Fungal isolates from natural pectic substrates for polygalacturonase and multienzyme production

Pectin rich wastes and waste dump yard soils were screened and eighty pectinolytic fungal isolates were obtained by enrichment culturing and ruthenium red plate assay. Eight isolates with higher zones of pectin hydrolysis were selected and tested for polygalacturonase production. One isolate identified as Aspergillus awamori MTCC 9166 with highest polygalacturonase activity was tested for utilization of raw pectins for enzyme production. Polygalacturonase production was high in raw pectin sources like Orange peel (16.8 U/ml) Jack fruit rind (38 U/ml) Carrot peel (36U/ml) and Beet root peel (24U/ml). Selected Aspergillus awamori MTCC 9166 was found to be having good polygalacturonase, xylanase, cellulase and weak amylase and protease activities. This isolate with multi-enzyme production could have application for enzymes production and degradation of fruit and vegetable waste in the process of urban waste disposal.     

Optimization of conditions for the production of lignocellulolytic enzymes by Sphingobacterium sp. ksn-11 utilizing agro-wastes under submerged condition

Abstract

The present work was aimed at studying the production of lignocellulolytic enzymes, namely cellulase, xylanase, pectinase, mannanase, and laccase by a newly isolated bacterium Sphingobacterium sp. ksn-11, utilizing various agro-residues as a substrate under submerged conditions. The production of lignocellulolytic enzymes was found to be maximum at the loading of 10%(w/v) agro-residues. The enzyme secretion was enhanced by two-fold at 2?mM CaCO_3, optimum pH 7, and temperature 40°. The Field Emission Gun-Scanning Electron Microscope (FEG-SEM) results have shown the degradative effect of lignocellulases; cellulase, xylanase, mannanase, pectinase, and laccase on corn husk with 3.55?U/ml, 79.22?U/ml, 12.43?U/ml, 64.66?U/ml, and 21.12?U/ml of activity, respectively. The hydrolyzed corn husk found to be good adsorbent for polyphenols released during hydrolysis of corn husk providing suitable conditions for stability of lignocellulases. Sphingobacterium sp. ksn is proved to be a promising candidate for lignocellulolytic enzymes in view of demand for enzymes in the biofuel industry.     

Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production

In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8?g:10?g:2?g yielded the highest enzyme production of 201.6?U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5?×?10⁶ spores/mL inoculum, which gave the highest enzyme activity of 389.5?U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2?g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300?g raw cassava chips/L with cane molasses.     

Extractive cultivation of xylanase by Penicillium janthinellum in a poly(ethylene glycol)/cashew-nut tree gum aqueous two-phase system

Cultivation of the fungus Penicillium janthinellum for xylanase production was studied in a poly(ethylene glycol)/cashew-nut tree gum aqueous two-phase system, using a two-level fractional factorial design. The parameters studied were initial pH, cultivation time, type of agro-industrial residue (oat husk or corn cob), agitation, temperature, and phase-forming polymers. The xylanase produced during fermentation partitioned into the top phase. The agitation and temperature (negative), cultivation time and initial pH (positive) effects proved statistically significant for xylanase production. The highest percentage yield of the xylanase in the top and its production in the top phase, about 97% and 160.7 U/mL, were obtained in cultures of 120 h, 40 rpm, 25 degrees C, and pH 5.0.     

Improving the quality of agro-wastes by solid-state fermentation: enhanced antioxidant activities and nutritional qualities

Solid substrate fermentations of some agro-wastes, namely cocoa pod husk (CPH), cassava peel (CP), and palm kernel cake (PKC) were carried out for the production of fructosyltransferase (FTase) by a newly isolated fungal strain Rhizopus stolonifer LAU 07. The fermented substrate were studied for improved nutritional quality by determining the crude protein, crude fibre, ash and lipid contents, and antioxidant activities. The cyanide content of cassava peels was also determined. Some levels of value-addition occured as a result of the fermentation. The protein contents of the substrates increased by 33.3, 55.4, and 94.8%, while the crude fibre contents decreased by 44.5, 8.6, and 7.2% in PKC, CP, and CPH, respectively. The cyanide content of cassava peel was reduced by 90.6%. Generally, fermentation of the substrates by R. stolonifer LAU 07 increased the antioxidant activity in a DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. The IC₅₀ (mg/ml) values of the methanolic extracts (fermented/unfermented) were obtained as 7.0/14.9, 4.4/10.6, and 5.5/14.7 mg/ml for PKC, CP, and CPH, respectively. Results herein reported showed that the nutritional qualities and antioxidant activities of all the investigated solid substrates were enhanced by fungal fermentation. Thus, scope exists for microbial upgrading of these low-quality agro-wastes and development of healthy animal feed supplements.     

Production, optimization growth conditions and properties of the xylanase from Aspergillus carneus M34

Growth conditions, including incubation times, temperature, agitation rate and initial pH of medium, that affect xylanase production by Aspergillus carneus M34 were studied sequentially use the classical “change-one-factor-at-a-time” method. Our results showed that there was a similar trend between cellular xylanase activity and extracellular xylanase activity. The optimal conditions for xylanase production, different from their cell growth, were on the third day, 30 °C, 100 rpm and pH 4, respectively, in this test. Response surface methodology (RSM) was further introduced to optimize the cultivation conditions and to evaluate the significance of these factors. The optimal cultivation conditions predicted from canonical analysis of this model were achieved by incubation at 35.08 °C with an agitation rate of 111.9 rpm and an initial pH of 5.16. In addition, temperature was the most critical factor for xylanase production by A. carneus M34. Xylanase activity of 22.2 U/mL was verified using the predicted optimal conditions and confirmed the fitness and applicability of the model. The optimal temperature and pH of the crude xylanase activity was observed at 60 °C and acidic pH, respectively. Sustained xylanase activity in the crude extract was also detected over a broad range of pH from 3 to 10. Considering its higher specificity toward agricultural wastes, especially corn cob and coba husk, this strain can be used to develop low-cost media for the mass-production of xylanase.     

Discoloration of the azo dye Congo Red by manganese-dependent peroxidase from <Emphasis Type="Italic">Pleurotus sajor caju</Emphasis>

This paper describes the production of ligninolytic enzymes by the white-rot fungus Pleurotus sajor caju under solid-state fermentation conditions using a cost-effective medium consisting of agro-industrial wastes. From the different agro-industrial wastes tested (i.e. orange, banana, mango and cantaloupe peels), banana peels led to the highest manganese-dependent peroxidase (MnP) activity (6.3 U/mL on the 10 day). MnP from banana peel cultures was purified and applied to the discoloration of the azo dye Congo Red (CR). The optimum temperature, pH and enzyme concentration for maximum discoloration (i.e. 95% in 1 h) were found to be 35°C, 4.0, and 1.4 U/mL, respectively. In addition, the phytotoxicity (with respect to Sorghum vulgare and Phaseolus radiatus seeds) of CR was considerably reduced after the treatment of plant material with MnP produced by P. sajor caju. The products obtained after discoloration of CR were characterized using GC/MS as 8-amino naphthol 3-sulfonic acid, 3-hydroperoxy 8-nitrosonaphthol, p-p'-dihydroxybiphenyl. Therefore, this approach holds promise for the production and application of MnP from P. sajor caju on a larger scale.     

An extracellular α-l-arabinofuranosidase fromSclerotium rolfsii

Activity of -l-arabinofuranosidae (EC 3.2.1.55) ranged between 0.03 and 1.63 U/ml whenSclerotium rolfsii was grown in a synthetic medium containing different nitrogen and carbon sources. Agricultural and forest residues such as rice straw, corn cob, groundnut husk, and apple pomace were suitable substrates for enzyme production. Maximum activity, 4.2 U/ml, was with pre-treated corn cobs.The authors are with the National Chemical Laboratory, Division of Biochemistry, Pune 411 008, India     

Trametes sp. LS-10C固态发酵产漆酶培养基优化及其对双酚A的降解

漆酶是一种绿色高效的多酚氧化酶,在降解双酚A方面具有巨大潜力。为降低发酵产漆酶的成本及考察漆酶在双酚A降解中的能力,本研究以麸皮和柚皮为主要基质,优化了栓菌固态发酵产漆酶条件,对优化后获得的漆酶在双酚A降解中的应用进行了研究。结果表明,在培养基组分为：麸皮和柚皮粉比例为6:4（W/W）、固液比1:2.5（W/V）、铜离子2%（W/W）、蔗糖3%（W/W）、硝酸钾2%（W/W）、稻壳20%（W/W）的条件下,栓菌发酵产漆酶的酶活最高,发酵11d后,酶活可达到38.4U/gds。当双酚A初始浓度为10μg/mL时,在55℃条件下酶解140min后,双酚A基本降解完全。     

Agro-food wastes utilization by Blakeslea trispora for carotenoids production

The all-trans-β-carotene is a natural pigment used in various industrial fields (food, cosmetics, pharmaceuticals, etc) and possesses the higher provitamin A activity, in respect to other carotenoids. All-trans-β-carotene is produced industrially by chemical and biotechnological means. For β-carotene biotechnological production in industrial scale mated cultures of Blakeslea trispora, a heterothallic fungus, are mainly used. Despite the intense research for β-carotene production by B. trispora, natural substrate utilization has not been extensively studied. Solid agro-food wastes such as cabbage, watermelon husk and peach peels from northern Greece as main carbon source into submerged B. trispora cultures for carotenoids production, was examined. The media containing only the agro-food waste (2-4) gave a biomass accumulation 7.77 ± 0.4 g/L, while a reference medium 1 with glucose (10 g/L) gave 4.65 ± 0.21 g/L. In another experiments series agro-food wastes were used with corn steep liquor and thiamine (media 6-8), giving a biomass accumulation and total carotenoid volumetric production 10.2 ± 2.41 g/L and 230.49 ± 22.97 mg/L, respectively. These are the higher values reported for solid wastes so far in respect to those obtained from a synthetic medium, with higher glucose concentration of 50 g/L where the correspondent values were 9.41 ± 1.18 g/L and 45.63 mg/L respectively. The results support that B. trispora is able to utilize, almost equivalently, different origin agro-food wastes for carotenoids production. Furthermore, β-carotene percentage in all examined cases was over 76%, as it was estimated by HPLC analysis, suggesting that these agro food wastes may be used for high purity, large scale β carotene production.     

一株纤维素降解菌的分离、鉴定及对 玉米秸秆的降解特性

[目的]获得高产纤维素酶细菌菌株,探讨以氨化预处理玉米秸秆为底物时的纤维素酶产酶特性及底物降解特性,探讨纤维素酶作用机理,提高玉米秸秆利用率.[方法]用LB培养基分离并纯化菌株,羧甲基纤维素钠培养基培养、刚果红染色进行初步筛选.考察氨化预处理对底物降解率、产酶能力的影响.通过形态特征观察及16S rRNA、Biolog鉴定菌株.[结果]分离到一株高效纤维素降解菌NH11,经鉴定为枯草芽孢杆菌(Bacillus subtilis). 30℃、发酵5d时,预处理前后玉米秸秆降解率分别为14.24％和24.73％.30℃、pH 7.2时,处理组CMC酶活力峰值处为153.84 U/mL,FPA酶活力为197.24 U/mL,比未处理组分别高出11.45％和10.59％.[结论]NH11具有较高的纤维素酶产酶能力,氨化预处理能够提高菌株对玉米秸秆的降解率.该菌株在秸秆堆肥、制作食用菌培养基和制取反刍动物粗饲料方面具有很高的应用价值.     

New insulating particleboards prepared from mixture of solid wastes from tissue paper manufacturing and corn peel

New composite boards with low-thermal conductivity produced from a mixture of solid wastes from tissue paper manufacturing (solid waste TPM) and corn peel have been developed. The effects of solid waste TPM/corn peel ratio on the properties of the boards were investigated and the possibility of using recycled polystyrene packaging foam as a laminating agent to improve the quality of the boards was also evaluated. Our results show that the density of the particleboards decrease with increasing the amount of corn peel added in the mixture, leading to a decrease in thermal conductivity of the final product. In contrary, larger amount of solid waste TPM added in the mixture produced stronger boards. The lamination of recycled polystyrene on the surface of particleboards improves the mechanical properties and reduces the thickness swelling of the boards. The best improvement in mechanical properties and swelling resistance could be achieved when 15% polystyrene (w/v) was coated on the surface of the boards.     

Evaluation of the potential of cassava-based residues for biofuels production

Cassava is the third significant source of calories after rice and maize in tropical countries. The annual production of cassava crop is approximately 550 million metric tons (MMT) which generates about 350 MMT of cassava solid residues, including peel, bagasse, stem, rhizome, and leaves. Cassava peel, bagasse, stem, and rhizome can be exploited for solid, liquid and gaseous biofuels production. Biofuels production from cassava starch started in the 1970s and researchers are now extensively studying cassava residues like peel, bagasse, stem, rhizome, and leaves to unravel their applications in biofuels production. However, there are technical and economic challenges to overcome the problems existing in the production of biofuels from cassava-based residues. This review provides a comprehensive summary of the techniques used for biofuels production from various cassava-based residues.     

红曲菌利用生物柴油废液生产红曲色素

对生物柴油废液作简单处理,利用红曲茵发酵生物柴油废液中副产物甘油生产红曲色素。通过响应面方法确定最佳发酵培养基为：甘油48．49g/L,蛋白胨3．12g／L,K2HPO4·3H202．01g/L,MgSO4 0．48g／L,ZnSO4·7H2O 0．04g／L,MnSO4·H2O 0．03g／L,玉米浆13mL／L,植物油10mL／L,起始pH为6。发酵结果表明：在接种量6％（v／v）,转速140r／min,35℃的条件下发酵培养6d,红曲色素最高产量到达204U／mL。说明用生物柴油废液中的粗甘油为原料生产红曲色素是基本可行的。可望为生物柴油废液的资源化提供一条环境友好型的途径。     

Kinetics of Gibberella fujikuroi growth and gibberellic acid production by solid-state fermentation in a packed-bed column bioreactor

In this work the growth of Gibberella fujikuroi and gibberellic acid (GA3) production were studied using coffee husk and cassava bagasse as substrates in a packed-bed column bioreactor connected to a gas chromatograph for exit gas analysis. With the respirometric data, a logarithmic correlation between accumulated CO2 and biomass production was determined, and the kinetics of the fungal growth was compared for estimated and experimental data. The solid medium consisted of coffee husk (pretreated with alkali solution), mixed with cassava bagasse (7:3 dry weight basis), with a substrate initial pH of 5.2 and moisture of 77%. Cultivation was carried out in glass columns, which were packed with preinoculated substrate and with forced aeration of 0.24 L of air/[h (g of substrate)] for the first 3 days, and 0.72 L of air/[h (g of substrate)] for the remaining period. The maximum specific growth rate (microm) obtained was 0.052 h(-1) (between 24 and 48 h of fermentation). A production of 0.925 g of GA3/kg of substrate was achieved after 6 days of fermentation.     

Improvement of lipase obtaining system by orange waste-based solid-state fermentation: production,characterization and application

Abstract

Lipases are an economic important group of biocatalysts that can be produced by some fungal under solid-state fermentation. Orange wastes are source of lipases and potential substrates for lipases production. This work assessed 19 fugal strains cultivated in Citrus sinensis cv. Hamlin orange wastes (peel, frit and core) for production of lipases in order to generate compounds with antioxidant, antimicrobial and cytotoxic properties. Fifteen of those fungi grew and produced lipases, mainly the Aspergillus brasiliensis [National Institute of Quality Control (INCQS) 40036]/frit system, which showed 99.58?U/g total lipase. The substrate with the highest production of lipase was frit with 26.67 and 78.91?U/g of total lipases produced on average by the 15 microorganisms. Aspergillus niger 01/frit (33.53?U/g) and Aspergillus niger (INCQS 40015)/frit (34.76?U/g) systems showed the highest specificity values in all the herein tested synthetic substrates with 4, 12 and 16 carbons. Analysis of the fatty acid profile of hydrolysis products obtained in the most prominent systems applied to corn and sunflower oils showed: palmitic acid, linoleic acid, oleic acid, and stearic acid. These acids showed antioxidant capacity of up to 58% DPPH (2,2-diphenyl-1-pierylhydrazyl) radical reduction and antibacterial activity against Escherichia coli, Listeria monocytogenes, Pseudomonas aureginosa, Salmonella Enteritidis and Staphylococcus aureus, as well as cytotoxicity to SCC9 cells (squamous cancer cells).     

Production of Gluconobacter oxydans cells from low-cost culture medium for conversion of glycerol to dihydroxyacetone

Gluconobacter oxydans could be immobilized as a biocatalyst for the conversion of glycerol to dihydroxyacetone. To reduce the production cost, the cells were produced from agricultural byproducts. Corn meal hydrolysate and corn steep liquor were employed to replace of sorbitol and yeast extract as medium for G. oxydans cell production. The optimal medium contained 80 g/L reducing sugar, 25 g/L corn steep liquor, and 10 g/L glycerol. The cell mass was about 4.22 g/L and the glycerol dehydrogenase activity was about 5.23 U/mL. For comparison, the cell mass was about 4.0 g/L and the glycerol dehydrogenase activity was about 5.35 U/mL cultured in sorbitol and yeast extract medium. These studies shown the corn meal hydrolysate and corn steep liquor medium was similar in performance to a nutrient-rich medium, but the cost of production was only 15% of that cultured in sorbitol and yeast extract medium. It was an economical process for the production of G. oxydans cells as biocatalyst for the conversion of glycerol to dihydroxyacetone in industry.     

产木聚糖酶厌氧真菌菌株筛选及产酶培养条件研究*

从12株分离自反刍动物瘤胃及粪样的厌氧真菌中筛选到一株木聚糖酶高产菌,编号为A4,初步鉴定为Neocallimastix属菌。以稻草秸、玉米秸、花生秸、滤纸片段为发酵底物,经39℃厌氧培养,A4菌产生的木聚糖酶活分别为14.31U/mL、11.39U/mL、6.99U/mL和13.38U/mL。对A4菌产生木聚糖酶的条件进行优化,结果发现,培养基中无细胞瘤胃液浓度对A4菌产生的木聚糖酶活无显著影响;但酵母膏浓度从1.0g/L降至0.5g/L后,A4菌产生的木聚糖酶活显著下降(P<0.05)。     

Galactomyces geotrichum Y25产脂肪酶条件的优化

应用响应面法对Galactomyces geotrichumY25液体发酵产脂肪酶的条件进行了优化。首先采用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出黄豆粉、玉米浆和发酵时间3个对产酶影响显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面设计对显著因素进行优化,得出黄豆粉、玉米浆最佳质量分数分别为2.51%、2.12%,最佳发酵时间101.95 h。优化后液体发酵液中脂肪酶活力提高到34.65 U/mL,比初始酶活力9.6 U/mL提高了3.61倍。表明响应面法可显著优化Galactomyces geotrichumY25液体发酵产脂肪酶条件。     

Fermentation optimization and industrialization of recombinant Saccharopolyspora erythraea strains for improved erythromycin a production

Improvement of Erythromycin A (Er-A) production and purity by metabolic engineering of the industrial erythromycin-producing strains Saccharopolyspora erythraea strians ZL1004 and ZL1007, in which the amounts of tailoring enzymes EryK (a P450 hydroxylase) and EryG (an S-adenosylmethionine-dependent O-methyltransferase) for biotransformation of Erythromycin D to Er-A were modulated, was performed in a 50 L fermentor. Addition of 15 g/L of corn steep liquor to the medium increased Er-A production; maximum Er-A production was 8,196 U/mL at 191 h, which was 81.8% higher than that of control (4,507 U/mL at 184 h). Er-B impurities were completely eliminated, whereas Er-C impurities were only 153 U/mL at 191 h. Analysis of intra- and extracellular metabolites and key enzyme activities in central carbon metabolism revealed that the pool of TCA cycle intermediates was enhanced by the addition of corn steep liquor and induced an increase in erythromycin biosynthesis. There were no significant differences between strains ZL1004 and ZL1007 regarding Er-A production and impurity accumulation. Compared to wild type strain, Er-A production was improved by 23.9% while Er-C was reduced by 83.9% and Er-B was completely eliminated. Furthermore, fermentation of recombinant strain ZL1004 was successfully scaled up from laboratory scale (50 L fermentor) to industrial scale (25 and 132 m³), with similar levels of Er-A production and purity obtained.