Influence of some environmental factors on Rhizopus nigricans submerged growth in the form of pellets

Morphological changes of a steroid transforming filamentous fungus Rhizopus nigricans were studied by altering submerged growth conditions at inoculum sizes previously determined to favor pelleted growth. Beside the main classification between pelleted and clumpy growth forms, the size, concentration and structure of pellets were characterized at different cultivating temperature, initial pH value, medium composition, additives, and aeration conditions. Initial pH below 4 and above 7, the presence of Ca²⁺ and Tween-80 gave rise to the clumpy growth, otherwise pelleted growth prevailed. Among tested factors the pellet size was mainly influenced by the inoculum size and the presence of baffles and Ca²⁺ in cultivation medium. The formation of smooth pellets, prerequisite for further application in the process of steroid biotransformation, resulted in cultivations at lower temperature, high agitation rates in shaken cultures without baffles and at high nitrogen concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation and characterisation of resistant-to-fermentation carbohydrate polymers from cultures of Rhizopus nigricans grown on agrofood waste materials

Rhizopus nigricans was cultivated in a liquid medium using lemon, mandarin, orange, pear and melon peel or artichoke bracts as the carbon source. In all cultures, a carbohydrate polymer fraction remained resistant to fermentation. These fractions were isolated in gram amounts and characterised. The molecular weight distribution of the fractions and its sugar composition resembles those of the hairy-regions of the pectins. In the fractions, four main carbohydrates were found: 4-7 mol% Rha, 42-59 mol% Ara, 7-14 mol% Gal, 17-33 mol% GalA.     

Removal of heavy metals from stainless steel effluents by waste biomass from Brazilian alcoholic beverage production

The capacity of waste biomasses from sugar-cane aguardente, a traditional Brazilian spirit, for metal biosorption was assessed. Free biomass and biomass immobilized onto chitin and Dowex (ion-exchange resin) were utilized to remove chromium, iron and nickel from both synthetic solutions and stainless steel effluents. The best performance in terms of metal sorbed was observed in with free biomass, with the following adsorption capacity: 70% chromium, 50% iron and 20% nickel at pH 4.0.     

Effect of some heavy metal ions on copper-induced metallothionein synthesis in the yeast Saccharomyces cerevisiae

Copper-induced metallothionein (MT) synthesis in Saccharomyces cerevisiae was investigated in order to associate this exclusively with Cu²⁺ in vivo, when cultured in nutrient medium containing other heavy metal ions. Expression of the CUP1 promoter/lacZ fusion gene was inhibited by all heavy metal ions tested, especially Cd²⁺ and Mn²⁺. By adding Cd²⁺ and Mn²⁺ at 10 M concentration, the -galactosidase activity decreased by about 80% and 50% of the maximum induction observed with 1 mM CuSO₄, respectively. Furthermore, cell growth was markedly inhibited by combinations of 1 mM-Cu²⁺ and 1 M-Cd²⁺. Therefore, the yeast S. cerevisiae could not rely on MT synthesis as one of the copper-resistance mechanisms, when grown in a Cd²⁺ environment. In contrast, the presence of Mn²⁺ in the nutrient medium showed alleviation rather than growth inhibition by high concentrations of Cu²⁺. The recovery from growth inhibition by Mn²⁺ was due to decreased Cu²⁺ accumulation. Inhibitory concentrations of Co²⁺, Ni²⁺ and Zn²⁺ on expression of the CUP1p/lacZ fusion gene were at least one order of magnitude higher than that of Cd²⁺ and Mn²⁺. These results are discussed in relation to Cu²⁺ transport and Cu-induced MT synthesis in the copper-resistance mechanism of the yeast S. cerevisiae.     

Rhizopus oligosporus grown on natural rubber waste serum for production of single cell protein: a preliminary study

Maximum production of mycelium and utilization of total organic carbon byR. oligosporus grown on natural rubber waste serum was achieved at 28°C with an inoculum size of 7.5% (v/v) and grown for 144 h with an initial pH of 4.0. The maximum production of total crude protein, however, was when culture medium was inoculated with 2% (v/v) of spore suspension under the same conditions. Natural rubber waste serum may be a potential substrate for the production of single cell protein.     

Screening of waste biomass from Saccharomyces cerevisiae,Aspergillus oryzae and Bacillus lentus fermentations for removal of Cu,Zn and Cd by biosorption

Three by-products of fermentations containing Bacillus lentus, Aspergillus oryzae or Saccharomyces cerevisiae biomass were tested for the capacity to absorb Cu, Cd and Zn. The composition of the three biomasses was first determined and showed high contents of ashes in both B. lentus and A. oryzae biomass and high amounts of lipids in the bacterial biomass. Metal ion binding experiments were performed by contact of 0.1 g of biomass (protonated for all the metal tests and not protonated only for the Cd test) with 50 ml of solutions containing each of the metals in the concentration range from 10 to 500 mg/ml, at pH 4.5, 3.5 and 2.5. The final metal ion concentrations were determined using a plasma absorption spectrometer, and the metal removal levels for isotherm plots were determined using the Langmuir model. The results showed that B. lentus protonated biomass had the best sorption capacity for Cu and Cd, followed by protonated A. oryzae and S. cerevisiae biomass. The sorption of Zn was low for all tested biomasses, as also was the binding of all metals at acidic pH (2.5 and 3.5). A significant increase in Cd sorption was obtained using non-protonated biomass from B. lentus and A. oryzae.     

Environment friendly modified chitosan hydrogels as a matrix for adsorption of metal ions, synthesis and characterization

Environment friendly modified chitosan hydrogels for metal ions absorption from aqueous systems were designed using simple technology. Chitosan was modified with [N,N′-bi-α-azidosuccinimide and N-phthalimido-α-azide succinimide] under different reaction conditions to prepare new hydrogels with high metal ion absorption efficiency. The hydrogels were characterized by FTIR, thermal stability, crystallography, solubility and swelling capacity. Promising results were obtained from this preliminary study to evaluate the efficiency of the new hydrogels to uptake copper and cobalt ions from aqueous systems.     

Differential pulse polarography: a method for the direct study of biosorption of metal ions by live bacteria from mixed metal solutions

The technique of differential pulse polarography is shown here to be applicable to the monitoring directly the biosorption of metal ions from solution by live bacteria from mixed metal solutions. Biosorption of Cd(II), Zn(II) and Ni(II) by P. cepacia was followed using data obtained at the potential which is characteristic of the metal ion in the absence and presence of cells. Hepes buffer (pH 7.4, 50 mM) was used as a supporting electrolyte in the polarographic chamber and metal ion peaks in the presence of cells of lower amplitude were obtained due to metal-binding by the cells. Well defined polarographic peaks were obtained in experiments involving mixtures of metal ions of Cd(II)-Zn(II), Cu(II)-Zn(II), Cu(II)-Cd(II) and Cd(II)-Ni(II). Biosorption of Cd(II), Zn(II) increased with solution pH. The method was also tested as a rapid technique for assessing removal of metal ions by live bacteria and the ability of the polarographic technique in measuring biosorption of metal ions from mixed metal solutions is demonstrated. Cu(II) was preferentially bound and removal of metals was in the order Cu(II) > Ni(II) > Zn(II), Cd(II) by intact cells of P. cepacia. This revised version was published online in August 2006 with corrections to the Cover Date.     

Construction of a <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis> glucoamylase gene suitable for expression in distinct host: introns spliced artificially by PCR

Glucoamylase is an industrially extremely important enzyme in the fermentative production of ethanol, used in the enzymatic conversion of starch into high glucose and fructose syrups. The aim of this study is to construct a Rhizopus arrhizus glucoamylase gene (RaGA)—introns artificially spliced by PCR—suitable for expression in S. cerevisiae host and tried expressing in Picha pastoris. In previous work, we failed in amplifying glucoamylase gene from R. arrhizus by RT-PCR, so several primers were designed to splicing the introns by PCR in vitro. Sequence analysis shown that all introns in the RaGA were deleted correctly and no mutant was induced in the extrons compared with the RaGA gene originally cloned. The RaGA gene artificially constructed was transferred into P. pastoris integrative expression vectors pPIC9 (containing а-factor). Consequently, the plasmids pPIC9-RaGA was lineared by SacI and inserted into P. pastoris GS115 (His⁻) genome downstream of the 5′AOX1 promoter by the method of electroporation. Induction by 0.75% methanol for 72 h led to synthesis of secreted glucoamylase. So it is demonstrated that the glucoamylase gene has been expressed in and secreted from P. pastoris.     

苦竹-杉木混交林界面区克隆分株秆形和地上生物量分配的适应策略

苦竹(Pleioblastus amarus)是优质笋材兼用竹种,分布广。为探究界面区苦竹分株秆形及地上构件生物量分配格局的变化特征,解析苦竹对异质生境适应机制,该研究选取了相邻的苦竹林和苦竹-杉木(Cunninghamia lanceolata)混交林两种林分类型,分别测定了苦竹林和混交林中心区及界面区不同龄级立竹秆形和秆、枝、叶的生物量,分析立竹秆形及地上构件生物量积累、分配、异速生长关系的差异。结果表明:(1)界面区1 a立竹生物量积累及分配差异增大,其中苦竹林界面区各构件相对生物量和叶生物量分配比例提高,而混交林界面区各构件相对生物量和叶生物量分配比例降低; 2 a立竹生物量积累及分配比例的差异缩小,界面区两边2 a立竹各构件相对生物量和生物量分配比例均无明显差异。(2)界面区立竹秆形特征及1 a立竹各构件生物量异速生长关系均无明显变化,而苦竹林界面区2 a立竹秆的增长速率提高,枝、叶的增长速率降低。综上认为,苦竹通过权衡资源分配关系,明显改变界面区立竹秆形及生物量分配格局,以提高克隆分株对异质环境的适合度。     

浙江慈溪不同利用年限水稻土微生物生物量与酶活性比较

测定了浙江慈溪5个不同利用年限水稻土(50～2000 a)的微生物生物量C、N和过氧化氢酶、转化酶、脲酶、磷酸酶的活性,分析了水稻土的生物化学环境质量与利用年限的相关性.结果发现,随着利用年限的延长,水稻土的微生物生物量C与N均趋于下降,其中微生物生物量N能较灵敏地反映利用年限造成的差异(p=0.091);水稻土的过氧化氢酶与转化酶活性均呈下降趋势,其中过氧化氢酶活性与利用年限之间确有线性回归关系(p = 0.042);水稻土的脲酶活性不呈显著变化规律,而磷酸酶活性显著上升,其与利用年限之间确有线性回归关系(p = 0.022).总体表明,利用年限延长虽然会降低水稻土中微生物的生物量和整体活性,但对水稻土的肥力并没有产生不良影响,相反对水稻土的供磷能力有保育和增强作用,而对水稻土的供氮能力没有定向影响.     

A novel method for in vitro culture of plants: Cultivation of barley in a floating hydroponic system

We describe a system for hydroponically growing plants that is based on using a polyethylene (PE) granulate as the floating body. The procedure can be performed in all suitable vessels (e.g., bowls, basins, glasses) and is independent of the culture area. Barley plants (Hordeum vulgare) grown in this hydroponic system had no difference in the uptake of mercuric and cadmium ions when compared with plants grown on a defined agar medium. The simple and inexpensive method can be used for the isolation of leaf tissue and large amounts of root tissue. In addition, numerous experiments can be run in parallel.     

Effects of tungstate on the growth of Desulfovibrio gigas NCIMB 9332 and other sulfate-reducing bacteria with ethanol as a substrate

Growth of Desulfovibrio gigas NCIMB 9332 in mineral, vitamin-supplemented media with ethanol as substrate was strongly stimulated by the addition of tungstate (optimal level approximately 10^-7 M). At suboptimal tungstate concentrations, up to 1.0 mM acetaldehyde was detected in the culture supernatant and growth was slow. Omission of both tungstate and molybdate from the media prevented growth and ethanol utilization. Tungstate-deprived cultures that were grown on lactate had much lower aldehyde dehydrogenase (benzylviologen as acceptor; BV-AIDH) levels than tungstate-supplemented cultures. These data suggest that tungstate is required for the synthesis of active BV-AIDH. The characteristics of the enzyme activities in cell-free extracts show that the BV-AIDH activity present in tungstate-supplemented cultures is not due to the recently characterized molybdenum-containing aldehyde dehydrogenase of D. gigas. Out of 13 other strains of ethanol-oxidizing, gram-negative, sulfate-reducing bacteria tested, most strains grew well with either tungstate or molybdate supplementation. In contrast to a recent report, good growth on ethanol of two D. baculatus (Desulfomicrobium) strains (DSM 1741 and DSM 1743) was observed.Abbreviations BV-AIDH Benzylviologen-linked aldehyde dehydrogenase - DCPIP-AIDH 2,6-dichlorophenolindophenol-linked aldehyde dehydrogenase - DTT dithiothreitol     

纳米铁联合氧化石墨烯对垃圾堆肥基质高羊茅生理生态的影响

通过室内盆栽试验,在生活垃圾堆肥中按质量比添加1%氧化石墨烯(GO)和不同比例的纳米铁(nZVI,1%、3%、5%),研究了GO和nZVI对高羊茅生长和生理特性的影响。结果表明:单独添加GO及共同添加GO和nZVI促进了高羊茅种子萌发,发芽率和发芽指数均在GO+5%nZVI处理达到最大。除1%nZVI处理外,其他添加剂处理显著提高了高羊茅地上生物量和叶绿素含量,在GO+5%nZVI处理达到最大,较对照分别提高25%和31%。添加3%、5%nZVI显著提高了超氧化物歧化酶(SOD)和过氧化物酶(POD)活性,且随nZVI添加比例的增加而升高。与对照相比,单独添加nZVI以及联合GO处理过氧化氢酶(CAT)与丙二醛(MDA)含量显著降低,随nZVI添加比例的增加而减少,分别在GO+5%nZVI和GO+3%nZVI处理中达到最低,较对照分别降低52%和48%。高羊茅地上部重金属含量随nZVI添加比例的增加而降低,在共同添加GO和nZVI的处理中效果显著,均显著低于对照。可见,nZVI和GO两种材料对堆肥重金属具有固定作用,减少了植物对重金属的吸收,促进了草坪植物的生长。     

Measuring maximum root growth instead of longest root elongation in metal tolerance tests for grasses (Agrostis capillaris,Agrostis delicatula and Agrostis castellana)

Longest root elongation diminished significantly in the three species tested from 6 mm d^-1 to 3 mm d^-1 in 3 weeks. During this period S.D. increased considerably (from 49% to 112%, A. castellana), and accounted on the average for 68% (A. capillaris) till 94% (A. castellana) of the mean. Maximum root growth stabilized at 6 mm d^-1 and showed less variation in the measurements (S.D. 52% of the mean). Growth of the originally longest root approaches zero in all three species, in accordance with the natural cease of growth of roots in grasses fascicular root system. Measuring maximum root growth instead of longest root elongation is proposed for testing metal tolerance of grasses in sequential experiments.     

川西亚高山不同林龄粗枝云杉人工林土壤微生物生物量及酶活性

林分结构随林龄增加发生变化,致使土壤微环境变化,进而对土壤微生物生物量和酶活性产生直接或间接影响。以川西亚高山米亚罗林区25、40、50、60年生粗枝云杉（Picea asperata）人工林表层（0-20 cm）土壤为研究对象,分析不同林龄表层土壤微生物生物量碳（MBC）、氮（MBN）和酶活性的变化以及驱动土壤酶活性变化的主要环境因子,为人工林生态系统恢复效果评价及经营管理提供科学依据。结果表明：随着林龄的增加,MBC和MBN先上升后下降,在40年生达到最大;β-葡萄糖苷酶（βG）、β-N-乙酰氨基葡萄糖苷酶（NAG）和多酚氧化酶（PHO）活性与微生物生物量碳氮变化趋势相似,其中βG、NAG活性在50年生人工林达到最大,PHO活性于40年生人工林达到最大;50年生人工林过氧化物酶（PEO）活性显著低于其他林龄人工林;纤维素水解酶（CBH）活性随林龄增加而显著增加。冗余分析显示,碱解氮（AN）、pH和可溶性有机碳（DOC）是影响土壤酶活性变化的主导因子,分别解释了土壤酶活性变异的65.4%、9.7%和7.6%。综上可知,林龄对粗枝云杉人工林表层土壤微生物生物量和酶活性变化产生了显著影响,60年粗枝云杉林地力呈现衰退态势。为此,可对近60年的粗枝云杉人工林增加碳氮元素（尤其是氮素）的输入,以提升土壤质量和酶活性。     

Seasonal changes in composition and biomass of epiphytic algae on the macrophyte Ranunculus penicillatus in a chalk stream,with estimates of production,and observations on the epiphytes of Cladophora glomerata

The epiphytic algae on surfaces of the macrophyte Ranunculus penicillatus (Dumort.) Bab. var. calcareus (R. W. Butcher) C. D. K. Cook and on Cladophora glomerata (L.) Kütz growing in the River Itchen at Otterbourne near Southampton were studied between February 1984 and June 1985. The river at this site has a mean flow rate of 0.33 m s^–1, and is about 16 m wide and on average 20 cm deep, with a discharge ranging through the year between 0.34 and 2.46 m³ s^–1. The pH of the river varies little around 8.2, with a mean alkalinity of 236 mg HCO _inf3 ^sup1 l^–1, because of its origin from chalk springs. Ranunculus grows throughout the year, with peaks of biomass in spring and autumn. It forms a very large surface for attachment of epiphytes, and covers on average 40% of the stream bed. Numbers of epiphyte cells removed from Ranunculus ranged through the year between 52 × 10³ and 271 × 10³ cells mm^–2 stream floor, with maximum numbers in April, and a secondary peak in October. This pattern partly reflects fluctuations in the biomass of Ranunculus; the number of cells per unit area of plant surface showed a broader spring peak and lower fluctuations in other seasons. Diatoms formed 65 to 98% of these epiphyte cells, with chlorophytes reaching their peak (10%) in summer and cyanophytes (25%) in autumn. Estimates of biomass of these epiphytic forms, derived from measurements of chlorophyll c, indicate a range between 30 and 100 g dry weight m^–2 of weed bed. Colonisation studies showed that the algae grow and reproduce throughout the year, with a mean generation time of about 5 days, suggesting an annual production of about 3 kg dry weight m^–2 of weed bed, which makes epiphytic algae the principal primary producers in the stream. The numbers and biomass of epiphytic algae on Cladophora are considerably less.The species of epiphytic algae found on Ranunculus were generally similar to those growing on Cladophora, and to epilithic algae on pebbles of the stream bed, but different species were dominant on the different substrata. Algal cells in the water column were all derived from benthic habitats, although their relative abundance was very different.     

Streptomyces neyagawaensis as a control for the hazardous biomass of Microcystis aeruginosa (Cyanobacteria) in eutrophic freshwaters

An aquatic bacterium capable of eliminating the cyanobacterium Microcystis aeruginosa was isolated from the sediment of an eutrophic lake (Lake Juam, Korea). On the basis of 16S rDNA sequences and biochemical and morphological characteristics, the isolate was determined to be Streptomyces neyagawaensis. It grew optimally at 40 °C and pH 7. In the presence of this bacterium, the biomass of cyanobacterium M. aeruginosa NIES-298 was strongly suppressed, by up to 84.5% in abundance compared to the control. The antialgal activity of S. neyagawaensis depended on the growth phase of the cyanobacterium, but not of the antialgal bacterium. The antialgal activity of S. neyagawaensis was effective against a wide range of algae, including the green alga Chlorella sp., the diatoms Aulacoseira granulata and Stephanodiscus hantzschii, and four cyanobacteria, M. aeruginosa NIES-44, Anabaena cylindrica, Anabaena flos-aquae, and Oscillatoria sancta. S. neyagawaensis indirectly attacked M. aeruginosa by secretion of extracellular antialgal substances that were localized in the bacterial periplasm and had a specific activity of 7.7 U/μg. These results suggest that indigenous bacteria isolated from sediments may have potential application in controlling harmful cyanobacterial blooms in freshwaters.     

Decaying wood in tree trunk hollows as a natural substrate for Cryptococcus neoformans and other yeast-like fungi of clinical interest

The occurrence of Cryptococcus neoformans var. neoformans and other yeast-like fungi of clinical interest in decaying wood inside tree trunk hollows, bark and other plant materials is reported. The var. neoformans was isolated from 3 of 45 (6.6%) wood and one of 390 Eucalyptus bark samples. Two of the positive wood samples came from a tree trunk hollow of Butea monosperma (Family: Papilionaceae) growing in Roshan Ara Garden, Old Delhi whereas the third was from a trunk hollow of Tamarindus indica (Family: Papilionaceae) growing outside of Talkatora Garden, New Delhi. The solitary positive Eucalyptus bark sample originated from Amritsar. The isolations of var. neoformans from decaying wood inside trunk hollows of B. monosperma and T. indica constitute the first record of the natural occurrence of this pathogen in association with these trees. The observation reinforces the recent evidence for decaying wood inside trunk hollows of some trees to be a new natural habitat of the variety neoformans. Besides, in consonance with their essentially saprobic character, a number of other yeast-like fungi were sporadically isolated. This includes, Cryptoccus laurentii Cryptococcus albidus Candida lusitaniae C. guilliermondii C. krusei C. tropicalis C. zeylanoides Trichosporon cutaneum Rhodotorula mucilaginosa R. glutinis Geotrichum capitatum G. klebahnii and Sporobolomyces salmonicolor. Cryptococcus neoformans var. gattii was not found in any of the 702 samples of plant materials, including the bark and detritus of Eucalyptus camaldulensis and E. tereticornis trees. A more extensive environmental survey, covering divergent climatic regions, is warranted to identify the natural reservoirs of var. gattii in India. This revised version was published online in August 2006 with corrections to the Cover Date.     

Alginate from the macroalgae <Emphasis Type="Bold">Sargassum sinicola</Emphasis> as a novel source for microbial immobilization material in wastewater treatment and plant growth promotion

Alginate extracted from the macroalgae Sargassum sinicola was used as the raw material for co-immobilization of the microalgae Chlorella sorokiniana and growth-promoting bacterium Azospirillum brasilense for wastewater treatment and as an inoculant carrier of A. brasilense for plant growth promotion. The composition, structure, viscosity, color, and phenolic compound content of the alginate were analyzed and compared with commercially available alginate produced from the macroalgae Macrocystis pyrifera. From ¹H NMR analysis of alginate, S. sinicola was found to have more guluronic acid (F _G=0.64) than it had mannuronic acid (F _M=0.38) and had a viscosity of 13.5 m Pa s compared to 50 m Pa s for M. pyrifera. The S. sinicola alginate had dark brown color, reducing light penetration, with more phenolic compounds than M. pyrifera alginate. Nonetheless, growth of C. sorokiniana and A. brasilense in S. sinicola alginate was not significantly different than the growth in M. pyrifera alginate beads. Nutrient removal from wastewater by the co-immobilized microorganisms was similar for both types of alginate beads, and so was the growth enhancement of tomato plants inoculated with microbeads containing A. brasilense. This study shows the potential use of S. sinicola alginate as a raw material for cell immobilization for wastewater treatment and plant growth promotion.