A Comparison of the Unfolded Protein Response in Solid-State with Submerged Cultures of Aspergillus oryzae

The unfolded protein response (UPR) is a regulatory system to maintain the homeostasis of ER functions. Here we report a comparison of express levels of UPR relevant genes in Aspergillus oryzae between solid-state and submerged cultivation. The results were that up-regulation of the UPR mechanism in solid-state culture was higher than in submerged culture (heat-shock or non-stress conditions). This might have been a result of changing culture conditions.     

Production of extracellular proteinases—protein C activators of blood plasma—by the micromycete Aspergillus ochraceus during submerged and solid-state fermentation

The conditions for the submerged and solid-state fermentation of the micromycete Aspergillus ochraceus VKM F-4104D, producing extracellular proteinases that activate protein C of human blood plasma, were optimized. It is shown that the protein C-activating activity of the micromycete in a solid-state culture was 1.5-3.5 times higher than in a submerged culture (as calculated per milliliter of culture medium). Among the extracellular proteins secreted by A. ochraceus VKM F-4104D during submerged and solid-state fermentation, a protein C-activating proteinase with a pI of 6.0–6.3 was identified.     

华根霉固态与液态培养产胞外蛋白的比较蛋白质组学分析

【目的】考察固态和液态两种培养方式对丝状真菌华根霉(Rhizopus chinensis)CCTCC M201021产胞外蛋白的影响,以加深对微生物固、液态培养特异性产酶的认识。【方法】利用成分相同的培养基对华根霉分别进行平板固态培养和液态培养,提取华根霉所产胞外蛋白,采用二维凝胶电泳(2-DE)结合质谱分析,分离鉴定差异蛋白。【结果】固态培养下华根霉所产胞外蛋白的蛋白酶活性是液态培养的9.2倍。胞外蛋白质组数据分析表明,华根霉固态和液态培养所产胞外蛋白存在显著差异,其中约70%是固态和液态培养下各自产生的特有蛋白。质谱鉴定进一步表明,固、液态培养对华根霉产胞外蛋白的种类和表达量都有显著影响,其中水解酶类所占比例较大,主要是与蛋白质降解相关的蛋白。【结论】固态和液态不同培养方式影响了华根霉产胞外蛋白的组成,有些基因只在特定培养方式下表达。固态培养下华根霉产胞外蛋白酶种类相对更多以及大部分蛋白酶的上调表达,可能是固态培养下胞外蛋白酶活性很高的原因。研究结果提示需要注意固液态不同培养方式下丝状真菌胞外酶的筛选和生产可能存在的不一致性。     

Optimization of process for the production of fungal pectinases from deseeded sunflower head in submerged and solid-state conditions

Pectinase production studies were carried out in submerged and solid-state conditions from deseeded sunflower head employing Aspergillus niger. The two potential strains of A. niger, DMF 27 for submerged and DMF 45 for solid-state were isolated by multi-step screening technique based on coefficient of pectolysis and capability of pectinase production. Process variables such as size of inoculum, pH, temperature, particle size and moisture content were optimized with an aim to achieve the maximum production of pectinases. The increased level of pectinase production was recorded at pH 5.0 and temperature 34 degrees C in submerged and solid-state conditions. The optimum inoculum size was 1x10(5)ml(-1) for submerged and 1x10(7)g(-1) for solid-state conditions. Five hundred micrometer particle size and 65% moisture content of the substrate were optimum for the maximum production of pectinases in solid-state condition. Under optimum conditions, maximum production of exo-pectinase was 34.2U/g in SSF and endo-pectinase was 12.6U/ml in SmF.     

Establishment of an effective oligotrophic cultivation system for Rhodococcus erythropolis N9T-4

Rhodococcus erythropolis N9T-4 grows on an inorganic solid-state medium with no additional carbon and energy sources; however, it is unable to grow well in a liquid culture medium under the oligotrophic conditions. We examined submerged cultivations of N9T-4 using a polyurethane foam sponge to achieve approximately 10 times of the oligotrophic growth of the bacterium in the liquid culture medium.     

Cellulase production by Aspergillus niger in biofilm, solid-state, and submerged fermentations

Cellulase production by Aspergillus niger was compared in three different culture systems: biofilm, solid-state, and submerged fermentation. Biofilm and solid-state fermentations were carried out on perlite as inert support, and lactose was used as a carbon source in the three culture systems. In cryo-scanning electron microscopy, biofilm and solid-state cultures gave similar morphological patterns and confirmed that both spore first attachment and hyphal adhered growth are helped by the production of an adhesive extracellular matrix. Biofilm cultures produced higher cellulase activities than those in submerged and solid-state cultures (1,768, 1,165, and 1,174 U l⁻¹, respectively). Although biofilm cultures grew less than the other cultures, they produced significantly higher cellulase yields (370, 212, and 217 U g⁻¹ lactose, respectively) and volumetric productivities (24, 16, and 16 U l⁻¹ h⁻¹, respectively). Likewise, endoglucanase and xylanase activities were higher in biofilm cultures. Under the conditions tested, it seems that fungal attached growth on perlite may favor better enzyme production. Biofilms are efficient systems for cellulase production and may replace solid-state fermentation. Biofilm fermentation holds promise for further optimization and development. The results of this work reveal that fungal biofilms may be used for the commercial production of cellulase employing the technology developed for submerged fermentation at high cell densities.     

不同栽培基质对糙皮侧耳不同发酵方式下产漆酶活性的影响

为了比较糙皮侧耳栽培种在不同常规栽培基质上的漆酶活性,分析更适合糙皮侧耳生长的栽培基质,以1株糙皮侧耳Pleurotus ostreatus栽培菌株为研究材料,研究在固态和液态发酵条件下添加木屑、玉米芯和棉籽壳这3种栽培基质后,对其产漆酶活性的影响。结果表明,不同栽培基质对糙皮侧耳漆酶活性具有极显著的影响（P<0.001）;不同发酵方法对糙皮侧耳漆酶活性也具有极显著的影响（P<0.001）,仅第2天差异不显著。固体发酵与液体发酵条件下,糙皮侧耳在棉籽壳培养基上所检测到的漆酶活性均高于在木屑或者玉米芯培养基上,表明棉籽壳对提高糙皮侧耳漆酶活性的诱导能力更强。此外,糙皮侧耳在棉籽壳培养基上能够快速分泌漆酶,表明棉籽壳对缩短糙皮侧耳漆酶分泌时间的诱导能力更强。     

Biosynthesis of tannin acyl hydrolase from tannin-rich forest residue under different fermentation conditions

Caesalpinia digyna, a tannin-rich forest residue, was used as substrate for production of tannase and gallic acid. Media engineering was carried out under solid-state fermentation, submerged fermentation and modified solid state fermentation conditions for optimum synthesis of tannase and gallic acid (based on 58% tannin content in the raw material). Tannase vis-à-vis gallic acid recovery under modified solid-state fermentation condition was maximum. Conversions of tannin to gallic acid under solid-state fermentation, submerged fermentation and modified solid-state fermentation conditions were 30.5%, 27.5% and 90.9%, respectively. Journal of Industrial Microbiology & Biotechnology (2000) 25, 29–38. Received 02 November 1999/ Accepted in revised form 12 February 2000     

High Level Secretion of Calf Chymosin Using a Glucoamylase-prochymosin Fusion Gene in Aspergillus oryzae

A recombinant chymosin was secreted at high levels using fusion genes with A. oryzae glucoamylase gene (glaA) and a wheat bran solid-state culture system. Two portions of the A. oryzae glucoamylase, one with almost the entire glucoamylase (GA1–603) lacking 9 amino acids at the carboxyl terminal, and the other (GA1–511) lacking the starch binding-domain, were fused in frame with prochymosin cDNA. Western blot analysis indicated that the mature chymosin was released from the secreted fusion protein by autocatalytic processing. The transformant harboring the GA1-511-prochymosin construct showed about 5-fold chymosin production of the transformant in which the chymosin gene was directly expressed under the control of the glaA promoter in submerged culture. Moreover, wheat bran solid-state culture gave about 500-fold higher yield of the chymosin (approximately 150 mg/kg wheat bran) compared with the submerged culture.     

Production and properties of three pectinolytic activities produced byAspergillus niger in submerged and solid-state fermentation

Three extracellular pectinases were produced byAspergillus niger CH4 by submerged and solid-state fermentation, and their physicochemical and kinetic properties were studied. The highest productivities of endo- and exo-pectinase and pectin lyase were obtained with solid-state fermentation. The kinetic and physicochemical properties of these enzymes were influenced by the type of culture method used. All activities were very different in terms of pH and temperature optima, stability at different pH and temperature values and affinity for the substrate (K _m values). In solid-state fermentation, all pectinase activities were more stable at extreme pH and temperature values but theK _m values of endo-pectinase and pectin lyase were higher with respect to those activities obtained by the submerged-culture technique. The pectin lyase activity obtained by the submerged-culture technique showed substrate inhibition but the enzyme obtained by solid-state fermentation did not. Electrophoresis, using sodium dodecyl sulphate/polyacrylamide gel with enzymatic extracts obtained for both culture methods, showed the same number on protein bands but some differences were found in their electrophoretic position. The results obtained in this work suggest that the culture method (submerged or solid-state) may be responsible for inducing changes in some of the pectinolytic enzymes produced byA. niger.     

Enhancement glucose oxidase production by solid-state fermentation of Aspergillus niger on polyurethane foams using mussel processing wastewaters

In the present work, studies of glucose oxidase (GOD) production in solid-state fermentation on polyurethane foams, using mussel processing wastewaters (MPW) as culture media, were carried out. Initially, the results generated in this experimental modality were compared with those that were obtained in conventional submerged fermentation. Later on, cultures were tested in solid state with fed-batch performance, defining suitable conditions of operation. These conditions were finally corroborated in experiments with a bioreactor of own design, in which GOD production was improved 13 times regarding to the initial cultures.     

Induction of laccase activity in the edible straw mushroom,Volvariella volvacea

Volvariella volvacea, strain V14, produces multiple forms of extracellular laccase when grown in submerged culture in a defined medium with glucose as sole carbon source, and on cotton waste 'compost' representative of the conditions used for industrial-scale mushroom cultivation. In liquid culture, enzyme synthesis is associated with the onset of secondary growth, and is positively regulated by copper (up to 200 microM CuSO(4)) and by various aromatic compounds. In solid-state systems, only low levels of laccase are detectable during the vegetative growth phase but enzyme activity increases sharply at the onset of fruiting and during sporophore development.     

Isolation and Screening of Alkaline Lipase-producing Fungi from Brazilian Savanna Soil

Summary Fifty-nine lipase-producing fungal strains were isolated from Brazilian savanna soil by employing enrichment culture tecniques. An agar plate medium containing bile salts and olive oil emulsion was employed for isolating and growing fungi in primary screening assay. Twenty-one strains were selected by the ratio of the lipolytic halo radius and the colonies radius. Eleven strains were considered good producers under conditions of submerged liquid fermentation (shaken cultures) and solid-state fermentation. The most productive strain, identified as Colletotrichum gloesporioides, produced 27,700 U/l of lipase under optimized conditions and the crude lipase preparation was capable of hydrolysing a broad range of substrates including lard, natural oils and tributyrin.     

Isolation of a novel promoter for efficient protein production in Aspergillus oryzae

A novel protein overexpression system of Aspergillus oryzae was constructed. Five promoters which originate from A. oryzae expressed sequence tag (EST) clones in submerged culture were obtained by genome walking. These were subjected to beta-glucuronidase (GUS) reporter assays. The promoter of manganese superoxide dismutase-encoding gene (sodM) showed the most GUS production. The sodM gene was abundantly expressed in submerged culture but little expressed in solid-state culture. The sodM promoter was approximately 3-fold induced by the addition of 0.01% H2O2. Glucoamylase production in A. oryzae using the sodM promoter led to secretion of approximately 1 g/l-broth in Czapek-Dox medium for 3 d. Fucose lectin production in A. oryzae using the sodM promoter led to overexpression as a specific and abundant intracellular protein.     

Growth and lipid production of Umbelopsis isabellina on a solid substrate—Mechanistic modeling and validation

Microbial lipids are an interesting feedstock for biodiesel. Their production from agricultural waste streams by fungi cultivated in solid-state fermentation may be attractive, but the yield of this process is still quite low. In this article, a mechanistic model is presented that describes growth, lipid production and lipid turnover in a culture of Umbelopsis isabellina on κ-carrageenan plates containing the monomers glucose and alanine as C-source and N-source, respectively, and improves the understanding of the complex solid-state system. The model includes reaction kinetics and diffusion of glucose, alanine and oxygen. It is validated empirically and describes the different phases of the culture very well: exponential growth, linear growth because of oxygen limitation, accumulation of lipids and carbohydrates after local N-depletion and turnover of lipids after local C-depletion. Extending the model with an unidentified extracellular product improved the fit of the model to the data. The model shows that oxygen limitation is extremely important in solid-state cultures using monomers, and explains the difference in production rate with submerged cultures. However, the results also show that the specific lipid production rate in solid-state cultures is much lower than in submerged cultures, which results in a low lipid yield.     

Studies on biosynthesis of hydrolases by trichoderma sp. M7 on submerged and solid-state cultivation conditions

The biosynthesis of cellulases and xylanase by the mould strain Trichoderma sp. M₇ on submerged and solid-state cultivation conditions has been studied. The effect of different inducers on the enzyme biosynthesis on the conditions used was determined. The relation between the enzyme biosynthesis and the morphological state of the producing strain was studied. The advantages of the submerged cultivation conditions with regards to the efficiency of the enzyme inducer are shown.     

Pediocin production by Pediococcus acidilactici in solid state culture on a waste medium: process simulation and experimental results

The production of pediocin by Pediococcus acidilactici was comparatively studied in submerged and solid-state culture, using polyurethane foam particles soaked in commercial (MRS) and waste media with various supplements, where product concentrations were 15 times higher in MRS medium. For the solid state analysis, cultures were treated by successive compression and refilling of tubular minireactors equipped with a piston, without the need for reinoculation. This method was found to be simple, reproducible, and easily controllable, allowing culture productivity to be maintained for long periods of time without alterations in the basic properties of the system. In addition, yields were found to be superior compared to those from submerged culture. The system kinetics were modeled on the basis of widely accepted assumptions with a good fit to the experimental results and observed biomass fluctuations less evident than those predicted by the kinetic model.     

Acylceramides and lanosterol-lipid markers of terminal differentiation in cultured human keratinocytes: Modulating effect of retinoic acid

Summary Epidermal differentiation is accompanied by profound changes in the synthesis of a variety of intracellular proteins and intercellular lipids. In conventional, submerged culture keratinocytes have been shown to lose the ability to synthesize the protein markers of differentiation. They re-express them, however, when they are cultured in medium supplemented with delipidized [retinoic acid (RA)-depleted] serum or in air-exposed cultures using de-epidermized dermis (DED) as a substrate. Recent studies have revealed that acylceramides (AC) and lanosterol (LAN), which are present only in trace amounts in cultures of keratinocytes grown under submerged conditions on DED in medium supplemented with normal serum, become expressed in significant amounts when the culture is lifted to the air-liquid interface. Inasmuch as culture conditions may markedly affect the extent of keratinocyte differentiation, the present study aimed to investigate the effect of normal (RA-containing) or delipidized (RA-depleted) serum and of RA administration on lipid composition (especially of the AC and LAN contents) in cells cultured under submerged and air-exposed conditions. To test a possible effect of dermal substrate (used in the air-exposed model), the lipid composition of keratinocytes grown under submerged conditions on a plastic and on a dermal substrate (de-epidermized dermis, DED) has also been compared. The results revealed that under all culture conditions, RA deprivation of fetal bovine serum resulted in a marked increase of total ceramide content. Even under submerged conditions, the presence of both AC and LAN could be detected. In air-exposed culture, the content of these lipids was markedly increased. Addition of RA at 1 μM concentration to cultures grown in RA-depleted medium induced marked changes in lipid composition under all culture conditions tested. In cells grown under submerged conditions (both on plastic and on DED) AC and LAN were no longer present in detectable amounts. Also in air-exposed culture, a marked decrease in the content of these lipids was observed. These results suggest that liposoluble serum components, like RA, control the synthesis of lipids that are present in later stages of epidermal differentiation.