Production of high-quality edible protein from Candida yeast grown in continuous culture

Candida utilis NRRL Y-900 was grown in aerobic continuous culture with cane molasses as the source of the growth-limiting carbon. At 1% reducing sugar in the chemostal (10 liter working volume) feed medium, addition of Zn (25μM) to a minimal salts medium resulted in an increase in the biomass productivity of the chemostat from 1.7 to 2.6 g/liter/hr with a growth yield of 0.55 g dry biomass/g reducing sugar utilized at D_max. On the average, the yeast biomass was 50–55% protein. At S_R > 2% sugar, the biomass productivity was limited by the oxygen supply. With O₂-supplemented aeration (at S_R = 4.2%)the maximum biomass productivity Was 7.25 g/liter/hr. Aerobic ethanol production was not observed. A highquality undenatured protein fraction was isolate from the yeast homogenate by isoelectric precipitation at pH 4.5. Contaminating nucleic acid was removed as an insoluble complex by chelation with an organic cation (cetavlon). The final protein product contained about 3% RNA (DWB) and was suitable for use as a food additive.     

Effects of Some Environmental Factors on Growth Characteristics of Candida utilis on Peat Hydrolysates

Samples of peat from Pine Island and Brookston bogs in Minnesota were hydrolyzed with various concentrations of HCl or H₂SO₄ solutions, before or after debituminization (an extraction process used to remove waxy materials, bitumens, from peat), to produce peak hydrolysates as growth substrates for Candida utilis. Hydrolysates were neutralized with concentrated NaOH solution to pH 3.5, 4.5, 5.0, 5.5, 6.0, and 7.0. The precipitated humates were removed by filtration. The resulting peat hydrolysates were amended with reagent-grade K₂HPO₄, K₂SO₄, and MgSO₄, 200, 100, and 50 mg per liter of peat hydrolysate, respectively. The debituminized peat produced more total nitrogen (TN) and reducing substances (RS) than the nondebituminized peat. Peat hydrolysates produced by HCl solutions contained slightly higher RS and TN than those produced by H₂SO₄ solutions; however, the latter were better growth substrates than the former. The yield coefficients in both H₂SO₄ and HCl hydrolysates initially decreased at 12 to 24 h and then increased gradually over the remaining incubation period (24 to 96 h). As TN and RS were increased, an increase in cell density, biomass, and productivity was observed. In contrast, a decrease in specific growth rate occurred as the RS and TN were increased. The generation time of C. utilis was affected by the concentrations of RS and TN. A peak substrate yield coefficient was found at pH 5.0 in HCl hydrolysates and at pH 6.0 to 6.5 in H₂SO₄ hydrolysates. Good linear correlation coefficients were found between RS and biomass of C. utilis. The coefficients of correlation increased as the TN level in hydrolysates was increased.     

Studies on quantitative physiology of Trichoderma reesei with two-stage continuous culture for cellulose production

By employing a two-stage continuous-culture system, some of the more important physiological parameters involved in cellulose biosynthesis have been evaluated with an ultimate objective of designing an optimally controlled cellulose process. The two-stage continuous-culture system was run for a period of 1350 hr with Trichoderma reesei strain MCG-77. The temperature and pH were controlled at 32°C and pH 4.5 for the first stage (growth) and 28°C and pH 3.5 for the second stage (enzyme production). Lactose was the only carbon source for the both stages. The ratio of specific uptake rate of carbon to that of nitrogen, Q(C)/Q(N), that supported good cell growth ranged from 11 to 15, and the ratio for maximum specific enzyme productivity ranged from 5 to 13. The maintenance coefficients determined for oxygen, M_O, and for carbon source, M_C, are 0.85 mmol O₂/g biomass/hr and 0.14 mmol hexose/g biomass/hr, respectively. The yield constants determined are: Y_X/O = 32.3 g biomass/mol O₂, Y_X/C = 1.1 g biomass/g C or Y_X/C = 0.44 g biomass/g hexose, Y_X/N = 12.5 g biomass/g nitrogen for the cell growth stage, and Y_X/N = 16.6 g biomass/g nitrogen for the enzyme production stage. Enzyme was produced only in the second stage. Volumetric and specific enzyme productivities obtained were 90 IU/liter/hr and 8 IU/g biomass/hr, respectively. The maximum specific enzyme productivity observed was 14.8 IU/g biomass/hr. The optimal dilution rate in the second stage that corresponded to the maximum enzyme productivity was 0.026 ～ 0.028 hr^?1, and the specific growth rate in the second stage that supported maximum specific enzyme productivity was equal to or slightly less than zero.     

Production of lactic acid and ethanol by Rhizopus oryzae integrated with cassava pulp hydrolysis

Cassava pulp was hydrolyzed with acids or enzymes. A high glucose concentration (>100 g/L) was obtained from the hydrolysis with 1 N HCl at 121 °C, 15 min or with cellulase and amylases. While a high glucose yield (>0.85 g/g dry pulp) was obtained from the hydrolysis with HCl, enzymatic hydrolysis yielded only 0.4 g glucose/g dry pulp. These hydrolysates were used as the carbon source in fermentation by Rhizopus oryzae NRRL395. R. oryzae could not grow in media containing the hydrolysates treated with 1.5 N H₂SO₄ or 2 N H₃PO₄, but no significant growth inhibition was found with the hydrolysates from HCl (1 N) and enzyme treatments. Higher ethanol yield and productivity were observed from fermentation with the hydrolysates when compared with those from fermentation with glucose in which lactic acid was the main product. This was because the extra organic nitrogen in the hydrolysates promoted cell growth and ethanol production.     

The reduction of xylose to xylitol by Candida guilliermondii and Candida parapsilosis: Incidence of oxygen and pH

Summary The ability of C. guilliermondii and C. parapsilosis to ferment xylose to xylitol was evaluated under different oxygen transfer rates in order to enhance the xylitol yield. In C. guilliermondii, a maximal xylitol yield of 0.66 g/g was obtained when oxygen transfer rate was 2.2 mmol/l.h. Optimal conditions to produce xylitol by C. parapsilosis (0.75 g/g) arose from cultures at pH 4.75 with 0.4 mmoles of oxygen/l.h. The response of the yeasts to anaerobic conditions has shown that oxygen was required for xylose metabolism.Nomenclature max maximum specific growth rate (per hour) - qSmax maximum specific rate of xylose consumption (g xylose per g dry biomass per hour) - qpmax maximum specific productivity of xylitol (g xylitol per g dry biomass per hour) - Qp average volumetric productivity of xylitol (g xylitol per liter per hour) - Y_P/S xylitol yield (g xylitol per g substrate utilized) - Y_P'/S glycerol yield (g glycerol per g substrate utilized) - Y_X/S biomass yield (g dry biomass per g substrate utilized)     

Increased production of cellulase of Trichoderma sp. by pH cycling and temperature profiling

Cultivation of Trichoderma reesei QM 9414 on 3% (w/v) cellulose medium (C/N ratio = 8.5) produced 4.5 IU/ml celulase 180 hr at a cell growth of 8.0 g/liter (0.266 g cell/g cellulose). It corresponded to an average cellulase productivity 25.0 IU/liter/hr (3.5 IU/g cell/hr). In the same medium 9.5 g/liter cell mass (0.316 g cell/g cellulose), 6.2 IU/ml cellulase, and 38.75 IU/liter/hr (4.0 IU/g cell/hr) cellulase productivity could be obtained using pH cycling condition during cultivation. Cell mass, cellulase yield, and productivity were further increased to 10.0 g/liter, 7.2 IU/ml, and 44.0 IU/liter/hr (4.5 IU/g cell/hr), respectively, by simultaneous pH cycling and temperature profiling strategy. Results are described.     

Flocculant production from kerosene

Cultivation of Corynebacterium hydrocarboclastus, which is capable of synthesizing an extracellular polymer and utilized hydrocarbons, has been reported. Growth studies in shake flasks and fermenters were made to obtain maximum polymer production. Polymer formation was found to be growth associated. The highest level of polymer accumulation was attained after 50–60 hr cultivation in the fermenter and it amounted to approximately 5.5–6 g/liter of fermentation broth. The medium contained initially 2% (v/v) kerosene as a carbon source. The maximum yield obtained corresponds to 37–40% (w/w) of kerosene supplied. At the same time the cell concentration was 10–13 g/liter which represents the yield of 67–87% (w/w). The rate of polymer production in the exponential phase was 0.25 g/liter hr and cell production rate was 0.27 g/liter hr. Sodium nitrate, 0.5%, and yeast extract, 0.3%, (w/w) were the best nigrogen sources for polymer formation. The highest level of polymer produced in broth was 6 g/liter.     

Production of a Thermostable Lipase by Humicola lanuginosa Grown on Sorbitol-Corn Steep Liquor Medium

For thermostable lipase production by Humicola lanuginosa No. 3, a simple optimized medium consisting of (%, w/v): sorbitol, 1.0; corn steep liquor, 1.0; NaCl, 0.5; CaCl₂–2H₂0, 0.01; Silicone Km-70 (antifoamer), 0.2; and whale oil or castor oil as a lipase inducer, 0.3, was used. The yield of the lipase was about 80 — 120U/ml after 25 hr aerobic cultivation at 45°C when the pH was maintained at 7 to 8. The acetone powder preparation of the enzyme was most active at pH 7.0 and 45°C. The enzyme retained 100% activity on incubation for 20 hr at 60°C. The enzyme was able to hydrolyze almost all forms of natural fats tested (14 kinds), coconut oil being the most rapidly hydrolyzed.     

Continuous fermentation to produce fungal protein. Effect of growth rate on the biomass yield and chemical composition of Fusarium moniliforme

Fusarium moniliforme was grown on a carob aqueous extract in a chemostat for fungal protein production. The substrate was adjusted to provide 0.5% carob sugars supplemented with inorganic salts. The dilution rate varied from 0.086 to 0.227 hr^?1 under constant conditions of temperature (30°C), pH (4.5), and oxygen saturation (60–80%). A yield of 0.709 g dry mycelium/g consumed carob sugar and a productivity value of 0.687 g dry mycelium/liter hr^?1 were obtained at μ = 0.205 hr^?1. The maintenance coefficient was 0.077 g carob sugar/g dry mycelium hr^?1. While the carbohydrate and purine content of dry mycelium increased at μ values from 0.114 to 0.205 hr^?1 both true (Lowry) and crude (N × 6.25) protein contents decreased at the same μ range. Maximum values of 36.3% true and 47.9% crude protein of dry mycelium were obtained at μ = 0.114 hr^?1, whereas a minimum purine content of 99.8 μmol/g corresponding to 6.42% nucleic acids was recorded at μ = 0.086 hr^?1. It was concluded that a continuous fermentation of carob aqueous extract using F. moniliforme should be operated at growth rates of approximately 0.205 hr^?1 in order to maximize protein production.     

Submerged growth of Morchella esculenta in peat hydrolysates

Summary Sphagnum peat acid hydrolysates have been successfully tested as a culture medium for the submerged growth of the fungus Morchella esculenta in batch fermentations. When glucose and (NH₄)₂HPO₄ are added to the hydrolysate, the concentration of biomass produced is lower than that obtained from nonsupplemented hydrolysates having an equivalent total carbohydrate (TCH) content. Therefore, it appears that the concentration of some essential nutrients (s), as well as the TCH content, is affected by the conditions of hydrolysis.     

不同磷、硫及二氧化碳浓度对标志链带藻生长和碳水化合物积累的影响

【目的】为了研究不同磷、硫及二氧化碳浓度对标志链带藻(Desmodesmus insignis)生长与碳水化合物积累的影响,本实验以改良BG11培养基为基础,设计了8种不同初始K_2HPO_4浓度、8种不同初始MgSO_4浓度及4种二氧化碳浓度培养标志链带藻。【方法】采用干重法和苯酚-硫酸法分别测定其生物质浓度与总碳水化合物的含量。【结果】实验结果显示,在高磷浓度(0.460 mmol/L)下生物量达到最高为6.37 g/L,磷浓度为0.230 mmol/L (对照组)时总碳水化合物含量及单位体积产率达到最高,分别为45.40%(%干重)和0.20 g/(L·d)。不同初始MgSO_4浓度实验结果显示,高硫浓度有利于标志链带藻生长及碳水化合物的积累,生物量、总碳水化合物含量及单位体积产率分别在硫浓度为1.217 mmol/L、0.609 mmol/L和1.824 mmol/L时达到最高,分别为7.02 g/L、51.6%(%干重)及0.26 g/(L·d)。当二氧化碳浓度为3%(V/V)时,标志链带藻生物量、总碳水化合物含量及单位体积产率均达到最高,分别为6.81 g/L、44.03%和0.20 g/(L·d)。【结论】因此,磷浓度为0.230 mmol/L、硫浓度为1.824 mmol/L和二氧化碳浓度为3%时最有利于标志链带藻生长及碳水化合物的积累。     

Carbon balance of rewetted and drained peat soils used for biomass production: a mesocosm study

Rewetting of drained peatlands has been recommended to reduce CO₂ emissions and to restore the carbon sink function of peatlands. Recently, the combination of rewetting and biomass production (paludiculture) has gained interest as a possible land use option in peatlands for obtaining such benefits of lower CO₂ emissions without losing agricultural land. This study quantified the carbon balance (CO₂, CH₄ and harvested biomass C) of rewetted and drained peat soils under intensively managed reed canary grass (RCG) cultivation. Mesocosms were maintained at five different groundwater levels (GWLs), that is 0, 10, 20 cm below the soil surface, representing rewetted peat soils, and 30 and 40 cm below the soil surface, representing drained peat soils. Net ecosystem exchange (NEE) of CO₂ and CH₄ emissions was measured during the growing period of RCG (May to September) using transparent and opaque closed chamber methods. The average dry biomass yield was significantly lower from rewetted peat soils (12 Mg ha^?1) than drained peat soils (15 Mg ha^?1). Also, CO₂ fluxes of gross primary production (GPP) and ecosystem respiration (ER) from rewetted peat soils were significantly lower than from drained peat soils, but net uptake of CO₂ was higher from rewetted peat soils. Cumulative CH₄ emissions were negligible (0.01 g CH₄ m^?2) from drained peat soils but were significantly higher (4.9 g CH₄ m^?2) from rewetted peat soils during measurement period (01 May–15 September 2013). The extrapolated annual C balance was 0.03 and 0.68 kg C m^?2 from rewetted and drained peat soils, respectively, indicating that rewetting and paludiculture can reduce the loss of carbon from peatlands.     

Effect of initial bacteria concentration on hydrogen gas production from cheese whey powder solution by thermophilic dark fermentation

Dark fermentative hydrogen gas production from cheese whey powder solution was realized at 55°C. Experiments were performed at different initial biomass concentrations varying between 0.48 and 2.86 g L^?1 with a constant initial substrate concentration of 26 ± 2 g total sugar (TS) per liter. The highest cumulative hydrogen evolution (633 mL, 30°C), hydrogen yield (1.56 mol H₂ mol^?1 glucose), and H₂ formation rate (3.45 mL h^?1) were obtained with 1.92 g L^?1 biomass concentration. The specific H₂ production rate decreased with increasing biomasss concentration from the highest value (47.7 mL g^?1 h^?1) at 0.48 g L^?1 biomass concentration. Total volatile fatty acid concentration varied beetween 10 and 14 g L^?1 with the highest level of 14.2 g L^?1 at biomass concentration of 0.48 g L^?1 and initial TS content of 28.4 g L^?1. The experimental data were correlated with the Gompertz equation and the constants were determined. The most suitable initial biomass to substrate ratio yielding the highest H₂ yield and formation rate was 0.082 g biomass per gram of TS. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 931–936, 2012     

Characteristics of transformedPanax ginseng C.A. Meyer hairy roots: Growth and nutrient profile

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, which are established via the infection of ginseng root discs withRhizobium rhizogenes, have been used to construct profiles of both biomass growth and nutrient consumption in flask cultures. In a 250 mL shake flask culture, the maximum biomass was observed on the 59th day of the culture period, at 216.8 g (fresh wt) per liter or 11.4 g (dry wt) per liter. The hairy roots were determined to have a growth rate of 0.355 g-DW/g cells/day during the exponential growth phase and a maximum specific growth rate on day 7. Total ginseng saponin and phenolic compound contents were noted to have increased within the latter portion of the culture period. Linear correlations between increases in biomass weight and nutrient uptake were used to imply the conductivity yield 2.60 g-DW/(L·mS) and carbon yield 0.45 g-DW/(g sugar) in the 250 mL flask cultures. The biomass yield when two different nitrogen sources were used (ammonia and nitrate) was shown to remain approximately constant, at 0.47 g-DW/(l·mM NH₄) and 0.33 g-DW/(L·mM NO₃); it remained at these levels for 16 days with the ammonia, and for 24 days with the nitrate. The biomass yield when a phosphate source was used was also shown to remain approximately constant for 9 days, at 3.17 g-DW/(L·mM PO₄), with an R² of 0.99.     

Biomethanation: Anaerobic fermentation of CO2, H2 and CO to methane

Studies to examine the microbial fermentation of coal gasification products (CO₂, H₂ and CO) to methane have been done with a mixed culture of anaerobic bacteria selected from an anaerobic sewage digestor. The specific rate of methane production at 37°C reached 25 mmol/g cell hr. The stoichiometry for methane production was 4 mmol H₂/mol CO₂. Cell recycle was used to increase the cell concentration from 2.5 to 8.3 g/liter; the volumetric rate of methane production ran from 1.3 to 4 liter/liter hr. The biogasification was also examined at elevated pressure (450 psi) and temperature to facilitate interfacing with a coal gasifier. At 60°C, the specific rate of methane production reached 50 mmol/g cell hr. Carbon monoxide utilization by the mixed culture of anaerobes and by a Rhodopseudomonas species was examined. Both cultures are able to carry out the shift conversion of CO and water to CO₂ and hydrogen.     

法夫酵母PLX-All发酵纤维素酶水解物合成虾青素

法夫酵母（Phaffia rhodozyma)PLX-All菌株能够发酵纤维素酶水解物进行虾青素的生物合成。纤维素的酶解物主要为纤维二糖和葡萄糖,在另外添加适量其它营养物后可被法夫酵母发酵用于生长及合成虾青素。摇瓶试验结果表明,培养108h,法夫酵母的生物量可达2.3g／L,虾青素的产率达913.4g／g干细胞,虾青素体积产率为2.1mg／L。在2L罐的发酵试验中,法夫酵母的生物量可达3.23g／L（第96h）,虾青素的产率达581.4g／g干细胞,虾青素体积产率达1.88mg／L。     

法夫酵母PLX-All发酵纤维素酶水解物合成虾青素*

法夫酵母（Phaffia rhodozyma)PLX-All菌株能够发酵纤维素酶水解物进行虾青素的生物合成。纤维素的酶解物主要为纤维二糖和葡萄糖,在另外添加适量其它营养物后可被法夫酵母发酵用于生长及合成虾青素。摇瓶试验结果表明,培养108h,法夫酵母的生物量可达2.3g／L,虾青素的产率达913.4g／g干细胞,虾青素体积产率为2.1mg／L。在2L罐的发酵试验中,法夫酵母的生物量可达3.23g／L（第96h）,虾青素的产率达581.4g／g干细胞,虾青素体积产率达1.88mg／L。     

Bioconversion of cheese whey permeate into fungal oil by <Emphasis Type="Italic">Mucor circinelloides</Emphasis>

Background

Oleaginous fungi are efficient tools to convert agricultural waste streams into valuable components. The filamentous fungus Mucor circinelloides was cultivated in whey permeate, a byproduct from cheese production, to produce an oil-rich fungal biomass. Response surface methodology was used to optimize the fermentation conditions such as pH and temperature for increased biomass yield and lipid accumulation. Quantification and characterization of the fungal biomass oil was conducted.

Results

Upstream lactose hydrolysis of the whey permeate increased the biomass yield from 2.4 to 7.8 (g dry biomass/L) compared to that of non-hydrolyzed whey permeate. The combination of low pH (4.5) and pasteurization minimized microbial competition, thus favoring fungal growth. A central composite rotatable design was used to evaluate the effects of temperature (22.4–33.6 °C) and a lower pH range (3.6–4.7) on biomass yield and composition. The highest biomass yield and oil content was observed at high temperature (33.6 °C), while the pH range evaluated had a less pronounced effect. The predictive model was validated at the optimal conditions of 33.6 °C and pH 4.5. The fungal biomass yield plateaued at 9 g dry cell weight per liter, while the oil content and lipid yield reached a maximum of 24% dry biomass and 2.20 g/L, respectively, at 168 h. Triacylglycerides were the major lipid class (92%), which contained predominantly oleic (41%), palmitic (23%), linoleic (11%), and γ-linolenic acid (9%).

Conclusions

This study provided an alternative way of valorization of cheese whey permeate by using it as a substrate for the production of value-added compounds by fungal fermentation. The fatty acid profile indicates the suitability of M. circinelloides oil as a potential feedstock for biofuel production and nutraceutical applications.

     

Production of microbial protein from tree bark by Phanerochaete chrysosporium

Phanerochaete chrysosporium was grown in fermentors on NaOH-extracted maple, pine, and cedar barks at the optimum substrate concentration of 1% (w/v). The yields (mg protein/liter) on maple, pine, and cedar were 1500, 1200, and 880, respectively, which are probably due to the different lignin contents of the barks. Lignin is not utilized. The productivities at 30°C obtained for pine (4.07 × 10^?2 g protein/liter hr) and cedar (2.63 × 10^?2 g protein/liter hr) barks were greater than for maple (2.63 × 10^?2 g protein/liter hr). The substrate (bark) was the limiting component of the fermentation. Over the 26–38°C temperature range protein productivity increased by a factor of three (1.55 × 10^?2 vs. 4.61 × 10^?2 g protein/liter hr) for maple bark. Low agitation rates resulted in an overproduction of cellulase and reduced levels of microbial protein.     

Comparative evaluations of cellulosic raw materials for second generation bioethanol production

Aims: To evaluate sugar recoveries and fermentabilities of eight lignocellulosic raw materials following mild acid pretreatment and enzyme hydrolysis using a recombinant strain of Zymomonas mobilis. Methods and Results: Dilute acid pretreatment (2% H₂SO₄) with 10% (w/v) substrate loading was performed at 134°C for 60 min followed by enzyme hydrolysis at 60°C. The results demonstrated that hydrolysis of herbaceous raw materials resulted in higher sugar recoveries (up to 60–75%) than the woody sources (<50%). Fermentation studies with recombinant Z. mobilis ZM4 (pZB5) demonstrated that final ethanol concentrations and yields were also higher for the herbaceous hydrolysates. Significant reduction in growth rates and specific rates of sugar uptake and ethanol production occurred for all hydrolysates, with the greatest reductions evident for woody hydrolysates. Further studies on optimization of enzyme hydrolysis established that higher sugar recoveries were achieved at 50°C compared to 60°C following acid pretreatment. Conclusions: Of the various raw materials evaluated, the highest ethanol yields and productivities were achieved with wheat straw and sugarcane bagasse hydrolysates. Sorghum straw, sugarcane tops and Arundo donax hydrolysates were similar in their characteristics, while fermentation of woody hydrolysates (oil mallee, pine and eucalyptus) resulted in relatively low ethanol concentrations and productivities. The concentrations of a range of inhibitory compounds likely to have influence the fermentation kinetics were determined in the various hydrolysates. Significance and Impact of the Study: The study focuses on lignocellulosic materials available for second generation ethanol fermentations designed to use renewable agricultural/forestry biomass rather than food‐based resources. From the results, it is evident that relatively good sugar and ethanol yields can be achieved from some herbaceous raw materials (e.g. sugarcane bagasse and sorghum straw), while much lower yields were obtained from woody biomass.