Growth enhancement of effective microorganisms for bioremediation of crude oil contaminated waters

The bioremediation of polluted groundwater, wastewater aeration pond and biopond sites was investigated using bacteria isolated from these sites located at the oil refinery Terengganu Malaysia. Out of 62 isolates, only 16 isolates from groundwater (8) and wastewater aeration pond (3) and biopond (5) were chosen based on growth medium containing 1% (v/v) Tapis crude oil. Only four isolates; Acinetobacter faecalis, Staphylococcus sp., Pseudomonas putida and Neisseria elongata showed percentage biodegradation of crude oil more than 50% after 5 days using Mineral Salts Medium (MSM). The effect of physical parameters (temperature, pH and agitation) on growth by all four strains showed a maximum growth in MSM medium with 1% Tapis crude oil at 37 degrees C with pH 7 and agitation of 130 rpm.     

Production and characterization of a mesophilic lipase isolated from Bacillus stearothermophilus AB-1

Using Bacillus stearothermophilus AB-1 isolated from air, the production of lipase was attempted along with its purification and characterization studies. When different carbon and nitrogen sources were supplemented in the culture medium, xylose, tryptophan, alanine, phenylalanine and potassium nitrate were found to be the best. During cultivation, the strain secreted most of its lipase content after 48 h. In particular, the lipase produced in the culture broth showed 300 U mL(-1) when cultivated at optimal temperature and pH of 35 degrees C and 7.5, respectively. The enzyme was purified using 60% ammonium sulfate precipitation and sephadex G200 column chromatography. The enzyme was stable up to 40 degrees C and in the range of pH 7-8. This research reports for the first time the characterization of mesophilic lipase from Bacillus stearothermophilus AB-1 isolated from air.     

<Emphasis Type="Italic">Carica papaya</Emphasis> Lipase: A Naturally Immobilized Enzyme with Interesting Biochemical Properties

Triacylglycerol (TAG) lipases have been thoroughly characterized in mammals and microorganisms, whereas very little is known about plant TAG lipases. The lipolytic activity occurring in all the laticies is known to be associated with sedimentable particles, and all attempts to solubilize the lipolytic activity of Carica papaya latex have been unsuccessful so far. However, some of the biochemical properties of the lipase from Carica papaya latex (CPL) were determined from the insoluble fraction of the latex. The activity was optimum at a temperature of 37°C and a pH of 9.0, and the specific activities of CPL were found to be 2,000 ± 185 and 256 ± 8 U/g when tributyrin and olive oil were used as substrates, respectively. CPL was found to be active in the absence of any detergent, whereas many lipases require detergent to prevent the occurrence of interfacial denaturation. CPL was inactive in the presence of micellar concentrations of Triton X-100, sodium dodecyl sulfate (SDS) and tetradecyl trimethylammonium bromide (TTAB), and still showed high levels of activity in the presence of sodium taurodeoxycholate (NaTDC) and the zwitterionic Chaps detergent. The effects of various proteases on the lipolytic activity of CPL were studied, and CPL was found to be resistant to treatment with various enzymes, except in the presence of trypsin. All these properties suggest that CPL may be a good candidate for various biotechnological applications.     

Effects of lipase on poly(lactic acid) fibers

Two types of lipases, L3126 and Lipex 100L, were used to modify the surface of poly(lactic acid) (PLA) fiber by measuring weight loss percentage and wettability of the fiber in this work. The influence factors were discussed and optimized based on single-factor experiments. The optimal conditions for the modification of poly(lactic acid) fiber with lipases were determined as follows: incubation with lipase L3126 of 0.5 g/l at 45 °C and pH 8.5 for 8 hours and incubation with lipase of 10 ml/l at 40 °C and pH 7.5 for 10 hours. Lipase L3126 showed higher biodegradation ability to poly(lactic acid) fiber than lipase Lipex 100L. The scanning electron microscopy confirmed that both of the two lipases could lead to the formation of etching characters on treated poly(lactic acid) fibers in comparison with the blank samples. Furthermore, the wettability of the fibers treated with the lipases was evidently improved, especially Lipase L3126.     

Production and partial characterization of uric acid degrading enzyme from new source Saccharopolyspora sp. PNR11

The strain PNR11 was isolated from gut of termite during the screening for uric acid degrading actinomyces. This strain was able to produce an intracellular uricase when cultured in fermentation medium containing uric acid as nitrogen source. Base on its morphological characters and 16S rDNA sequence analysis, this strain belong to the genus Saccharopolyspora. This is the first report ofuricase produced from the genus Saccharopolyspora. The aim of this study was to investigate the effects of different factors on uricase production by new source of Saccharopolyspora. Saccharopolyspora sp. PNR11 was cultured in production medium in order to determine the best cultivation period. The result showed that the time period required for maximum enzyme production was 24 h on a rotary shaker operating at 180 rpm. Optimized composition of the production medium consisted of 1% yeast extract, 1% maltose, 0.1% K2HPO4, 0.05% MgSO4 7H2O, 0.05% NaCl and 1% uric acid. The optimum pH and temperature for uricase production in the optimized medium were pH 7.0 and 30 degrees C, respectively. When the strain was cultured at optimized condition, the uricase activity reached to 216 mU mL(-1) in confidential level of 95%. The crude enzyme had an optimum temperature of uricase was 37 degrees C and it was stable up to 30 degrees C at pH 8.5. The optimum pH ofuricase was 8.5 and was stable in range of pH 7.0-10.0 at 4 degrees C. This strain might be considered as a candidate source for uricase production in the further studies. Present finding could be fulfill the information ofuricase produce from actinomycetes.     

Isolation and identification of a lipase producing Bacillus sp. from soil

Lipase production in an indigenous lipolytic Bacillus sp. was detected in media containing Tributyrin-Tween 80 and Rhodamine B-Olive oil. The statistical Taguchi model was used to predict the optimum experimental conditions for bacterial growth and lipase production. Partial optimization was carried out for selection of salt base, oil, glucose, NH4Cl and yeast extract concentrations, inoculum density, pH and agitation. Maximum lipase activity was detected in the cell free supernatants of cultures grown in a medium containing 10 g L(-1) yeast extract, 15 g L(-1) NH4Cl, 3 g L(-1) K2HPO4, 1 g L(-1) KH2PO4, 0.1 g L(-1) MgSO4 x 7H2O, 2 g L(-1) glucose, 0.6 mM MgCl2 and 15 ml L(-1) olive oil, pH 8.5 at 30 degrees C for 24 h and low agitation. The amount oflipase produced in the designed medium was in agreement with the predicted values by the statistical method. 16S rRNA cloning and sequencing identified the test organism as Bacillus pumilus.     

Chemical and Physical Culture Conditions Significantly Influence the Cell Mass and Docosahexaenoic Acid Content of Aurantiochytrium limacinum Strain PKU#SW8

Thraustochytrids are well-known unicellular heterotrophic marine protists because of their promising ability to accumulate docosahexaenoic acid (DHA). However, the implications of their unique genomic and metabolic features on DHA production remain poorly understood. Here, the effects of chemical and physical culture conditions on the cell mass and DHA production were investigated for a unique thraustochytrid strain, PKU#SW8, isolated from the seawater of Pearl River Estuary. All the tested fermentation parameters showed a significant influence on the cell mass and concentration and yield of DHA. The addition of monosaccharides (fructose, mannose, glucose, or galactose) or glycerol to the culture medium yielded much higher cell mass and DHA concentrations than that of disaccharides and starch. Similarly, organic nitrogen sources (peptone, yeast extract, tryptone, and sodium glutamate) proved to be beneficial in achieving a higher cell mass and DHA concentration. PKU#SW8 was found to grow and accumulate a considerable amount of DHA over wide ranges of KH₂PO₄ (0.125–1.0 g/L), salinity (0–140% seawater), pH (3–9), temperature (16–36 °C), and agitation (140–230 rpm). With the optimal culture conditions (glycerol, 20 g/L; peptone, 2.5 g/L; 80% seawater; pH 4.0; 28 °C; and 200 rpm) determined based on the shake-flask experiments, the cell mass and concentration and yield of DHA were improved up to 7.5 ± 0.05 g/L, 2.14 ± 0.03 g/L, and 282.9 ± 3.0 mg/g, respectively, on a 5-L scale fermentation. This study provides valuable information about the fermentation conditions of the PKU#SW8 strain and its unique physiological features, which could be beneficial for strain development and large-scale DHA production.     

Isolation of a novel mutated strain of Xanthomonas campestris for xanthan production using whey as the sole substrate

This study was conducted to isolate novel lactose utilizing Xanthomonas campestris mutants. Such a mutant will assist the utilization of whey as the sole carbon source for xanthan gum production, lower costs of fermentation process and set a precise application for whey as a waste. In this study, a mutant strain (NA1) was isolated from Xanthomonas campestris cells exposed to nitrous acid mutagenesis Environmental conditions were optimized and maximum activity of the beta-galactosidase enzyme was obtained at pH 5.5 and 38 degrees C following which the beta-galactosidase activity in NA1 culture was increased 9.5 folds, compared to that of the wild type culture (336.1 U vs. 35.4 U). Xanthan gum production by NA1 using whey as carbon source was also studied. Using the experimental design of Plackett-Burman and statistical analysis, whey, as the main substrate and pH were the first factors affecting gum production among the seven parameters tested. Gum production using significant factors (such as substrate concentration and pH) was carried out in a lab-scale fermentor and 10 g L(-l) xanthan was obtained.     

Extraction of oil from Jatropha curcas L. seed kernels by enzyme assisted three phase partitioning

Three phase partitioning has been evaluated for extraction of oil from Jatropha curcas L. seeds. This process consisted of simultaneous addition of t-butanol (1:1, v/v) and 30% (w/v) ammonium sulphate to the slurry prepared from Jatropha seed kernels. Combination of sonication and enzyme treatment with a commercial preparation of fungal proteases at pH 9, led to 97% oil yield within 2 h.     

Moderate quantity of lard mixed with sunflower oil attenuate lipid accumulation in mice

Lard, a fat rich in saturated fatty acids (SFAs), is regarded as a risk factor for metabolic diseases. In the present study, effect of different lard blended with sunflower oil diets on lipid accumulation in adipose tissue, liver, and serum by mouse model was researched. Body weight, body fat percentage, cross-sectional area of adipocytes, liver triglycerides (TGs), and oil red stained area in mice liver of lard blend sunflower oil (L-SFO) group were significantly lower than those of sunflower oil (SFO) group, whereas no significant differences were observed between mice of lard and L-SFO groups. Serum TG and free fatty acid levels were significantly lower in L-SFO group than in other two groups. Furthermore, data showed that sunflower oil decreased contents of hormone-sensitive lipase and carnitine palmitoyl transferase 1 (CPT-1) and increased fatty acid synthase activity in liver tissue. A mixture of lard and sunflower oil rather than only sunflower oil or lard might promote body fat loss and reduce lipid accumulation in adipose tissue, serum, and liver by promoting hydrolysis of TG, increasing β-oxidation of fatty acids. These data suggested that mixing lard and vegetable oil (e.g. sunflower oil) for cooking, or alternate using lard and vegetable oil could be beneficial for reducing body fat.     

Impact of lipases with different substrate specificity in wheat flour separation on the properties of the resultant gluten

Wheat gluten was isolated in a laboratory dough-batter flour separation process in the presence or absence of lipases differing in hydrolysis specificity. The obtained gluten was blended with wheat starch to obtain gluten-starch (GS) blends of which the water and oil binding capacities were investigated. Furthermore, GS blends were mixed into dough and processed into model breads, of which dough extensibility and loaf volume were measured, respectively. In comparison to GS blends prepared with control gluten, oil binding capacity was higher when GS blends contained gluten isolated with Lecitase Ultra (at 5.0 mg enzyme protein/kg flour), a lipase hydrolyzing both non-polar and polar lipids. Additionally, dough extensibility and total work needed for fracture were lower for dough prepared from GS blends containing gluten isolated with Lipolase (at 5.0 mg enzyme protein/kg flour), a lipase selectively degrading non-polar lipids. In GS blend bread making, this resulted in inferior loaf volumes. Comparable GS blend properties were measured when using control gluten and gluten isolated with YieldMAX, a lipase mainly degrading N-acyl phosphatidylethanolamine. In conclusion, properties of GS blend model systems are altered when gluten prepared in the presence of lipases is used to a degree which depends on lipase specificity and concentration.     

Jatropha curcas seed cake as substrate for production of xylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentation

Jatropha curcas seed-cake was evaluated for use as a solid state fermentation substrate for production of cellulolytic and xylanolytic enzymes by Aspergillus niger. Supplementation of the seedcake with 10% thatch grass (Hyperrhaenia sp.) resulted in a fivefold increase in xylanase production. Ammonium chloride supplementation increased production of xylanase by 13%. Under the same conditions, cellulase production was not influenced by supplementation with grass or the nitrogen sources used. Maximum xylanase was produced at 25 °C whilst cellulase was maximally produced at 40 °C. Highest xylanase activity was obtained when the cultures had an initial pH of 3 whereas cellulase was maximally produced at an initial pH of 5. Under optimised conditions, 6087 U and 3974 U of xylanase and cellulase respectively were obtained per gram of substrate. Zymograms of crude enzyme extracts showed six active bands ranging from 20 kDa to 43 kDa for cellulase and a 31 kDa active band for xylanase.     

Optimization of temperature, moisture content and inoculum size in solid state fermentation to enhance mannanase production by Aspergillus terreus SUK-1 using RSM

Optimization of three parameters, temperature (25-35 degrees C), moisture content (40% (w/v)-60% (w/v) and inoculum sizes (5% (w/v)-15% (w/v) were investigated and optimized by Response Surface Methodology (RSM) for optimal mannanase production by Aspergillus terreus SUK-1. A second order polynomial equation was fitted and the optimum condition was established. The result showed that the moisture content was a critical factor in terms of its effect on mannanase. The optimum condition for mannanase production was predicted at 42.86% (w/v) initial moisture (31 C) temperature and 5.5% (w/v) inoculum size. The predicted optimal parameter were tested in the laboratory and the mannanase activity 45.12 IU mL-1 were recorded to be closed to the predicted value (44.80 IU mL-1). Under the optimized SSF condition (31 degrees C, 42.86% moisture content (w/v) and 5.5% inoculum size (w/v)), the maximum mannanase production was to prevail about 45.12 IU mL-1 compare to before optimized (30 degrees C, 50% moisture content (w/v) and 10% inoculum size (w/v)) was only 34.42 IU mL-1.     

Direct measurement of rice bran lipase activity for inactivation kinetics and storage stability prediction

Rice bran is a rich source of valuable nutrients and has potential for high-value applications. Endogenous lipases catalyze the hydrolysis of rice bran oil to free fatty acids, which initiates lipid oxidation. The evaluation of the success of rice bran stabilization processes in terms of the degree of lipid oxidation and shelf-life has so far relied on the measurement of free fatty acid content over a storage period of 3–6 months. In the present study, a photometric and a titrimetric pH-stat method for direct lipase activity measurement immediately after debranning were adapted to rice bran. The photometric method was further applied to determine rice bran lipase/esterase inactivation kinetics, which are useful to optimize stabilization treatments in order to prevent overprocessing and retain maximum level of nutrients. Rice bran was heat-treated in a specialized, hermetically sealable reactor at controlled holding times (5–40 min), temperatures (70–145 °C) and moisture contents (10–20%). Temperature dependency of the lipase/esterase inactivation rate could be described by the Arrhenius equation. Empirical findings on the importance of moisture content for effective rice bran stabilization could be quantified. Furthermore, the results demonstrate the great potential of the method to predict the shelf-life of stabilized rice bran without time-consuming storage tests.     

鹤望兰叶斑病菌的鉴定及生物学特性

鹤望兰（Srelizia regne）叶斑病菌鉴定为柯氏帚梗柱孢霉菌（Cylindroclalium collouniperdl colouni）。该病原菌的菌丝在不同培养基里生长情况不一致,其中以Richards培养基最佳。在5种培养基中Richards培养基和PCA培养基不产孢。菌丝生长的适宜温度为15~35℃,适宜pH是5.01~6.49,分生孢子萌发的适宜温度为25~30℃.病原菌分生孢子在碳源的利用上,D-葡萄糖、蔗糖、淀粉明显优于麦芽糖、D-果糖和乳糖∶;对氮源的利用以酵母浸膏、牛肉浸膏、硝酸铵明显优于蛋白陈、L-天冬氨酸和硫酸铵。     

Production of Chondroitin Sulphate from Head,Skeleton and Fins of Scyliorhinus canicula By-Products by Combination of Enzymatic,Chemical Precipitation and Ultrafiltration Methodologies

This study illustrates the optimisation of the experimental conditions of three sequential steps for chondroitin sulphate (CS) recovery from three cartilaginous materials of Scyliorhinus canicula by-products. Optimum conditions of temperature and pH were first obtained for alcalase proteolysis of head cartilage (58 °C/pH 8.5/0.1% (v/w)/10 h of hydrolysis). Then, similar optimal conditions were observed for skeletons and fin materials. Enzymatic hydrolysates were subsequently treated with a combination of alkaline hydroalcoholic saline solutions in order to improve the protein hydrolysis and the selective precipitation of CS. Ranges of 0.53–0.64 M (NaOH) and 1.14–1.20 volumes (EtOH) were the levels for optimal chemical treatment depending on the cartilage origin. Finally, selective purification and concentration of CS and protein elimination of samples obtained from chemical treatment, was assessed by a combination of ultrafiltration and diafiltration (UF-DF) techniques at 30 kDa.     

Analysis of the relationships of environmental factors with seed oil and fatty acid concentrations of wild annual sunflower

Seed oil and fatty acid concentrations of wild annual sunflower (Helianthus annuus L.) seed vary greatly depending on the environmental conditions during development. Previous research has shown seed oil and fatty acid concentrations' response to temperature has been variable in wild and cultivated sunflower. The objective of the present study was to examine environmental factors, specifically temperature (maximum and minimum), total solar radiation and daylength for their direct and indirect effects on seed oil and fatty acid concentrations in seed oil of wild annual and cultivated sunflower using correlation and path-coefficient analyses. Ten populations of wild annual sunflower indigenous to areas from 29° to 46° N Lat. and 81° to 122° W Long. were grown in a randomized block design with three replications on Pullman clay loam (fine, mixed, thermic Torrertic Paleustoll) soils in 1980 and 1981. Three heads per genotype at early anthesis were sibbed or interpollinated at six different dates, and mature seeds were collected 28 days after sibbing for determination of seed oil and fatty acid concentrations. Hybrid ‘894’ was grown as a check for comparisons. Path-coefficient analyses indicated that minimum temperature and total solar radiation have the greatest direct effect on seed oil concentration in wild annual sunflower, though the influence was very low. In the cultivated hybrid, minimum temperature and daylength had the highest direct effect on seed oil concentration. Path-coefficient analyses also indicated that minimum temperature and solar radiation had the primary influence on oleic acid concentration in the wild and cultivated sunflower, with maximum temperature being less important. Linoleic acid concentration was primarily influenced (negatively) by minimum temperature and solar radiation as indicated by path-coefficient analyses in the wild and cultivated sunflower. The highest indirect effects of other environmental factors on fatty acid concentrations in the wild and cultivated sunflower were via minimum temperature followed by total solar radiation. There was a strong negative relation between linoleic and oleic fatty acid concentrations in the study. Path-coefficient analyses indicated that the wild annual sunflower reacted similarly to the cultivated sunflower to the environmental factors examined. This information will be useful to sunflower plant breeders when they incorporate the wild germplasm into commercial sunflower breeding lines.     

Chemical, physical and microbiological changes during composting of the water hyacinth

An investigation of the physical, chemical and microbial population changes that occurred during the composting of water hyacinth was carried out. After 11 weeks of composting, the compost turned black, had decomposed and had no smell. The pH was 7 and the highest temperature reached, of 40 degrees C occurred in the first week. The initial carbon/nitrogen ratio was 17.61 and this increased to 18.12 by the end of the composting. The coliform population declined greatly from 8.11 to 5.85 MPN (log)/g and fecal coliforms and Escherichia coli were not detected in the final product. Bacteria were the dominant microbes in the compost followed by actinomycetes and fungi. Mesophillic microorganisms were present in higher numbers than thermophillic microorganisms throughout the composting. The highest cellulase and xylanase activities in the compost of 6.67 and 10.24 U/kg DW, respectively were detected in the second week which was related to the temperature. Bacillus sp. strain B4 was isolated and investigated for cellulase and xylanase using agro-industrial residues as substrates during Solid-State Fermentation (SSF) processes. Corncob and rice straw were good substrates for the production of the enzymes with a maximum cellulase of 1.19 U/gDW and xylanase activity of 2.54 U/g DW, respectively. The activities of both enzymes were stable and maximum at 50 degrees C. This study indicated that agro-industrial residues should be mixed with water hyacinth for composting to facilitate the development of a thermophilic phase during the composting process and to improve the product. Bacillus sp. strain B4 can be used as a starter strain.     

茉莉花芳樟醇β-D吡喃葡萄糖苷酶的分离与纯化

茉莉花芳樟醇 β_D吡喃葡萄糖苷酶 (LGA)丙酮粉经过pH6 0柠檬酸缓冲液萃取、 3 0 %和 80 %硫酸铵分步沉淀 ,以及CM_Toyopearl65 0M柱层析 ,用 0 - 0 5MNaCl柠檬酸缓冲液梯度洗脱酶蛋白 ,最后用快速蛋白液相色谱 (FPLC)的SephacrylS_2 0 0柱分离系统纯化出了LGA ,LGA蛋白的SDS_PAGE图谱在分子量 1 4 4万道尔顿处显示了一条谱带。它可以催化芳樟基 β_D_吡喃葡萄糖苷分解释放芳樟醇     

Genetic variability in the response of fatty acid composition to minimum night temperature during grain filling in sunflower

It is not known if the response of the oil fatty acid composition to temperature is similar among sunflower hybrids, especially among traditional hybrids. The objective of this work was to asses the genetic variability of the response of the oil fatty acid composition to temperature during the period 100–300 °C day after flowering among sunflower hybrids. Seven traditional hybrids and a high oleic hybrid were grown in several locations of Argentina. With the same hybrids two experiments were carried out in growth chambers where the plants were exposed to different day–night temperatures during grain filling. From these field and growth chamber experiments wide ranges of variation of temperature and oil fatty acid compositions for each hybrid were obtained. The concentration of oleic acid in sunflower oil showed a sigmoidal response to minimum night temperature from 100 to 300 °C day after flowering, increasing almost lineally within a given range of temperature. Outside this range, the concentration of this fatty acid remained merely constant. The same mathematical expressions characterized the response of oleic acid concentration to temperature in both, traditional and high oleic hybrids. The same expression also described the response of other fatty acids to temperature in all the hybrids. Differences between hybrids were observed for the minimum and maximum concentration of oleic acid and also for the maximum slope and range of the response. Since the sum of oleic + linoleic acids increased with temperature, increasing temperature reduced the concentration of saturated fatty acids and behenic acid. Genetic variability in the response of these fatty acids to temperature was observed. The results presented here show that it is possible to obtain sunflower oils of very different qualities combining the genetic variability in the response of the fatty acid composition to temperature and the climatic diversity under which sunflower is cultivated.