Effect of ammonium-N on malic enzyme and lipid production in Rhodotorula glutinis grown on monosodium glutamate wastewater

Rhodotorula glutinis, an oil producing strain, can utilize monosodium glutamate (MSG) wastewater as a raw material for lipid production. The effects of ammonium-N in the MSG wastewater (ammonium 15,000–25,000?mg/L, COD 30,000–50,000?mg/L) on cell growth, lipid accumulation and malic enzyme activity of R. glutinis have been studied. Four initial ammonium sulfate concentrations in the medium were set, which were 20, 60, 100, and 140?g/L. With an increase in the ammonium sulfate concentration, the uptake of ammonia nitrogen and lipid accumulation increased while the biomass decreased at 72?h. The maximum value of ammonia nitrogen consumption reached 5.77?g/L for an initial ammonium sulfate concentration of 140?g/L at 72?h. In addition, 60?g/L ammonium sulfate concentration may be an appropriate concentration for R. glutinis cultivation. The activity of the malic enzyme was measured and the results showed that there was a linear relationship between the intracellular lipid content and the total malic enzyme activity.     

Biotechnological conversions of bio‐diesel‐derived crude glycerol by Yarrowia lipolytica strains

In the present report, crude glycerol, waste discharged from bio‐diesel production, was used as carbon substrate for three natural Yarrowia lipolytica strains (LFMB 19, LFMB 20 and ACA‐YC 5033) during growth in nitrogen‐limited submerged shake‐flask experiments. In media with initial glycerol concentration of 30 g/L, all strains presented satisfactory microbial growth and complete glycerol uptake. Although culture conditions favored the secretion of citric acid (and potentially the accumulation of storage lipid), for the strains LFMB 19 and LFMB 20, polyol mannitol was the principal metabolic product synthesized (maximum quantity 6.0 g/L, yield 0.20–0.26 g per g of glycerol consumed). The above strains produced small quantities of lipids and citric acid. In contrast, Y. lipolytica ACA‐YC 5033 produced simultaneously higher quantities of lipid and citric acid and was further grown on crude glycerol in nitrogen‐limited experiments, with constant nitrogen and increasing glycerol concentrations (70–120 g/L). Citric acid and lipid concentrations increased with increment of glycerol; maximum total citric acid 50.1 g/L was produced (yield 0.44 g per g of glycerol) while simultaneously 2.0 g/L of fat were accumulated inside the cells (0.31 g of lipid per g of dry weight). Cellular lipids were mainly composed of neutral fraction, the concentration of which substantially increased with time. Moreover, in any case, the phospholipid fraction was more unsaturated compared with total and neutral lipids, while at the early growth step, microbial lipid was more rich in saturated fatty acids (e.g. C16:0 and C18:0) compared with the stationary phase.     

Effects of 20 Standard Amino Acids on the Growth,Total Fatty Acids Production,and γ-Linolenic Acid Yield in Mucor circinelloides

Twenty standard amino acids were examined as single nitrogen source on the growth, total fatty acids production, and yield of γ-linolenic acid (GLA) in Mucor circinelloides. Of the amino acids, tyrosine gave the highest biomass and lipid accumulation and thus resulted in a high GLA yield with respective values of 17.8 g/L, 23 % (w/w, dry cell weight, DCW), and 0.81 g/L, which were 36, 25, and 72 % higher than when the fungus was grown with ammonium tartrate. To find out the potential mechanism underlying the increased lipid accumulation of M. circinelloides when grown on tyrosine, the activity of lipogenic enzymes of the fungus during lipid accumulation phase was measured. The enzyme activities of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and ATP-citrate lyase were up-regulated, while NADP-isocitrate dehydrogenase was down-regulated by tyrosine during the lipid accumulation phase of the fungus which suggested that these enzymes may be involved in the increased lipid biosynthesis by tyrosine in this fungus.     

Kinetic modeling of Candida shehatae ATCC 22984 on xylose and glucose for ethanol production

Candida shehatae ATCC 22984, a xylose-fermenting yeast, showed an ability to produce ethanol in both glucose and xylose medium. Maximum ethanol produced by the yeast was 48.8?g/L in xylose and 52.6?g/L in glucose medium with ethanol yields that varied between 0.3 and 0.4?g/g depended on initial sugar concentrations. Xylitol was a coproduct of ethanol production using xylose as substrate, and glycerol was detected in both glucose and xylose media. Kinetic model equations indicated that growth, substrate consumption, and product formation of C. shehatae were governed by substrate limitation and inhibition by ethanol. The model suggested that cell growth was totally inhibited at 40?g/L of ethanol and ethanol production capacity of the yeast was 52?g/L, which were in good agreement with experimental results. The developed model could be used to explain C. shehatae fermentation in glucose and xylose media from 20 to 170?g/L sugar concentrations.     

Modeling of oleaginous fungal biofilm developed on semi-solid media

An oleaginous fungus, Mortierella isabellina, able to transform efficiently sugar to storage lipid, was used as a model microorganism which develops a biofilm structure during the semi-solid fermentation process for the production of biodiesel from sweet sorghum. A mathematical model was developed to describe the fungal oil production in M. isabellina biofilm. The model describes diffusion and consumption of sugars and nitrogen of sweet sorghum and single cell oil production in a biofilm, which grows according to the kinetics of double-substrate limitation (sugars and nitrogen) with sugar inhibition. Experimental data from a previous experimental study were used to determine the kinetic parameters of the model. Maximum biofilm thickness and the percentage of lipid inside the biofilm were estimated using the model at 1892 μm and 15%, respectively. The proposed mathematical model could prove a useful tool for designing semi-solid fermentation processes.     

霍山石斛类原球茎二步法培养细胞生长和多糖合成的动力学研究

磷是调控类原球茎细胞生长和多糖积累的有效因素。为了获得较高的多糖产量,根据霍山石斛类原球茎生长和多糖积累的动力学特性,提出了二步培养方式,采用了补料策略,研究了其培养过程的动力学特性,并建立了相关模型。结果表明,采用二步法培养,生物量从28.7g DW/L提高到44.2g DW/L, 多糖产量从1.86g/L提高到5.22g/L,多糖含量从6.4%提高到11.9%。建立的模型基本反映了类原球茎生长和多糖积累的动力学机制。     

β-Mannanase production and kinetic modeling from carob extract by using recombinant Aspergillus sojae

The main objectives of this study were to optimize β-mannanase fermentation conditions by using Response Surface Methodology (RSM) and to model kinetically using the kinetic models. Based on the results, the optimum fermentation conditions were found to be initial sugar concentration of 10°Bx, whey concentration of 0.75% [w/v], and inoculum size of 8% (v/v). Under optimized conditions, β-mannanase activity (P), sugar consumed (ΔS), maximum β-mannanase production rate (Q_P), and sugar utilization yield (SUY) were 687.89 U/mL, 47.38 g/L, 118.54 U mL^–1 day^–1, and 69.73%, respectively. Kinetic models were employed to describe the optimum β-mannanase fermentation process. The kinetic analysis of β-mannanase fermentation showed that β-mannanase fermentation is growth associated because the α value (U/mgX) is approximately 330-fold higher than the β value (U/mgX·hr). Nevertheless, maintenance value (Z) was lower than γ value, thus showing that Aspergillus niger mainly utilizes the sugars for β-mannanase production and fungal growth. Consequently, carob extract and whey powder could be used to be cost-effective carbon and organic nitrogen sources, respectively. It was clearly indicated that the suggested kinetic models can successfully describe the fungal growth, β-mannanase production, and substrate consumption.     

Single-cell oil production by Mortierella isabellina DSM 1414 using different sugars as carbon source

Pure carbon sources, especially carbohydrates which are raw materials deriving from agro-industrial processes, are often used for small-scale single-cell oil production by fermentation. The aim of this study was to investigate the effects of different pure carbon sources on cell growth, lipid accumulation, and γ-linolenic acid (GLA) production by the filamentous fungus Mortierella isabellina DSM 1414 (Deutsche Sammlung von Mikroorganismen). The sugars utilized in this study are found extensively and abundantly in nature, especially in food raw materials and, in consequence, in agro-food industry wastes or surpluses. Thus, the potential of many waste materials containing these sugars to be used in the production of single-cell oil by fermentation could also be evaluated. The effects of the sugars utilized on cell growth, biomass production, and lipid production were investigated. Fatty acids were also analysed in the lipids produced at the end of the fermentations. Results showed that the maximum biomass production was 10.80 g/L in lactose-based media, while the maximum oil production was 5.44 g/L in maltose-based media. Oleic (20.42%–42.94%), palmitic (14.96%–22.19%), and stearic (9.00%–26.92%) acids were the major fatty acids along with linoleic acid (11.35%–18.67%) and GLA (3.56%–8.04%). The production of GLA as the target fatty acid was remarkable. This study indicates that agro-industrial waste including most of the sugars utilized (except for arabinose and sucrose with lipid production of 0.81 and 0.28 g/L, respectively) can be employed for production of single-cell oil by M. isabellina DSM 1414 which contains a high amount of GLA.     

Single cell oil production from rice hulls hydrolysate

Rice hull hydrolysate was used as feedstock for microbial lipids production using the oleaginous fungus Mortierella isabellina. Kinetic experiments were conducted in C/N ratios 35, 44 and 57 and the oil accumulation into fungal biomass was 36%, 51.2% and 64.3%, respectively. A detailed mathematical model was used in order to describe the lipid accumulation process. This model was able to predict reducing sugar and nitrogen consumption, fat-free biomass synthesis and lipid accumulation. Neutral lipids constitute the predominant lipid fraction, while the major fatty acids were oleic, palmitic and linoleic acid. Fatty acids of long aliphatic chain were not detected, thus the microbial oil produced is a promising feedstock for biodiesel production.     

Mathematical modeling of production of microbial lipids

In the microbial lipid production system using the yeast Rhodotorula gracilis, CFR-1, kinetics of lipid accumulation and substrate utilisation at initial substrate concentrations in the range of 20–100 kg/m³ were investigated using shake flask experiments. A mathematical representation based on logistic model for biomass and Luedeking-Piret model for lipid accumulation gave reasonably good agreement between the theoretical and experimental values for substrate concentration less than 60 kg/m³. The kinetic expressions and parameters obtained through shake flask studies were directly applied to experiments in the laboratory fermentors also and the models were found to hold good for the prediction of the change of biomass, product as well as substrate with time. The attainment of a saturation in the intracellular lipid accumulation with time, however, was not predicted by the model which was shown to be an inherent feature of the Luedeking-Piret model.List of Symbols S ₀, P ₀ kg/m³ Initial concentrations of sugar and lipid respectively - S, S(t) kg/m³ Concentrations of sugar and lipid respeclively at any timet - p,p(t) L kg/m³ Maximum concentration of lipid produced - E % Maximum sugar utilised - dP/dt kg/(m³ · h) Rate of lipid production - -dS/dt kg/(m³ · h) Rate of sugar utilisation - _max h^–1 Maximum specific growth rate - X _max kg/m³ Maximum biomass reached in a run - P _max kg/m³ Maximum product concentration - m, n Constants used in Luedeking-Piret model in eq. (7) - , Constants used to predict residual sugar - k _e maintainance coefficient - Y _x g/g Biomass yield based on sugar consumed - Y _p g/g Lipid yield based on sugar consumed - (dP/d t)_stat kg/(m³ · h) Rate of lipid production at stationary phase - (dS/dt)_stat kg/(m³ · h) Rate of sugar utilisation at stationary phase     

Statistical media optimization for cell growth and azadirachtin production in Azadirachta indica (A. Juss) suspension cultures

Azadirachtin is one of the most widely used biopesticide originating from a plant source. Its production from plant cell cultivation was viewed to overcome constraints associated with its regular supply from the seed kernels. In order to select the effective carbon and nitrogen source, different concentrations of carbon (sucrose/glucose) and nitrogen (NO₃⁻/NH₄⁺ ratio) were studied in A. indica suspension culture. Glucose turned out to be a better carbon source over sucrose yielding high biomass (6.32 g/L) and azadirachtin (11.12 mg/L) content. Nitrate alone as nitrogen source was favorable for both biomass and azadirachtin accumulation. Plackett–Burman design was adopted to select the most important nutrients influencing the growth and azadirachtin accumulation in suspension culture. After identifying effective nutrients, Central Composite Design (CCD) was used to develop mathematical model equations, study responses and establish the optimum concentrations of the key nutrients for higher growth and azadirachtin production. A maximum of 15.02 g/L biomass and 2.98 mg/g azadirachtin was produced using optimum nutrient concentrations representing 99 and 96% validity of the model prediction with respect to biomass and azadirachtin, respectively. This optimized media can be used for cultivation of A. indica cells in bioreactor for mass production of azadirachtin.     

Modeling of lutein production by heterotrophic <Emphasis Type="Italic">Chlorella</Emphasis> in batch and fed-batch cultures

Chlorella is a promising alternative resource of lutein (xanthophyll) production as it can be cultivated heterotrophically in fermentors. In this paper, a kinetic model for lutein production by heterotrophic Chlorella pyrenoidosa was developed based on batch cultivations in 250-ml flasks and a 19-l fermentor. The model was validated by experimental data from two fed-batch cultivations performed in the same fermentor. The dynamic behavior of lutein production by C. pyrenoidosa with various concentrations of glucose and nitrogen was analyzed based on the kinetic model. Model-based analyses suggested that glucose concentrations between 5 and 24 g/l and nitrogen concentrations between 0.7 and 12 g/l during the cultivation were favorable for lutein production by heterotrophic C. pyrenoidosa. It also showed that fed-batch cultivations are more suitable for efficient production of lutein than batch ones. The results obtained in this study may contribute to commercial lutein production by heterotrophic Chlorella.     

Influence of cultivation conditions on citrate production by Aspergillus niger in a semi-pilot-scale plant

The influence of some fermentation parameters on the semi-pilot scale (alteration of growth conditions,e.g., sugar concentration, incubation temperature and initial pH) on citrate production was demonstrated in parent and mutant strains ofAspergillus niger. Raw material from sugar industry (cane molasses) was examined as basal fermentation medium in a stirred stainless-steel 15-L fermentor. After growth on medium with 150 g/L sugar, the parent strain produced 51.2 g/L citric acid; the mutant strain achieved production maximum of 96.2 g/L. Comparing the growth, kinetic (volumetric substrate uptake rate, rate of substrate consumption and volumetric productivity rate) and production parameters it was found that the mutant strain grows more rapidly, with slightly changed morphology (intermediate, shiny round pellets with diameter 0.6–0.7 mm), and exhibits a higher citrate production and higher efficiency of sugar utilization.     

Organic nitrogen of tomato waste hydrolysate enhances glucose uptake and lipid accumulation in Cunninghamella echinulata

Aims: To investigate the effect of organic nitrogen on lipogenesis during growth of Cunninghamella echinulata on tomato waste hydrolysate (TWH) media. Methods and Results: Cunninghamella echinulata grown on a TWH medium rapidly took up glucose and produced large amounts of lipids. However, when some quantities of the organic nitrogen were removed from TWH (by acid followed by alkaline precipitation of proteins) the uptake of glucose was dramatically reduced and large quantities of fungal biomass having low lipid content were produced. Nevertheless, when glycerol was used as carbon source instead of glucose, the uptake rate as well as the biomass production and the lipid accumulation processes were unaffected by the TWH organic nitrogen removal. Finally, when the fungus was grown on a glucose supplemented TWH medium that contained no assimilable organic nitrogen (after further precipitation of proteins with methanol), the produced biomass contained non-negligible quantities of lipids, although glucose uptake remained low. Lipid analysis showed that the produced lipids comprised mainly of neutral lipids, which were preferentially consumed during lipid turnover. Lipid production on the original TWH medium having glucose as carbon source was 0·48 g of lipid per gram of dry biomass, corresponding to 8·7 g of lipid per litre of growth medium. The produced lipids contained 11·7%γ-linolenic acid (GLA), hence the GLA yield was more than 1 g l⁻¹. Conclusions: Organic nitrogen compounds found in TWH favour glucose (but not glycerol) uptake and lipid accumulation in C. echinulata. Significance and Impact of the Study: Agro-industrial wastes containing organic nitrogen, such as tomato waste, are produced in vast amounts causing severe environmental problems. These wastes could be used as fermentation feedstock to produce microbial lipids.     

Effects of medium composition on the growth and lipid production of microplasmodia of Physarum polycephalum

Physarum polycephalum is a plasmodial slime mold. One of the trophic stages in the life cycle of this organism is a plasmodium. In submerged culture, plasmodia are fragmented into microplasmodia. The latter both lack cell walls and are capable of rapid growth. There has been limited information on the effects of medium composition on the growth and lipid accumulation of microplasmodia. In this study, optimization of medium components by response surface methodology showed that tryptone and yeast extract concentrations had the most significant effects on lipid and biomass production; significant synergistic interactions between glucose and tryptone concentration on these responses were also recorded. The optimal medium was composed of 20 g/L of glucose, 6.59 g/L of tryptone, and 3.0 g/L of yeast extract. This medium yielded 13.86 g/L of dry biomass and 1.97 g/L of lipids. These amounts are threefold higher than those of the American Type Culture Collection (ATCC) medium. In addition, biomass and lipid production reached maximal values between only 4 and 5 days. Fatty acid compositions analysis by gas chromatography-mass spectrometer (GC–MS) revealed that P. polycephalum lipids consisted mainly of oleic acid (40.5%), linoleic acid (10%), and octadecynoic (15.8%). This is the first report on the fatty acid composition of P. polycephalum microplasmodia. These results suggest that the biomass of microplasmodia could be used as a source of material for direct conversion into biodiesel because of the absence of cell walls or it could also be used as a supplemental source of beneficial fatty acids for humans, albeit with some further evaluation needed.     

霍山石斛类原球茎二步法培养细胞生长和多糖合成的动力学研究

磷是调控类原球茎细胞生长和多糖积累的有效因素。为了获得较高的多糖产量,根据霍山石斛类原球茎生长和多糖积累的动力学特性,提出了二步培养方式,采用了补料策略,研究了其培养过程的动力学特性,并建立了相关模型。结果表明,采用二步法培养,生物量从28·7gDW/L提高到44·2gDW/L,多糖产量从1·86g/L提高到5·22g/L,多糖含量从6·4%提高到11·9%。建立的模型基本反映了类原球茎生长和多糖积累的动力学机制。     

Influence of Glucose and Saturated Free-Fatty Acid Mixtures on Citric Acid and Lipid Production by Yarrowia lipolytica

In the present report, the effect of glucose and stearin (substrate composed by saturated free-fatty acids) on the production of biomass, reserve lipid, and citric acid by Yarrowia lipolytica ACA-DC 50109 was investigated in nitrogen-limited cultures. Numerical models that were used in order to quantify the kinetic behavior of the above Yarrowia lipolytica strain showed successful simulation, while the optimized parameter values were similar to those experimentally measured and the predictive ability of the models was satisfactory. In nitrogen-limited cultures in which glucose was used as the sole substrate, satisfactory growth and no glucose inhibition occurred, although in some cases the initial concentration of glucose was significantly high (150 g/l). Citric acid production was observed in all trials, which was in some cases notable (final concentration 42.9 g/l, yield 0.56 g per g of sugar consumed). The concentration of unsaturated cellular fatty acids was slightly lower when the quantity of sugar in the medium was elevated. In the cases in which stearin and glucose were used as co-substrates, in spite of the fact that the quantity of cellular lipid inside the yeast cells varied remarkably (from 0.3 to 2.0 g/l – 4 to 20% wt/wt), de novo fatty acid biosynthesis was observed. This activity increased when the yeast cells assimilated higher sugar quantities. The citric acid produced was mainly derived from the catabolism of sugar. Nevertheless, citric acid yield on sugar consumed and citrate specific production rate, as evaluated by the numerical model, presented substantially higher values in the fermentation in which no fat was used as glucose co-substrate compared with the cultures with stearin used as co-substrate.     

高糖和氮源对鼠李糖乳杆菌(Lactobacillus rhamnosus)L-乳酸发酵的影响

鼠李糖乳杆菌经实验室耐高糖高酸选育,能够在高糖浓度下高效高产L-乳酸。以酵母粉为氮源和生长因子,葡萄糖初始浓度分别为120 g/L和146 g/L,摇瓶培养120h,L-乳酸产量分别为104g/L和117.5g/L,L-乳酸得率分别为86.7%和80.5%。高葡萄糖浓度对菌的生长和乳酸发酵有一定的抑制。增加接种量,在高糖浓度发酵条件下,可以缩短发酵时间,但对增加乳酸产量效果不明显。乳酸浓度对鼠李糖乳杆菌生长和产酸有显著的影响。初始乳酸浓度到达70g/L以上时,鼠李糖乳杆菌基本不生长和产酸,葡萄糖消耗也被抑制。酵母粉是鼠李糖乳杆菌的优良氮源,使用其它被测试的氮源菌体生长和产酸都有一定程度的下降。用廉价的黄豆粉并补充微量维生素液,替代培养基中的酵母粉,可以使产酸浓度和碳源得率得以基本维持。     

不同氮源及其浓度对标志链带藻合成淀粉和油脂的影响北大核心CSCD

【目的】以标志链带藻(Desmodesmus insignis)为实验材料,研究不同氮源及其浓度对该藻生长、总脂和淀粉(碳水化合物)含量的影响,为该藻在生物能源方面的应用提供一定的理论依据。【方法】以硝酸钠、碳酸氢铵或尿素为氮源,5个氮浓度(3、6、9、12和18 mmol/L)的BG-11培养基培养标志链带藻,采用干重法测定生物质浓度、重量法测定总脂、苯酚-硫酸法测定、总碳水化合物和淀粉的含量。【结果】标志链带藻在3种氮源下均能很好的生长。最高油脂含量出现在3 mmol/L硝酸钠实验组,达到32.61%(d.w)。当18 mmol/L碳酸氢铵作为氮源时,总碳水化合物与淀粉的含量以及产率都达到最高,分别为56.54%(d.w)和55.33%(d.w)、0.24和0.23 g/(L·d)。以尿素为氮源时,其生物质浓度和各组分含量与其它氮源实验组差别不大,均有利于该藻的生长及各生化组分含量的积累。【结论】以该藻种生产生物能源的成本等综合考虑,以18 mmol/L碳酸氢铵和尿素为氮源培养标志链带藻最优。     

Lactic acid production with Lactobacillus casei ssp. rhamnosus NBIMCC 1013: Modeling and optimization of the nutrient medium

The medium needed to perform a fermentation process with viable cells of Lactobacillus casei ssp. rhamnosus NBIMCC 1013 for the production of lactic acid was modeled and optimized. On the basis of single‐factor experiments and statistical analysis, the significant factors affecting the fermentation process, i.e. the concentration of carbon source, concentrations of both yeast and meat extracts, and the range of variability of these components were determined. Modeling and optimization of the medium contents were performed using central composite design. The composition of the medium used for the production of lactic acid (g/L) was as follows: glucose 69.8, meat extract 17.07, yeast extract 10.9, CH₃COONa 10, K₂HPO₄ 0.25, KH₂PO₄ 0.25, MgSO₄·7H₂O 0.05, and FeSO₄ 0.05. The maximum specific growth rate of the lactic acid bacteria (μ=0.51 h⁻¹) and other kinetic parameters were determined during cultivation in a laboratory bioreactor using the logistic equation and the Luedeking–Piret model. The obtained medium allows the production of lactic acid under optimum conditions, at high specific sugar assimilation rates and high lactic acid accumulation rates. The positive results of the paper are the new nutrient medium for lactic acid production and the process kinetic model, enabling scaling up and switching to a continuous process.