Valorization of solid olive mill wastes by cultivation of a local strain of edible mushrooms

Olive oil industry generates huge quantities of solid olive mill wastes (SOMW), causing environmental damage. Cultivation of edible mushrooms, such as Pleurotus ostreatus is a valuable approach for SOMW valorization. A local strain mycelium (Tizi-Ouzou, Algeria) of P. ostreatus (LPO) was isolated from castor oil plants. Oyster mushroom spawn, produced on barley grains, was used to inoculate wet SOMW, steamed in a traditional steamer during 45 min. The mycelium growth rate on SOMW was first estimated in Petri dish by measuring the surface colonized by the mycelium. The fruit body yields were estimated on culture bags containing 2 kg each of SOMW inoculated at 7% (w/w). The local strain potential was compared with that of a commercial one. Both strains produced high-quality mushrooms, but with low yields. The supplementation of the SOMW with wheat straw at the rate of 10% and 2% of CaCO₃ had significantly enhanced the productivity of the two strains, multiplying it by 3.2 for LPO and by 2.6 for CPO.     

Kinetics of batch single cell protein production from rice polishings with Candida utilis in continuously aerated tank reactors

Single cell protein was produced from the defatted rice polishings by fermentation with Candida utilis in an aerated 14-L fermentor to optimize bioprocess variables. Maximum values of specific growth rate coefficient (mu, h(-1)), cell mass yield (Y(X/S), g/g) and cell mass productivity (g/Lh) were 0.31, 0.65, and 1.24, respectively under optimized conditions of aeration rate (1 v.v(-1) m(-1)), dissolved oxygen (50%), corn steep liquor (5%), temperature (35 degrees C), and substrate concentration (90 g rice polishings/L) in yeast salt medium (pH 6.0). The kinetic parameters for 50-L fermentor under same conditions were 0.33 h(-1), 0.66 g/g, 1.33 g/Lh, 2.25 g/Lh, 1.23 g/Lh, 0.45 g/g substrate and 0.20 g/g cell h for mu, Y(X/S), Q(X), Q(S), Q(CP), Y(TP/S), and q(CP), respectively and were significantly higher than their respective values reported on C. utilis in batch culture studies. This biomass protein contained 23.6%, 32.75%, 11.50%, 12.95%, 10.5%, and 0.275% true protein, crude protein, crude fiber, ash, cellulose and RNA content respectively. This implied that the fermentation process could be up scaled to manufacture animal feed. Gross metabolizable energy content of dried SCP was 29,711 kcal/kg and indicated that the SCP could serve both as energy as well as a protein source. Yeast can replace expensive feed ingredients currently being incorporated in poultry feed and can reduce cost of poultry ration by 0.33 US dollars-0.51 US dollars/100 kg bag and improve the economics of feed production in our country.     

Influence of specific growth rate on biomass yield, productivity, and compostion of Candida utilis in batch and continuous culture.

Candida utilis was grown in batch and continuous culture on prickly pear juice as sole carbon and energy source. In batch culture the maximum specific growth rate (mum) and the substrate yield coefficient (Yps) varied according to sugar concentration. When the fermentation was carried out with 1% sugar, mum and Ys were 0.47/h and 42.6%, respectively. The best yields occurred in a chemostat at the pH range of 3.5 to 4.5 and temperature of 30 C. A beneficial effect on Ys was observed when the dilution rate (D) was increased. At a D of 0.55/h, the productivity was 2.38 g/liter per h. The maintenance coefficient attained a value of 0.09 g of sugar/g of biomass per h. Increases of D produced higher protein contents of the biomass. The information obtained indicates that protein production with Candida utilis, using prickly pear juice, should be carried out a high dilution rates where the Ys and protein content of the cell mass are also higher.     

Biomass production from glutamate fermentation wastewater by the co-culture of Candida halophila and Rhodotorula glutinis

In this study, the biomass production and pollutant removal from high-strength glutamate fermentation wastewater (GFW) using yeast isolates was investigated. Following enrichment culture, two species of yeasts, Candida halophila and Rhodotorula glutinis, were isolated from raw GFW with chemical oxygen demand (COD) and ammonia-nitrogen levels of 40 and 16 g l(-1), respectively. The binary mixed yeast culture was cultivated batchwise in 2.5-fold diluted GFW from which 85% of COD and 96% of reducing sugar were removed. The resulting yeast biomass contained 56% crude protein, 36.0% carbohydrate and 0.4% crude lipid. The amino acid composition of mixed yeast cells was balanced and was comparable with that of C. utilis and soybean.     

Influence of specific growth rate on biomass yield, productivity, and compostion of Candida utilis in batch and continuous culture.

Candida utilis was grown in batch and continuous culture on prickly pear juice as sole carbon and energy source. In batch culture the maximum specific growth rate (mum) and the substrate yield coefficient (Yps) varied according to sugar concentration. When the fermentation was carried out with 1% sugar, mum and Ys were 0.47/h and 42.6%, respectively. The best yields occurred in a chemostat at the pH range of 3.5 to 4.5 and temperature of 30 C. A beneficial effect on Ys was observed when the dilution rate (D) was increased. At a D of 0.55/h, the productivity was 2.38 g/liter per h. The maintenance coefficient attained a value of 0.09 g of sugar/g of biomass per h. Increases of D produced higher protein contents of the biomass. The information obtained indicates that protein production with Candida utilis, using prickly pear juice, should be carried out a high dilution rates where the Ys and protein content of the cell mass are also higher.     

一种适用于产朊假丝酵母的基因敲除系统及其在gsh1基因敲除中的应用

报道一种适用于产朊假丝酵母Candida utilis的基因敲除系统,利用该敲除系统获得gsh1基因敲除杂合突变株。根据不同种属酵母菌γ-谷氨酰半胱氨酸合成酶(γ-GCS)蛋白质的保守序列,克隆C.utilis SZU 07-01的gsh1基因;以商品化质粒pPICZalpha A为基础,构建gsh1基因的敲除载体pPICZalpha A-kan 3,其中,kan基因的启动子TEF被替换为来自于C.utilis SZU 07-01的GAP启动子(pGAP:kan)。质粒电转化C.utilis,获得gsh1基因敲除杂合突变株C.utilis GSH-6。结合发酵培养得到的数据进行分析,突变株的γ-GCS酶活比出发菌株降低17.5%,GSH合成量降低61%,细胞干重降低18.5%。所构建敲除组件pGAP:kan的成功应用为从分子水平研究C.utilis中谷胱甘肽(GSH)的生理功能提供了一种新借鉴。     

Factors affecting the growth and the oil accumulation of marine microalgae, Tetraselmis suecica

Biomass production and oil productivity in microalgae culture are the most important key factors for algal biodiesel production. However, proper culture condition for the biomass production of microalgae is different from that for the oil production of microalgae. A study on the biomass production of Tetraselmis suecica using various light intensities and nitrate concentrations as growth factors was carried out to evaluate proper culture conditions in 20-L batch culture. The effect of nitrate depletion on the oil accumulation was also evaluated with two-stage culture. It took 5 days to reach the stationary phase for the cultures of T. suecica on the light intensities of 108.9 and 133.1 μmol m(-2 )s(-1) with biomass of 0.89 and 0.88 g dcw L(-1), respectively. Biomass productions of 1.07 and 1.00 g dcw L(-1) were obtained with the nitrate concentrations of 18.6 and 24.7 mg L(-1), respectively. The two-stage culture increased oil contents from 7.6 to 17.3% (w/w) and contents of C(16)-C(18) fatty acids from 540.2 to 720.5 mg g(-1) oil. The predominant fatty acid was palmitic acid (C(16:0)) in nitrate depletion group, however, oleic acid (C(18:1)) was predominated in nitrate added groups. The two-stage culture enhanced overall oil productivity of 18.7 mg g(-1) day(-1) which is higher than that of 12.2 mg g(-1) day(-1) in single-stage culture.     

Use of waste Chinese cabbage as a substrate for yeast biomass production

The possibility of using waste Chinese cabbage (Brassica campestris) as a substrate for microbial biomass production was investigated. The juice from waste Chinese cabbage contains relatively high amounts of reducing sugars suitable for yeast culture. The cell mass and protein content of four species of yeast, Candida utilis, Pichia stipitis, Kluyveromyces marxianus, and Saccharomyces cerevisiae, were determined when cultured in juice extracted from cabbage waste. Compared to YM broth containing the same level of sugar, all the strains except C. utilis showed higher total protein production in cabbage juice medium (CJM).     

Optimization of solid substrate fermentation of wheat straw

Optimal conditions for solid substrate fermentation of wheat straw with Chaetomium cellulolyticum in laboratory-scale stationary layer fermenters were developed. The best pretreatment for wheat straw was ammonia freeze explosion, followed by steam treatment, alkali treatment, and simple autoclaving. The optimal fermentation conditions were 80% (w/w) moisture content; incubation temperature of 37 degrees C; 2% (w/w) unwashed mycelial inoculum; aeration at 0.12 L/h/g; substrate thickness of 1 to 2 cm; and duration of three days. Technical parameters for this optimized fermentation were: degree of substance utilization, 27.2%; protein yield/substrate, 0.09 g; biomass yield/bioconverted substrate, 0.40 g; degree of bioconversion of total available sugars in the substrate, 60.5%; specific efficiency of bioconversion, 70.8%; and overall efficiency of biomass production from substrate, 42.7%. Mixed culturing of Candida utilis further increased biomass production by 20%. The best mode of fermentation was a semicontinuous fed-batch fermentation where one-half of the fermented material was removed at three-day intervals and replaced by fresh substrate. In this mode, protein production was 20% higher than in batch mode, protein productivity was maintained over 12 days, and sporulation was prevented.     

Optimizing the continuous production of Candida utilis and Saccharomycopsis fibuliger on potato processing wastewater.

The yeasts Candida utilis and Saccharomycopsis fibuliger were propagated as a source of single-cell protein in a continuous, mixed, aerobic, single-stage cultivation on blancher water generated during potato processing. A series of steady-state experiments based on a two-level factorial design, half-replicate modified with an intermediate experiment, was performed to determine the effect of pH, 3.8 to 4.8; dissolved oxygen, 42 to 80% saturation; dilution rate, 0.17 to 0.31 h(-1); and temperature, 27 to 32 degrees C on the amount of carbon consumed, the rate of carbon consumption (R(c)), the amount of reducing sugar consumed, the rate of sugar consumption (R(g)), the amount of protein produced, the rate of protein production (R(p)), the yield from carbon, and the yield from reducing sugar. The results were analyzed by stepwise multiple regression and Fisher's least significant difference test. Analyses showed that high dilution rates resulted in increased R(c), R(g), and R(p) and indicated that a rate of 0.31 h(-1) was below the critical dilution rate. A temperature of 32 degrees C increased the amount of carbon consumed by 34%. A pH of 4.3 to 4.8 increased the amount of protein produced. The yield from carbon was constant, and the relatively high yield from reducing sugar indicated that other substrates were consumed. Dissolved oxygen was in excess at 42% saturation and above. Since C. utilis predominated the mixed cultures and amylase production appeared to be limited, a single-stage fermentation lacked efficiency. The experimental design allowed preliminary optimization of major environmental variables with relatively few experiments and provided a basis for future kinetic studies.     

Light enables a very high efficiency of carbon storage in developing embryos of rapeseed

The conversion of photosynthate to seed storage reserves is crucial to plant fitness and agricultural production, yet quantitative information about the efficiency of this process is lacking. To measure metabolic efficiency in developing seeds, rapeseed (Brassica napus) embryos were cultured in media in which all carbon sources were [U-14C]-labeled and their conversion into CO2, oil, protein, and other biomass was determined. The conversion efficiency of the supplied carbon into seed storage reserves was very high. When provided with 0, 50, or 150 micromol m(-2) s(-1) light, the proportion of carbon taken up by embryos that was recovered in biomass was 60% to 64%, 77% to 86%, and 85% to 95%, respectively. Light not only improved the efficiency of carbon storage, but also increased the growth rate, the proportion of 14C recovered in oil relative to protein, and the fixation of external 14CO2 into biomass. Embryos grown at 50 micromol m(-2) s(-1) in the presence of 5 microM 1,1-dimethyl-3-(3,4-dichlorophenyl) urea (an inhibitor of photosystem II) were reduced in total biomass and oil synthesis by 3.2-fold and 2.8-fold, respectively, to the levels observed in the dark. To explore if the reduced growth and carbon conversion efficiency in dark were related to oxygen supplied by photosystem II, embryos and siliques were cultured with increased oxygen. The carbon conversion efficiency of embryos remained unchanged when oxygen levels were increased 3-fold. Increasing the O2 levels surrounding siliques from 21% to 60% did not increase oil synthesis rates either at 1,000 micromol m(-2) s(-1) or in the dark. We conclude that light increases the growth, efficiency of carbon storage, and oil synthesis in developing rapeseed embryos primarily by providing reductant and/or ATP.     

温度对谷胱甘肽分批发酵的影响及动力学模型

研究了24～32℃范围内产朊假丝酵母生产谷胱甘肽的分批发酵过程,发现较高温度对细胞生长有促进作用,而较低温度则更有利于谷胱甘肽产量的提高。应用改进的Logistic和LuedekingPiret方程分别对细胞生长动力学和谷胱甘肽合成动力学进行了模拟,得到不同温度下各种动力学参数。在此基础上,进一步研究了温度同细胞生长动力学参数之间的内在联系,得到谷胱甘肽分批发酵过程中细胞浓度的变化同温度以及底物浓度之间的一般关系式：dX-dt＝［0.0224(T+1.7)］2X(1-X/X_max)1+S｛8.26×10.6×exp［-31477/R/(T+273)］｝。验证实验结果表明,该模型具有很好的适用性。     

Enhanced intracellular glutathione synthesis and excretion capability of Candida utilis by using a low pH-stress strategy

AIMS: To study the effect of low pH stress on glutathione (GSH) synthesis and excretion capability of GSH fermentation production in Candida utilis. METHODS AND RESULTS: When C. utilis WSH 02-08 was cultivated in a glucose-ammonium sulfate medium without pH control, GSH leakage occurred when the pH of the medium decreased to 1.5. However, analysis of the cell viability indicated that the cells were not lysed. To further study the effect of low pH stress on GSH production, pH-controlled batch cultures were conducted, where the pH was switched from 5.5 to 1.2 at 24 h and maintained at 1.2 for 6 h. Nearly all intracellular GSH was leaked into the medium and the cell viability decreased dramatically, conceiving a long-term exposure of strain WSH 02-08 at low pH environment led to a complete cell lysis. A critical point (treated at pH 1.2 for 3 h) was experimentally determined, where most cells were alive but suffering a low pH stress. Low pH-stressed C. utilis cells displayed an increased intracellular GSH synthesis and export capability, which protected the cells against short-term low pH treatment. CONCLUSIONS: Using this knowledge, a low pH-stress strategy was developed and applied in fed-batch production of GSH and 197.3 mg l-1 of GSH was secreted into the medium. The GSH-specific production yield could be increased from 2.11 to 2.67% (w/w), and the total GSH concentration could reach 737.1 mg l-1 and increased by 24.9%. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of GSH secretion of C. utilis at low pH. This study demonstrated the importance of the physiology-based fermentation strategy in the production of useful metabolites.     

High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters

The aim of the study was to obtain high quality biodiesel production from a microalga Chlorella protothecoids through the technology of transesterification. The technique of metabolic controlling through heterotrophic growth of C. protothecoides was applied, and the heterotrophic C. protothecoides contained the crude lipid content of 55.2%. To increase the biomass and reduce the cost of alga, corn powder hydrolysate instead of glucose was used as organic carbon source in heterotrophic culture medium in fermenters. The result showed that cell density significantly increased under the heterotrophic condition, and the highest cell concentration reached 15.5 g L(-1). Large amount of microalgal oil was efficiently extracted from the heterotrophic cells by using n-hexane, and then transmuted into biodiesel by acidic transesterification. The biodiesel was characterized by a high heating value of 41 MJ kg(-1), a density of 0.864 kg L(-1), and a viscosity of 5.2 x 10(-4) Pa s (at 40 degrees C). The method has great potential in the industrial production of liquid fuel from microalga.     

Sunflower shells utilization for energetic purposes in an integrated approach of energy crops: laboratory study pyrolysis and kinetics

Sunflower is a traditional crop which can be used for the production of bioenergy and liquid biofuels. A study of the pyrolytic behaviour of sunflower residues at temperatures from 300 to 600 degrees C has been carried out. The experiments were performed in a captive sample reactor under atmospheric pressure and helium as sweeping gas. The yields of the derived pyrolysis products were determined in relation to temperature, with constant sweeping gas flow of 50 cm3 min(-1) and heating rate of 40 degrees Cs(-1). The maximum gas yield of around 53 wt.% was obtained at 500 degrees C, whereas maximum oil yield of about 21 wt.% was obtained at 400 degrees C. A simple first order kinetic model has been applied for the devolatilization of biomass. Kinetic constants have been estimated: E=78.15 kJ mol(-1); k(0)=1.03 x 10(3)s(-1).     

Yeast pyruvate decarboxylases: variation in biocatalytic characteristics for (R)-phenylacetylcarbinol production

Based on previous studies, Candida utilis pyruvate decarboxylase (PDC) proved to be a stable and high productivity enzyme for the production (R)-phenylacetylcarbinol (PAC), a pharmaceutical precursor. However, a portion of the substrate pyruvate was lost to by-product formation. To identify a source of PDC which might overcome this problem, strains of four yeasts -- C. utilis, Candida tropicalis, Saccharomyces cerevisiae and Kluyveromyces marxianus -- were investigated for their PDC biocatalytic properties. Biotransformations were conducted with benzaldehyde and pyruvate as substrates and three experimental systems were employed (in the order of increasing benzaldehyde concentrations): (I) aqueous (soluble benzaldehyde), (II) aqueous/benzaldehyde emulsion, and (III) aqueous/octanol-benzaldehyde emulsion. Although C. utilis PDC resulted in the highest concentrations of PAC and was the most stable enzyme, C. tropicalis PDC was associated with the lowest acetoin formation. For example, in system (III) the ratio of PAC over acetoin was 35 g g(-1) for C. tropicalis PDC and 9.2 g g(-1) for C. utilis PDC. The study thereby opens up the potential to design a PDC with both high productivity and high yield characteristics.     

Production of microbial lipid: Effects of growth rate and oxygen on lipid synthesis and fatty acid composition of Rhodotorula gracilis

Microbial lipids produced by Rhodotorula gracilis NRRL Y-1091 grown in continuous culture under nitrogen-limiting condition were evaluated and the effects of growth rate and oxygen concentration on the degree of unsaturatoin of fatty acids studied. As the growth rate increased the protein content of the biomass increased but cell biomass, lipid content, and lipid productivity decreased; the specific lipid production rate remained constant at about 0.012 g lipid/g dry biomass/h. The maximum lipid content recorded was 49.8% (w/w) of the cell mass at a growth rate of 0.02 h(-1). The growth rate also affected fatty acid composition; polyunsaturated fatty acids (C18:2 and C18:3) increaded with growth rate while other fatty acids (C16:0, C18:0, C18:1) decreased. Increase in oxygen concentration between 5 and 234muM increased the lipid content without significantly affecting its degree of unsaturation. On the other hand, the degree of unsaturation was significantly affected by specific oxygen uptake rate for this obligate aerobe, Rh. gracilis.     

The influence of biomass temperature on biostabilization-biodrying of municipal solid waste

A laboratory study was carried out to obtain data on the influence of biomass temperature on biostabilization-biodrying of municipal solid waste (initial moisture content of 410 g kg wet weight (w.w.)(-1)). Three trials were carried out at three different biomass temperatures, obtained by airflow rate control (A = 70 degrees C, B = 60 degrees C and C = 45 degrees C). Biodegradation and biodrying were inversely correlated: fast biodrying produced low biological stability and vice versa. The product obtained from process A was characterized by the highest degradation coefficient (166 g kg TS0(-1); TS0(-1) = initial total solid content) and lowest water loss (409 g kg W0(-1); W0 = initial water content). Due to the high reduction of easily degradable volatile solid content and preservation of water, process A produced the highest biological stability (dynamic respiration index, DRI = 141 mg O2 kg VS(-1); VS = volatile solids) but the lowest energy content (EC = 10,351 kJ kg w.w.(-1)). Conversely, process C which showed the highest water elimination (667 g kg W0(-1)), and lowest degradation rate (18 g kg TS0(-1)) was optimal for refuse-derived fuel (RDF) production having the highest energy content (EC = 14,056 kJ kg w.w.(-1)). Nevertheless, the low biological stability reached, due to preservation of degradable volatile solids, at the end of the process (DRI = 1055 mg O2 kg VS(-1)), indicated that the RDF should be used immediately, without storage. Trial B showed substantial agreement between low moisture content (losses of 665 g kg W0(-1)), high energy content (EC = 13,558 kJ kg w.w.(-1)) and good biological stability (DRI = 166 mg O2 kg VS(-1)), so that, in this case, the product could be used immediately for RDF or stored with minimum pollutant impact (odors, leaches and biogas production).     

Protein enrichment of potato processing waste through yeast fermentation

Potato starch obtained from waste waters of chips manufacturing was used as a fermentation substrate for yeast protein enrichment. Among 18 yeast strains, 6 strains were screened according to their biomass yield and protein content after fermentation for 16 h at 30 degrees C in an aerated glucose-based liquid media (4.5 Ls). Using concentrated media (25% solids) made from potato starch pre-hydrolyzed with malt flour and batch-fermented for 20 h at 26 degrees C under aerobic conditions, Candida utilis ATCC 9256 was the most efficient protein-forming strain. Scaled-up at the 100 Ls level, the aerobic batch process was improved under fed-batch conditions with molasses supplementation. After drying, fermented starch contained 11-12% protein, including 7-8% yeast protein.     

A theoretical evaluation of growth yields of yeasts

Growth yields of Saccharomyces cerevisiae and Candida utilis in carbon-limited chemostat cultures were evaluated. The yields on ethanol and acetate were much lower in S. cerevisiae, in line with earlier reports that site I phosphorylation is absent in this yeast. However, during aerobic growth on glucose both organisms had the same cell yield. This can be attributed to two factors: --S. cerevisiae had a lower protein content than C. utilis; --uptake of glucose by C. utilis requires energy whereas in S. cerevisiae it occurs via facilitated diffusion. Theoretical calculations showed that, as a result of these two factors, the ATP requirement for biomass formation in C. utilis is 35% higher than in S. cerevisiae (theoretical YATP values of 20.8 and 28.1, respectively). The experimental YATP for anaerobic growth of S. cerevisiae on glucose was 16 g biomass.mol ATP-1. In vivo P/O-ratios can be calculated for aerobic growth on ethanol and acetate, provided that the gap between the theoretical and experimental ATP requirements as observed for growth on glucose is taken into account. This was done in two ways: --via the assumption that the gap is independent of the growth substrate (i.e. a fixed amount of ATP bridges the difference between the theoretical and experimental values). --alternatively, on the assumption that the difference is a fraction of the total ATP expenditure, that is dependent on the substrate. Calculations of P/O-ratios for growth of both yeasts on glucose, ethanol, and acetate made clear that only by assuming a fixed difference between theoretical and experimental ATP requirements, the P/O-ratios are more or less independent of the growth substrate. These P/O-ratios are approximately 30% lower than the calculated mechanistic values.