Studies on influence of natural biowastes on cellulase production by Aspergillus niger

The objective of this study was to determine the influence of natural biowaste substrates such as banana peel powder and coir powder at varying environmental parameters of pH (4-9) and temperature (20-50 degrees C) on the cellulase enzyme production by Aspergillus niger. The cellulase enzyme production was analyzed by measuring the amount of glucose liberated in IU ml(-1) by using the dinitrosalicylic acid assay method. The substrates were pretreated with 1% NaOH (alkaline treatment) and autoclaved. The maximum activity of the enzyme was assayed at varying pH with temperatures being constant and varying temperatures with pH being constant. The highest activity of the enzyme at varying pH was recorded at pH 6 for banana peel powder (0.068 +/- 0.002 IU ml) and coir powder (0.049 +/- 0.002 IU ml(-1)) and the maximum activity of the enzyme at varying temperature was recorded at 35 degrees C for both banana peel powder (0.072 +/- 0.001 IU ml(-1)) and coir powder (0.046 +/- 0.003 IU ml(-1)). At varying temperatures and pH the high level of enzyme production was obtained at 35 degrees C and pH 6 by using both the substrates, respectively. However among the two substrates used for the production of cellulases by Aspergillus niger banana peel powder showed maximum enzymatic activity than coir powder as substrate.     

黑曲霉耐热木聚糖酶XYNB在毕赤酵母中的高效表达

高比活木聚糖酶的高效表达是进一步提高木聚糖酶发酵效价、降低生产成本的有效途径.将黑曲霉木聚糖酶基因XynB(不含信号肽)克隆到分泌型表达载体pPIC9K上,线性化后电击转化巴斯德毕赤酵母GS115,G418和PCR鉴定的阳性转化子经0.5%甲醇、在28℃诱导表达.SDS-PAGE分析表明,该蛋白相对分子质量为20×103左右.优化的诱导表达条件为,每隔12 h添加0.5%的甲醇,发酵5 d后,比活达4 757 U/mg;其最适温度为55℃,最适pH为5.0,80℃处理30min后仍有74%的残余酶活.     

生淀粉糖化酶产生菌Aspergillus niger(6#)的分离筛选及其产酶条件

从大曲中分离到了1株降解生淀粉能力较强的黑曲霉Aspergillusniger(6#).固体发酵酶活可达2461U/g,RDA值为21.47%;液体发酵酶活可达353U/mL,RDA值为20.30%.以6#菌为实验材料,进一步考察了氮源、碳源及pH对生淀粉糖化酶形成的影响.实验结果表明,无机氮源NaNO3较有机氮源蛋白胨更有利于生淀粉糖化酶的形成;pH是影响生淀粉糖化酶形成的重要因素,低pH会阻遏生淀粉糖化酶的形成;玉米粉和麦芽糖较其它碳源更有利于生淀粉糖化酶的形成.图4表2参12     

Utilization of banana agricultural waste: production of cellulases by soil fungi

Banana a major cash crop of Maharashtra is cultivated over 46900 hectares generating large amount of agro waste after the harvest. Attempts were made to utilize these agro wastes for production of cellulases. Of the 127 fungi isolated from the soil of banana field, 12 fungi were found to utilize cellulose as source of carbon. Trichoderma lignorum showed appreciable cellulolytic activity. It produced Cl, Cx and beta glucosidase in Carboxymethyl Cellulose Peptone medium as well as on agro waste based medium containing leaves, stem and rhizome powders. T. lignorum (0. 45 U/ml) produced maximum enzymes on leaf based medium.     

木聚糖酶基因xynB在不同大肠杆菌表达系统中的表达比较

从黑曲霉(Aspergillus niger)nl-1中克隆获得木聚糖酶基因xynB.该基因全长745 bp,含有67 bp内含子,与公布的黑曲霉xynB基因有较高的同源性.将PCR扩增的木聚糖酶成熟肽基因和含有信号肽的基因分别连接到表达载体肠杆菌Top10 F’、DH10B和两种BL21宿主中获得重组菌株.通过IPTG诱导,xynB基因在重组菌株中获得特异性表达.表达产物以胞内可溶性蛋白和不溶性包涵体形式存在.诱导4 h,重组菌株pTrc-99a-xynB[BL21 condon plus(DE3)]表达量最高,胞内酶活达到299 IU/L.重组质粒pTrc-99a-xyn B(S)在不同宿主胞内和胞外均能分泌目的蛋白,诱导10 h,胞外酶活pTrc-99a-xynB(S)[BL21 condon plus(DE3)]达到347 IU/L.而pET-20b-xynB(S)在两种BL21宿主中均不表达.经SDS-PAGE分析,以pTrc-99a和pET-20b作为表达载体时,重组蛋白相对分子质量分别约为45×103和30×103.与pET-20b相比,pTrc-99a以及自身信号肽的引导更有利于重组木聚糖酶的表达.     

Insights into the changes of amino acids,microbial community,and enzymatic activities related with the nutrient quality of product during the composting of food waste

● The highest seed germination index was achieved at 0.3 g/g total solids of food waste. ● Proline was identified as the key amino acid related with the composting process. ● Amino acid metabolism sequences predominated during the whole composting process. This study systematically investigated the changes of amino acids as the composting process of food waste proceeded. It is found that the addition of 0.3 g/g total solids of food waste achieved the highest seed germination index of the product (268 %). The microbial community results indicated that the abundance of amino acid metabolism sequences remained at high levels during the whole composting process. Proline was identified as the key amino acid related with the nutrient quality of product during the composting of food waste. Further plant germination and hydroponic experiments found, that compared with those without the addition of proline, the addition of 50 mg/L proline increased seed germination rate by 20 %, increased shoot length by 3 %, increased root biomass of seedlings by 82 %, and increased leaf biomass of seedlings by 76 %, respectively. Firmicutes, γ-Pseudomonadota, Chloroflexi and Planctomycetes were the key identified bacteria related with the increase of proline during the composting of food waste. Meanwhile, the enzymatic tests of the activities of superoxide dismutase, peroxidase and malondialdehyde indicated that proline did not cause oxidative damage on the growth of plants. This study provided novel insights into the changes of amino acids, microbial community, and enzymatic activities related with the nutrient quality of product during the composting of food waste.     

Biodegradation of crude petroleum and petroleum products by fungi isolated from two oil seeds (melon and soybean)

Crude petroleum oil degrading fungi were isolated from two oil seeds, Cucumeropsis mannii (melon) and Glycine max (soybean) seeds in the presence and absence of petroleum fumes. An assessment of the relative ability of each fungus to degrade crude petroleum, diesel and kerosene on minimum salt solution was done using change in optical density read on spectrophotometer. Twenty-one fungal species (14 genera) were isolated altogether during this experiment. These include eight species of Aspergillus; one species each of Botryodiplodia, Bipolaris, Cladosporium, Cunnighamella, Dreschlera, Fusarium, Helminthosporium, Macrophomina, Mucor, Paeciliomyces, Penicillium, Rhizopus and Talariomyces. It was evident that most of the fungi tested were able to biodegrade the crude petroleum oil, though at different rates. Bipolaris had a low rate of biodegradation of the petroleum oil of all the fungal species isolated Botryodiplodia theobromae had the highest degrading ability on the crude oil, while Aspergillus flavus had the least after 40 days of incubation. Aspergillus flavus had the highest ability to biodegrade diesel while A. niger had the least ability. In kerosene, Macrophomina phaseolina had the highest ability while A. niger had the least ability to biodegrade it. There was fluctuation in the growth pattern of the fungi in the petroleum oil medium. The implication of these are discussed.     

Effect of phosphogypsum amendment on soil physico-chemical properties, microbial load and enzyme activities

Phosphogypsum (PG) is produced as a solid waste from phosphatic fertilizer plants. The waste slurry is disposed off in settling ponds or in heaps. This solid waste is now increasingly being used as a calcium supplement in agriculture. This study reports the effectof PG amendmenton soil physico chemical properties, bacterial and fungal count and activities of soil enzymes such as invertase, cellulase and amylase over an incubation period of 28 days. The highest mean percent carbon loss (55.98%) was recorded in 15% PG amended soil followed by (55.28%) in 10% PG amended soil and the minimum (1.68%) in control soil. The highest number of bacterial colonies (47.4 CFU g(-1) soil), fungal count (17.8 CFU g(-1) soil), highest amylase activity (38.4 microg g(-1) soil hr(-1)) and cellulase activity (38.37 microg g(-1) soil hr(-1)) were recorded in 10% amended soil. Statistically significant difference (p<0.05) has been recorded in the activities of amylase and cellulase over the period of incubation irrespective of amendments. Considering the bacterial and fungal growth and the activities of the three soil enzymes in the control and amended sets, it appears that 10% PG amendment is optimal for microbial growth and soil enzyme activities.     

Radionuclide concentrations in raw and purified phosphoric acids from Brazil and their processing wastes: implications for radiation exposures

Radionuclides from the U and Th natural series are present in alkaline rocks, which are used as feedstock in Brazil for the production of raw phosphoric acid, which can be considered as a NORM (naturally occurring radioactive material). As a result of the purification of raw phosphoric acid to food-grade phosphoric acid, two by-products are generated, i.e., solid and liquid wastes. Taking this into account, the main aim of this study was to evaluate the fluxes of natural radionuclide in the production of food-grade phosphoric acids in Brazil, to determine the radiological impact caused by ingestion of food-grade phosphoric acid, and to evaluate the solid waste environmental hazards caused by its application in crop soils. Radiological characterization of raw phosphoric acid, food-grade phosphoric acid, solid waste, and liquid waste was performed by alpha and gamma spectrometry. The ²³⁸U, ²³⁴U, ²²⁶Ra, and ²³²Th activity concentrations varied depending on the source of raw phosphoric acid. Decreasing radionuclides activity concentrations in raw phosphoric acids used by the producer of the purified phosphoric acid were observed as follows: Tapira (raw phosphoric acid D) > Catal?o (raw phosphoric acids B and C) > Cajati (raw phosphoric acid A). The industrial purification process produces a reduction in radionuclide activity concentrations in food-grade phosphoric acid in relation to raw phosphoric acid produced in plant D and single raw phosphoric acid used in recent years. The most common use of food-grade phosphoric acid is in cola soft drinks, with an average consumption in Brazil of 72 l per person per year. Each liter of cola soft drink contains 0.5 ml of food-grade phosphoric acid, which gives an annual average intake of 36 ml of food-grade phosphoric acid per person. Under these conditions, radionuclide intake through consumption of food-grade phosphoric acid per year per person via cola soft drinks is not hazardous to human health in Brazil. Considering these annual additions of ²³⁸U, ²²⁶Ra, ²³²Th and ⁴⁰K, and since these radionuclide should be homogeneously distributed in the upper 10 cm of soils with an assumed apparent density of 1.5 g/cm³, a maximum increase of 0.19 ± 0.03 Bq kg⁻¹ of soil is expected for ²³⁸U and ²³⁴U. Thus, the addition of solid waste as phosphate fertilizers to Brazilian agricultural soils does not represent a hazard to the ecosystem or to human health.     

Growth and amylase production by Aspergillus oryzae during solid state fermentation using banana waste as substrate

Aspergillus oryzae, isolated from the starch rich litter soil, produced amylase when banana fruit stalk was used as substrate in a solid state fermentation system. The effects of incubation period, pH, temperature and various carbon sources on the production of amylase were studied. A maximum yield of 380U/mg was recorded on 96th hour of incubation. The amylase is tolerant to wide range of initial culture pH values (3 to 8) and temperature (25 to 35 degrees C), with an optimum pH of 5 and temperature of 35 degrees C. In SSF addition of starch (1%) increased the amylase production, when compared with other carbon sources used.     

三相鼓泡塔生物反应器培养黄孢原毛平革菌合成木素过氧化物酶系

利用三相鼓泡塔反应器固定化培养黄孢原毛平革菌，可以高效地合成木素过氧化物酶系，固定化载体为聚氨酯泡沫塑料．实验表明，合成木素过氧化物酶和锰过氧化物酶的最佳通气量均是1．0vvm。在此通气量下，最大木素过氧化物酶的酶活达367U/L，最大锰过氧化物酶的酶活达4．72U／mL。在使用相同的培养基和固定化载体单位体积用量条件下，与摇瓶培养相比，酶活分别增大1倍和1．2倍．一定条件下，在三相鼓泡塔中可以进行重复间歇培养生产木素过氧化物酶，连续进行了5批培养，每批最大木素过氧化物酶的活力均在250U／L以上，最高酶活出现在第二批为480U／L，总培养时间达22d．图9参15     

Extracellular amylase(s) production by fungi Botryodiplodia theobromae and Rhizopus oryzae grown on cassava starch residue

The fungi Botryodiplodia theobromae and Rhizopus oryzae produce extracellular amylase when grown on a liquid medium containing 2% (WN) soluble starch or cassava starch residue(CSR) (as starch equivalent), a waste generated after extraction of starch from cassava, as the sole carbon source. Using CSR as the sole carbon source, the highest amylase activity of 3.25 and 3.8 units (mg, glucose released x ml(-1) x h(-1)) were obtained in shake flask cultures during the late stationary phase of growth of B. theobromae and R. oryzae, respectively. These values were slightly lower than the values obtained using soluble starch as the carbon source. Maximum enzyme synthesis in CSR incorporated medium occurred at the growth temperature of 30 degrees C and pH 6.0. Presence of inorganic NH4+ salts like ammonium acetate and ammonium nitrate in culture medium yielded more amylase than the other nitrogen sources. Amylase(s) production in the controlled environment of a Table-Top glass Jar Fermenter (2-L capacity) was 4.8 and 5.1 units for B. theobromae and R. oryzae, respectively using CSR as the carbon substrate. It is concluded that CSR, a cheap agricultural waste obtained after starch extraction from cassava could replace soluble starch as carbon substrate for commercial production of fungal amylase(s).     

Antimicrobial activity of methanol extract of Origanum majorana L. (Sweet marjoram)

In-vitro microbicidal activity of the methanol extract of Origanum majorana L. was tested against seven fungi (Fusarium solani, Candida albicans, Aspergillus niger, A. parasiticus, Rhizopus oryzae, Rhizoctonia otyzae-sativae and Altemaria brassicicola) and six bacteria (Bacillus subtilis, B. megaterium, Escherichia coil, Proteus vulgaris, Pseudomonas aeruginosa and Staphylococcus aureus). The methanol extract of O. majorana can be used as an effective herbal protectant against different pathogenic bacteria and fungi. High toxicity against the growth of Aspergillus niger was diagnosed.     

Chromium (VI) biosorption by immobilized Aspergillus niger in continuous flow system with special reference to FTIR analysis

Aspergillus niger was treated with acid and immobilized in calcium alginate matrix. The dynamic removal of Cr (VI) ion was studied using continuously fed column packed with immobilized biosorbent beads. Column experiments were carried out to study the effect of various bed heights (20, 30, 40 cm) under different flow rates (5, 7.5, 10 ml min(-1)) on efficiency of biosorption. The maximum time (1020 minutes; 17 hr) before breakthrough point was observed in case of 40 cm bed height with flow rate of 5ml min(-1). FTIR analysis of acid treated immobilized A. niger was used fora qualitative and preliminary analysis of chemical functional groups present on its cell wall which provided the information on nature of cell wall and Cr (VI) interaction during the process of biosorption. The IR spectra of biosorbent recorded before and after chromium biosorption had shown some changes in the band patterns, which were finally analyzed and was found that chemical interaction such as ion-exchange between carboxyl (-COOH), hydroxyl (-OH) and amine (-NH2) group of biosorbent and Chromium ion were mainlyinvolved in biosorption of Cr (VI) onto A. niger cell wall surface. The biosorbed metal was eluted from biosorbent by using 0.1 M H2SO4 as eluant. Immobilized biosorbent could be reused for five consecutive biosorption and desorption cycles without apparent loss of efficiency after its reconditioning. Considering all above factors together this paper discusses the efficient chromium biosorption process carried out by immobilized A. niger biosorbent.     

黑胸散白蚁纤维素酶的体外酶学特性

采用DNS法研究了我国广泛分布的一种低等木食性白蚁——黑胸散白蚁纤维素酶的体外酶活特性以了解其纤维素降解机制.结果表明,内切β-1,4-葡聚糖酶(EG)、纤维二糖水解酶(CBH)和β-葡萄糖苷酶(BG)这3种酶的最佳反应时间均为15 min,最佳底物浓度为1%,最适反应pH为5.6,最适反应温度为35℃.在最适反应条件下,EG、CBH和BG的活性分别达到71.3(±13.9)U/mg、5.8(±0.8)U/mg和4.1(±0.7)U/mg.EG在体外的热稳定性较差,在50℃及更高温度酶活很低或完全失活,但该酶对pH稳定性较好,在pH 3.2～8.0范围内酶活力变化不大.Native-PAGE电泳检测到该白蚁体内至少有8种不同的EG活性条带,肠道不同部位纤维素酶活性条带种类不同.这些研究表明,木食性白蚁降解纤维素是一个复杂的过程,需要多种纤维素酶的共同作用.     

Studies on in vitro degradability of mixed crude enzyme extracts produced from Pleurotus spp

A preliminary investigation was conducted to assess lignocellulolytic efficiency of crude extracts from three white-rot fungi, Pleurotus florida PF05 (PF), Pleurotus sajor-caju PS07 (PS) and Pleurotus eryngii PE08 (PE). The activities of CMC-ase, xylanase, beta-glucosidase, beta-xylosidase, laccase and Mn peroxidase in extracts were evaluated. PF produced its highest CMC-ase (317 UL(-1)'), beta-glucosidase (62 UL(-1)), beta-xylosidase (37 UL(-1)) and laccase (347 UL(-1)) activities while, PS produced highest xylanase (269 UL-(1)) and Mn peroxidase (69 UL(-1)) activities. In addition, crude extracts extracted were employed for their in vitro degradability assessment; and were evaluated with mono and mixed extracts separately to corn cob substrate. The losses in cell wall components and dry matter during 5 and 10 days incubations were analyzed after treatments of extracts. Maximum 8.2, 4.4 and 2.8% loss were found respectivelyin hemicellulose (HC), cellulose (C) and lignin (L) with mono extract of PF within 10 days. The influence of mono extract of each strain (PF PS and PE) and their mixed extracts (PF+PS, PF+PE, PS+PE and PF+PS+PE) on degradation of cell wall constituents were remarkably differed. The mixed extract treatment proved maximum 13.6% HC loss by PF+PS+PE extract, 9.2% loss in C by PF+PS extract and 5.2% loss of L by the PF+PS+PE extract treatment. The highest dry matter loss (8.2%) was recorded with PF+PS+PE mixed extract combination.     

Impact of Ni(II), Zn(II) and Cd(II) on biogassification of potato waste

A study was conducted on anaerobic digestion of potato waste and cattle manure mixture, inoculated with 12% inoculum and diluted to 1:1 substrate water ratio at 37 +/- 1 degrees C. Initially pH of substrate was found to be 4.5 to 5.0. Lime and sodium bicarbonate solutions were employed to adjust the pH to 7.5. Biogas production continued up to 10 and 7 days, when lime and sodium bicarbonate solutions were used to adjust the pH, respectively. Biogassification potential was studied in response to different ratio of waste and cattle manure. Biogas production rate was higher when potato waste and cattle manure were used in 50:50 ratio. Effect of two different concentrations (2.5 and 5.0 ppm) of three heavy metals viz. (Ni (II), Zn (II) and Cd (II)) on anaerobic digestion of substrate (potato waste--cattle manure, 50:50) was studied. At 2.5 ppm, all the three heavy metals increased biogas production rate over the control value. The percentage increase in biogas production over the control was highest by Cd, followed by Ni and Zn. In all the treatments, methane content of biogas increased with increase in time after feeding. Various physico-chemical parameters viz. total solids, total volatile solids, total organic carbon and chemical oxygen demand considerably declined after 7 days of digestion and decline was greater in presence of heavy metals as compared to control. The physico-chemical parameters revealed maximum decrease in the presence of 2.5-ppm concentrations of heavy metals with the substrate. Among all the three heavy metals employed in the study, Cd++ at 2.5 ppm was found to produce maximum biogas production rate. The use of three heavy metals to enhance biogas production from potato and other horticultural waste is discussed.     

Pharmacological evaluation of crude methanolic extract of Kalanchoe pinnata leaves

Medicinal plants play a crucial role in maintaining human health. Kalanchoe pinnata has been used for treatment of different ailments like infections, rheumatism, and inflammation. Data showed that Kalancho pinnata methanolic extract (100 or 1000 μg/ml) significantly inhibited growth of shoots (hypocotyls) and roots (radicals) of rice compared to control after 3 or 7 days treatment. Further, the Kalancho pinnata methanolic extract (50–1000 μg/ml) exhibited antioxidant properties and scavenged free radicals in dose-dependent manner compared with a standard antioxidant (ascorbic acid). Kalancho pinnata methanolic extract also possesses antifungal property that inhibited approximately 76% and 51% growth of Aspergillus flavous and Aspergillus niger, respectively, using the extract at 200 μg/ml.     

Influence of glucose feeding on the ligninolytic enzyme production of the white-rot fungus Phanerochaete chrysosporium

The present work studied the influence of glucose feeding on the ligninolytic enzyme production of Phanerochaete chrysosporium in a nitrogen-limited (C/N ratio is 56/8.8 mmol/L) medium. Several sets of shaking flask experiments were conducted. The results showed that 2 g/L glucose feeding on the first day of the culture (24 h after the inoculation) simulated both fungal biomass growth and enzyme production. The manganese peroxidase (MnP) activity was 2.5 times greater than that produced in cultures without glucose feeding. Furthermore, the glucose feeding mode in fed-batch culture was also investigated. Compared to cultures with glucose feeding every 48 h, cultures with glucose feeding of 1.5 g/L (final concentration) every 24 h produced more enzymes. The peak and total yield of MnP activity were 2.7 and 3 times greater compared to the contrast culture, respectively, and the enzyme was kept stable for 4 days with an activity of over 200 U/L.     

复合垂直流人工湿地基质氧化还原酶活性研究

对复合垂直流人工湿地系统(IVCW)基质中5种常见的氧化还原酶活性进行了研究.结果表明,氧化还原酶活性存在显著性季节差异,过氧化物酶在春、夏、秋三季的酶活性显著高于冬季的酶活性(P＜0.05);多酚氧化酶和过氧化氢酶的活性最高在秋季;脱氢酶在夏、冬两季时酶活性显著高于秋季和春季的酶活性(P＜0.05);硝酸盐还原酶在春、冬季时显著高于夏、秋季(P＜0.01).空间分布上,除过氧化物酶外,湿地下行流池的酶活性显著高于上行流池,并且随着基质深度的增加递减;但基质硝酸盐还原酶各层之间差异不显著.图6表3参17