method from culture supernatant of the isolate degraded SEA. Two main proteolytic bands were observed in the sample by zymographic analysis with casein. These results suggested that the SEA in raw milk might be degraded by a protease(s) produced by the P. aeruginosa isolate. This finding might be the first report on SEA degradation by a proteolytic enzyme(s) derived from Pseudomonas bacteria to our knowledge.     

Raw starch-degrading enzyme from newly isolated strains of endophytic fungi

Four newly isolated strains of endophytic fungi namely Gibberella pulicaris, Acremonium sp., `Synnematous' sp. and Nodilusporium sp. were compared for their degradative activity on raw and gelatinized starches, substrate specificity and optimum pH. Results showed that the raw starch-degrading enzyme from Acremonium sp. had a broad activity towards both small and large granule size of raw starches while the enzyme from other strains showed high activity toward starches of smaller granule size. Analysis of the end product by TLC showed that enzyme from Gibberella pulicaris, Acremonium and Nodilusporium sp. hydrolysed raw sago starch to produce solely glucose but the enzyme of `Synnematous' sp. produced glucose and maltose. This revised version was published online in July 2006 with corrections to the Cover Date.     

The digestibility of raw and processed soybeans by Helmeted Guineafowl,Numida meleagris

Helmeted Guineafowl, Numida meleagris, populations have declined significantly within the Midlands of KwaZulu-Natal since the early 1980s. Because guineafowl have been observed feeding on harvested soybean lands and because raw soybeans are known to contain anti-nutritional factors, research into the digestibility of raw soybeans was conducted to investigate possible localized factors in suppressing populations of these birds. A digestibility study was carried out on the apparent metabolizable energy (AME) and the amino acids of both raw and processed soybeans, as well as a number of other feed ingredients, using Helmeted Guineafowl and adult roosters, Gallus gallus, as a control. The results of the energy balance studies were similar for both the guineafowl and the roosters. Soybean, both raw and processed, was found to be comparable with the other feed ingredients in terms of the digestibility of gross energy, but the amino acid digestibility of raw soybeans was considerably lower than that of processed soybean oilcake meal. The availability of raw soybean may thus only be contributing to localized dietary constraints within guineafowl populations.     

Direct production of L-lysine from raw corn starch by Corynebacterium glutamicum secreting Streptococcus bovis alpha-amylase using cspB promoter and signal sequence

Corynebacterium glutamicum is an important microorganism in the industrial production of amino acids. We engineered a strain of C. glutamicum that secretes α-amylase from Streptococcus bovis 148 (AmyA) for the efficient utilization of raw starch. Among the promoters and signal sequences tested, those of cspB from C. glutamicum possessed the highest expression level. The fusion gene was introduced into the homoserine dehydrogenase gene locus on the chromosome by homologous recombination. L-Lysine fermentation was conducted using C. glutamicum secreting AmyA in the growth medium containing 50 g/l of raw corn starch as the sole carbon source at various temperatures in the range 30 to 40°C. Efficient L-lysine production and raw starch degradation were achieved at 34 and 37°C, respectively. The α-amylase activity using raw corn starch was more than 2.5 times higher than that using glucose as the sole carbon source during L-lysine fermentation. AmyA expression under the control of cspB promoter was assumed to be induced when raw starch was used as the sole carbon source. These results indicate that efficient simultaneous saccharification and fermentation of raw corn starch to L-lysine were achieved by C. glutamicum secreting AmyA using the cspB promoter and signal sequence.     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> VPI 3266 using a synthetic medium and raw glycerol

Growth inhibition of Clostridium butyricum VPI 3266 by raw glycerol, obtained from the biodiesel production process, was evaluated. C. butyricum presents the same tolerance to raw and to commercial glycerol, when both are of similar grade, i.e. above 87% (w/v). A 39% increase of growth inhibition was observed in the presence of 100 g l^–1 of a lower grade raw glycerol (65% w/v). Furthermore, 1,3-propanediol production from two raw glycerol types (65% w/v and 92% w/v), without any prior purification, was observed in batch and continuous cultures, on a synthetic medium. No significant differences were found in C. butyricum fermentation patterns on raw and commercial glycerol as the sole carbon source. In every case, 1,3-propanediol yield was around 0.60 mol/mol glycerol consumed.     

Prevalence and fate of giardia cysts in wastewater treatment plants

The present study was conducted to review factors affecting the prevalence and concentration of Giardia in raw wastewater. The removal and inactivation efficiency of Giardia by wastewater treatment technologies was also reviewed. Data published for the prevalence of Giardia in wastewater and the removal by wastewater treatment plants was reviewed. Giardia cysts are highly prevalent in wastewater in various parts of the world, which may reflect the infection rate in the population. In 23 of 30 (76·6%) studies, all of the tested raw wastewater samples were positive for Giardia cysts at concentrations ranging from 0·23 to 100 000 cysts l^?1. The concentration of Giardia in raw wastewater was not affected by the geographical region or the socio‐economic status of the community. Discharge of raw wastewater or the application of raw wastewater for irrigation may result in Giardia transmission. Activated sludge treatment resulted in a one to two orders of magnitude reduction in Giardia, whereas a stabilization pond with a high retention time removed up to 100% of the cysts from wastewater. High‐rate sand filtration, ultrafiltration and UV disinfection were reported as the most efficient wastewater treatment methods for removal and disinfection of Giardia cysts. Wastewater treatment may not totally prevent the environmental transmission of Giardia cysts. The reviewed data show that a combination of wastewater treatment methods may results in efficient removal of Giardia cysts and prevent their environmental transmission.     

Efficacy of Chlorella pyrenoidosa and Scenedesmus abundans for Nutrient Removal in Rice Mill Effluent (Paddy Soaked Water)

Microalgae are product of sustainable development owing to its ability to treat variety of wastewater effluents and thus produced biomass can serve as value added product for various commercial applications. This paper deals with the cultivation of microalgae species namely Chlorella pyrenoidosa and Scenedesmus abundans in rice mill effluent (i.e., paddy soaked water) for nutrient removal. In order to investigate the nutrient removal capability, microalgae are subjected to cultivation in both raw and autoclaved samples. The maximum phosphate removal by Scenedesmus abundans and Chlorella pyrenoidosa in raw sample was 98.3% and 97.6%, respectively, whereas, the removal of ammoniacal nitrogen by Scenedesmus abundans and Chlorella pyrenoidosa in raw sample was 92% and 90.3%, respectively. The growth (measured in terms of chlorophyll content) of Scenedesmus abundans and Chlorella pyrenoidosa in raw sample was 3.88 mg/l and 5.55 mg/l, respectively. The results indicate the suitability of microalgae cultivation in rice mill effluent treatment for nutrient removal.     

基于DNA高通量测序分析生料酿醋过程中的真菌多样性

【目的】了解生料酿醋不同阶段的真菌群落结构及其变化规律,为生料酿醋工艺优化提供理论指导。【方法】从山西一家生料酿醋企业采集原料、麸曲、发酵缸醋醅、熏醋样、淋醋样等涉及生料酿醋各阶段的样品共51份,扩增真菌ITS1区序列并利用高通量测序技术分析真菌多样性。【结果】除5份样品未扩增成功外,在剩余46份样品中共检测到489个真菌OTU,以子囊菌为主(占88.3%)。原料、麸曲、发酵缸醋醅、熏醋样、淋醋样等不同组别间在真菌群落结构方面存在显著差异。原料和麸曲中的真菌物种丰富度最低,发酵缸醋醅的真菌物种丰富度最高,熏醋样和淋醋样中的真菌物种丰富度又有所降低。原料和麸曲中的优势真菌分别为酿酒酵母和黑曲霉,是发酵阶段真菌的重要来源,但发酵缸醋醅中也检测到大量可能来源于发酵室环境的真菌。发酵缸醋醅在不同发酵时期间也存在明显的真菌群落结构差异,并可据此划分成发酵前期(包括发酵第2–13天的样品)和发酵后期(包括发酵第17–46天的样品)。酿酒酵母和亮白曲霉的丰度在发酵前期显著高于发酵后期,而黑曲霉、一种小戴卫霉科真菌等的丰度在发酵后期显著高于发酵前期。【结论】生料酿醋的不同阶段和发酵缸醋醅发酵的不同时期,其真菌群落结构都存在明显差异。酿酒酵母和黑曲霉是发酵阶段的优势真菌。本研究为生料酿醋工艺优化提供了理论依据。     

Amplicon-based profiling of bacteria in raw and secondary treated wastewater from treatment plants across Australia

In this study, we investigated the use of Illumina high-throughput sequencing of 16S ribosomal RNA (rRNA) amplicons to explore microbial diversity and community structure in raw and secondary treated wastewater (WW) samples from four municipal wastewater treatment plants (WWTPs A–D) across Australia. Sequence reads were analyzed to determine the abundance and diversity of bacterial communities in raw and secondary treated WW samples across the four WWTPs. In addition, sequence reads were also characterized to phenotypic features and to estimate the abundance of potential pathogenic bacterial genera and antibiotic-resistant genes in total bacterial communities. The mean coverage, Shannon diversity index, observed richness (S _obs), and abundance-based coverage estimate (ACE) of richness for raw and secondary treated WW samples did not differ significantly (P > 0.05) among the four WWTPs examined. Generally, raw and secondary treated WW samples were dominated by members of the genera Pseudomonas, Arcobacter, and Bacteroides. Evaluation of source contributions to secondary treated WW, done using SourceTracker, revealed that 8.80–61.4% of the bacterial communities in secondary treated WW samples were attributed to raw WW. Twenty-five bacterial genera were classified as containing potential bacterial pathogens. The abundance of potentially pathogenic genera in raw WW samples was higher than that found in secondary treated WW samples. Among the pathogenic genera identified, Pseudomonas and Arcobacter had the greatest percentage of the sequence reads. The abundances of antibiotic resistance genes were generally low (<0.5%), except for genes encoding ABC transporters, which accounted for approximately 3% of inferred genes. These findings provided a comprehensive profile of bacterial communities, including potential bacterial pathogens and antibiotic-resistant genes, in raw and secondary treated WW samples from four WWTPs across Australia and demonstrated that Illumina high-throughput sequencing can be an alternative approach for monitoring WW quality in order to protect environmental and human health.

     

High performance liquid chromatography analysis of diarrhetic toxins inDinophysis spp. from the French coast

Diarrhetic shellfish poisoning has been a recurrent problem along the Brittany coast (France) since 1983. Okadaic acid (O.A.), the main toxin detected in mussels, is generally associated with the presence ofDinophysis cells in sea water. We report here the results of okadaic acid analyses by high performance liquid chromatography, on planktonic samples collected during the summer of 1991.D. sacculus, the majorDinophysis species, throughout this period, showed low okadaic acid content in raw extract, whereas toxic levels were 3- to 12-fold higher in sorted samples than in raw ones. A maximum O.A. concentration of 29.6 pg cell^-1 was found inD. sacculus/D. cf. acuminata extract. Similarly, higher O.A. levels were noted in raw samples when these two species were associated. Generally speaking, variations in raw samples were similar to those in sorted samples. Nevertheless, whenD. rotundata reached a concentration equal to that ofD. sacculus in sorted samples, the O.A. level was lower.     

Improved preparation of vitexin from hot water extract of Basella alba,the commercially available vegetable Malabar spinach (“Tsurumurasaki” in Japanese) and the application to semisynthesis of chafuroside B

ABSTRACT

Hot water extraction of D-arabinofuranosylvitexin from the raw leaves of commercially available Basella alba “Tsurumurasaki” and subsequent acidic hydrolysis was improved to be a procedure using a high-pressure steam sterilizer to afford vitexin. The amount was estimated to be 14.1 mg from 1 g of dry weight of the raw leaves, whose recovery was calculated to be 95% based on the estimated content of D-arabinofuranosylvitexin in B. alba raw leaves. The product was dehydratively cyclized between hydroxy groups on the carbohydrate and flavone skeletons under modified Mitsunobu reaction conditions in N,N-dimethylformamide to give chafuroside B, which is known to be a bioactive Oolong tea polyphenol. Through these transformations, 10.2 mg of chafuroside B could be semisynthesized from 1 g of dry weight of the raw leaves, and the efficiency was improved compared to that from the extraction from Oolong tea (3.4 μg from 1 g of dry weight).     

Archaeal and bacterial community structures of rural household biogas digesters with different raw materials in Qinghai Plateau

The present study aims to investigate microbial community structures household biogas digesters with different raw materials in Qinghai Plateau rural. High-throughput 16S rRNA gene sequencing analysis revealed that Firmicutes, Bacteroidetes, and Proteobacteria are the most abundant bacterial phyla (64.08%). Prevotella group 7 was the most abundant genus in digester YL9 and YL10 (69.72% and 26.96%, respectively) using vegetable waste raw materials. Trichococcus exhibited the highest abundance (14.55%) in YL1 digester using sheep and pig manure. Clostridium sensu stricto 1 (13.89%) and Synergistaceae_uncultured (15.52%) comprised the highest abundances in digester YL5 with mixed raw materials (i.e., dairy manure, sheep manure, and human feces). In addition, Proteiniphilum and Pseudomonas exhibited the highest abundances among bacterial genera in YL4 digester using pig manure. Methanomicrobiales was the most dominant archaeal communities, ranging from 13.35% to 81.34% in abundance. Methanocorpusculum exhibited dominant abundances in all digesters using various raw materials. Methanogenium was the most abundant archaeal genera in YL4 and YL6 digesters, which consume pig manure as primary raw material. In addition, Methanosarcina and Methanosaeta exhibited the highest abundances in digester YL1 (55.03%) and YL9 (51.40%), respectively. Moreover, fermentation temperatures and pH both contributed to the archaeal and bacterial community structures in all the investigated digesters. Specially, fermentation temperature showed positive correlation with the abundances of Synergistaceae_uncultured, Methanogenium, and Methanosaeta, and pH was positively correlated with the abundances of Prevotella group 7 and Methanosarcina abundances.

     

Production of multiple extracellular enzyme activities by novel submerged culture of <Emphasis Type="Italic">Aspergillus kawachii</Emphasis> for ethanol production from raw cassava flour

Cassava is a starch-containing root crop that is widely used as a raw material in a variety of industrial applications, most recently in the production of fuel ethanol. In the present study, ethanol production from raw (uncooked) cassava flour by simultaneous saccharification and fermentation (SSF) using a preparation consisting of multiple enzyme activities from Aspergillus kawachii FS005 was investigated. The multi-activity preparation was obtained from a novel submerged fermentation broth of A. kawachii FS005 grown on unmilled crude barley as a carbon source. The preparation was found to consist of glucoamylase, acid-stable α-amylase, acid carboxypeptidase, acid protease, cellulase and xylanase activities, and exhibited glucose and free amino nitrogen (FAN) production rates of 37.7 and 118.7 mg/l/h, respectively, during A. kawachii FS005-mediated saccharification of uncooked raw cassava flour. Ethanol production from 18.2% (w/v) dry uncooked solids of raw cassava flour by SSF with the multi-activity enzyme preparation yielded 9.0% (v/v) of ethanol and 92.3% fermentation efficiency. A feasibility study for ethanol production by SSF with a two-step mash using raw cassava flour and the multi-activity enzyme preparation manufactured on-site was verified on a pilot plant scale. The enzyme preparation obtained from the A. kawachii FS005 culture broth exhibited glucose and FAN production rates of 41.1 and 135.5 mg/l/h, respectively. SSF performed in a mash volume of about 1,612 l containing 20.6% (w/v) dry raw cassava solids and 106 l of on-site manufactured A. kawachii FS005 culture broth yielded 10.3% (v/v) ethanol and a fermentation efficiency of 92.7%.     

Influence of contamination by Penicillium brevicompactum and Trichoderma harzianum during Lentinula edodes spawn run on fruiting in sawdust-based substrates

 Substrates with two kinds of supplements, raw and deoiled rice bran, were artificially infected with Penicillium brevicompactum or Trichoderma harzianum on days 0, 26, 61, and 90 after inoculation with Lentinula edodes. With P. brevicompactum infection, there was no significant difference in the yield and size of the fruit-bodies among either infected and uninfected substrates, raw and deoiled rice bran substrates, or days when the substrates were infected. However, the irregularly shaped fruit-bodies, which were commercially of low value, yielded greatly on raw rice bran substrates infected on days 0 and 26, whereas the substrates infected with T. harzianum on any day were covered with conidia and fatally damaged. Received: August 29, 2001 / Accepted: February 15, 2002     

Orthogonal test design for optimization of the extraction of polysaccharides from Phascolosoma esulenta and evaluation of its immunity activity

Yield of polysaccharides from Phascolosoma esulenta obtained by phosphate buffer extraction through an orthogonal experiment (L₉(3)⁴) were investigated to get the best extraction conditions. The results showed that extraction temperature, ratio of phosphate buffer to raw material, extraction time, and ratio of trypsinase to raw material were the main four variables that influenced the yields of extracts. The highest yield was obtained when extraction temperature, ratio of phosphate buffer to raw material, extraction time and ratio of trypsinase to raw material were 40 °C, 2, 5.5 h and 1.6, respectively. The immunity-stimulating method showed that polysaccharides from P. esulenta could significantly raise liver, spleen and thymus index of mice and enhance Con A-stimulated mouse spleen cells proliferation. These results indicate that polysaccharides from P. esulenta had significantly higher immunity-stimulating activities.     

Production of multifunctional lipases by Penicillium verrucosum and Penicillium brevicompactum under solid state fermentation of babassu cake and castor meal

The main objective of this work was to optimize lipase production, in terms of hydrolytic and esterification activities, by Penicillium brevicompactum and Penicillium verrucosum in solid state fermentation using agroindustrial residues as raw material. Maxima hydrolytic activities of 48.6 and 87.7 U/g were achieved when P. brevicompactum was cultured in babassu cake and castor meal, respectively. Higher esterification activities (around 244 U/g) were achieved when P. brevicompactum was used as microorganism and babassu cake as raw material. Different experimental conditions led to these promising values, clearly showing that no correlation can be attributed between hydrolytic and esterification activities. In spite of the several applications of lipases which are capable of catalyze synthesis reactions, only few works in this subject are presented in the literature, especially when low cost raw materials are used.     

Raw Starch-digestive Glucoamylase Productivity of Protease-less Mutant from Aspergillus awamori var. kawachi

Mutation experiments were performed to decrease the protease productivity of Aspergillus awamori var. kawachi using ultraviolet light and N-methyl-N'-nitro-N-nitrosoguanidine. The selected mutant HF-15 showed reductions in protease productivity of 93%, 84% and 50% in solid wheat bran culture, shaking Medium B and wheat bran cultures, respectively, as compared with the parent. Protease-less mutant HF-15 failed to produce α-mannosidase, and N-acetyl-β-d-glucosaminidase productivity decreased by 35%. Mutant HF-15 specifically produced a high amount of raw starch-adsorbable and raw starch-digestive glucoamylase similar to GA I under all tested cultural conditions. On the contrary, high protease-producing mutant HF-10 produced a glucoamylase with very limited adsorption and digestion capacity on raw corn starch, and lower hydrolysis toward gelatinized potato starch and glycogen that was similar to GA I'.     

Improvement of ethanol yield from raw corn flour by <Emphasis Type="Italic">Rhizopus</Emphasis> sp.

A novel raw-starch-digesting glucoamylase producer, Rhizopus sp. W-08, and Saccharomyces cerevisiae Z-06 were used in a fed batch process for simultaneous saccharification and fermentation of raw corn flour. Ethanol concentration of 21% (v/v) was obtained after 48 h. The conversion efficiency of raw corn flour to ethanol was 94.5% of the theoretical ethanol yield.     

Physicochemical and microbiological characteristics of Kulek cheese made from raw and heat-treated milk

The effect of heat treatment and commercial starter culture utilization on the physicochemical and microbiological properties of Kulek cheese made from raw milk with or without starter culture and heated milk with starter culture were investigated during ripening. Titratable acidity (TA) was the highest in cheeses made from heated milk while total solids (TS), salt, and fat were the highest in cheeses made from raw milk. The heat treatment significantly decreased the counts of coliforms and Enterobacteriaceae in cheeses. At the beginning of the ripening period, cheeses manufactured from heated milk with starter exhibited significantly higher counts of lactococci and proteolytic organisms and lower counts of lactobacilli than the other cheeses. After the first day, raw milk cheeses without starter showed higher microbiological counts than the others. In fresh cheeses, Lactococcus was the main lactic acid bacterium, with Lc. lactis lactis being predominant. Lactobacillus plantarum and Lactobacillus paracasei paracasei dominated at the later stages of the ripening.