The effect of supplementation by different nitrogen sources on the production of lactic acid from date juice by Lactobacillus casei subsp. rhamnosus

Production of lactic acid from date juice by fermentation has been studied using Lactobacillus casei subsp. rhamnosus as the producer organism. The optimum substrate concentration, expressed in its glucose content, was 60 g l(-1). Various nitrogen sources were compared with yeast extract in terms of their efficiency for lactic acid production. None of these nitrogen sources gave lactic acid concentrations as high as that obtained with yeast extract. As yeast extract supplementation was not economically attractive, different proportions of (NH4)2SO4 and yeast extract were used. When the elemental nitrogen ratio of(NH4)2SO4 to yeast extract was 4:1, the substrate use and efficiency of lactic acid production were the same as in date juice supplemented with 20 g l(-1) yeast extract (0:5).     

Influence of an initial addition of lactic acid on growth, acid production and their coupling for batch cultures of Lactobacillus helveticus

Growth and production coupling for L. helveticus growing at constant pH?on a medium largely supplemented with yeast extract and peptons has been examined as a function of initial lactic acid addition: these conditions allowed to avoid side effects of cultivation pH?and nitrogen source deficiency on coupling. Growth of L. helveticus strain milano was reduced by half for a free lactic acid concentration of 0.34?g.l^?1; both growth and production were fully inhibited for a critical end-product concentration of 83?g.l^?1, despite the fact that lactose was still available. The extent of autolysis 8?h after growth ceased was practically independent of initial lactic acid concentration: the same conclusion held for the part played by a growth-associated mechanism in total acid production. During the slowing down phase of growth, production was non-growth-associated: production rate remained close to its maximal value, while growth rate declined.     

Optimization of submerged culture conditions for exopolysaccharide production in <Emphasis Type="Italic">Sarcodon aspratus</Emphasis> (Berk) S.lto TG-3

The effect of medium components (carbon, nitrogen, and mineral sources) and environmental factors (initial pH and temperature) for mycelial growth and exopolysaccharide (EPS) production in Sarcodon aspratus(Berk) S.lto TG-3 was investigated. The optimal temperature (25°C) and initial pH (5.0) for the EPS production in shake flask cultures of S. aspratus were determined using the two-dimensional contour plot. The most suitable carbon, nitrogen, and mineral sources for EPS production were glucose, yeast extract, CaCl₂ and KH₂PO₄, respectively. Notably, the EPS production was significantly enhanced by supplementation of calcium ion. Subsequently, the optimum concentration of glucose (30gl^–1), yeast extract (15gl^–1), CaCl₂ (1.1gl^–1), and KH₂PO₄ (1.2gl^–1) were determined using the orthogonal matrix method. The effects of nutritional requirement on the mycelial growth of S.aspratuswere in regular sequence of glucose>KH₂PO₄>yeast extract>CaCl₂, and those on EPS production were in the order of glucose>yeast extract>CaCl₂>KH₂PO₄. Under the optimal culture conditions, the maximum EPS concentration in a 5-l stirred-tank reactor was 2.68gl^–1 after 4days of fermentation, which was 6-fold higher than that at a basal medium. The two-dimensional contour plot and orthogonal matrix method allowed us to find the relationship between environmental factors and nutritional requirement by determining optimal operating conditions for maximum EPS production in S.asparatus. The statistical experiments used in this work can be useful strategies for optimization of submerged culture processes for other mushrooms.     

Batch and repeated batch production of l(+)-lactic acid by Enterococcus faecalis RKY1 using wood hydrolyzate and corn steep liquor

Lactic acid production was investigated for batch and repeated batch cultures of Enterococcus faecalis RKY1, using wood hydrolyzate and corn steep liquor. When wood hydrolyzate (equivalent to 50 g l⁻¹ glucose) supplemented with 15–60 g l⁻¹ corn steep liquor was used as a raw material for fermentation, up to 48.6 g l⁻¹ of lactic acid was produced with, volumetric productivities ranging between 0.8 and 1.4 g l⁻¹ h⁻¹. When a medium containing wood hydrolyzate and 15 g l⁻¹ corn steep liquor was supplemented with 1.5 g l⁻¹ yeast extract, we observed 1.9-fold and 1.6-fold increases in lactic acid productivity and cell growth, respectively. In this case, the nitrogen source cost for producing 1 kg lactic acid can be reduced to 23% of that for fermentation from wood hydrolyzate using 15 g l⁻¹ yeast extract as a single nitrogen source. In addition, lactic acid productivity could be maximized by conducting a cell-recycle repeated batch culture of E. faecalis RKY1. The maximum productivity for this process was determined to be 4.0 g l⁻¹ h⁻¹.     

Batch propagation of Lactobacillus helveticus for production of lactic acid from lactose concentrated cheese whey with microaeration and nutrient supplementation

Continuous mix batch bioreactors were used to study the kinetic parameters of lactic acid fermentation in microaerated-nutrient supplemented, lactose concentrated cheese whey using Lactobacillus helveticus. Four initial lactose concentrations ranging from 50 to 150 g l^–1 were first used with no microaeration and no yeast extract added to establish the substrate concentration above which inhibition will occur and then the effects of microaeration and yeast extract on the process kinetic parameters were investigated. The experiments were conducted under controlled pH (5.5) and temperature (42 °C) conditions. The results indicated that higher concentrations of lactose had an inhibitory effect as they increased the lag period and the fermentation time; and decreased the specific growth rate, the maximum cell number, the lactose utilization rate, and the lactic acid production rate. The maximum lactic acid conversion efficiency (75.8%) was achieved with the 75 g l^–1 initial lactose concentration. The optimum lactose concentration for lactic acid production was 75 g l^–1 although Lactobacillus helveticus appeared to tolerate up to 100 g l^–1 lactose concentration. Since the lactic acid productivity is of a minor importance compared to lactic acid concentration when considering the economic feasibility of lactic acid production from cheese whey using Lactobacillus helveticus, a lactose concentration of up to 100 g l^–1 is recommended. Using yeast extract and/or microaeration increased the cell number, specific growth rate, cell yield, lactose consumption, lactic acid utilization rate, lactic acid concentration and lactic acid yield; and reduced the lag period, fermentation time and residual lactose. Combined yeast extract and microaeration produced better results than each one alone. From the results it appears that the energy uncoupling of anabolism and catabolism is the major bottleneck of the process. Besides lactic acid production, lactose may also be hydrolysed into glucose and galactose. The -galactosidase activity in the medium is caused by cell lysis during the exponential growth phase. The metabolic activities of Lactobacillus helveticus in the presence of these three sugars need further investigation.     

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。     

The production of lactic acid from whey permeate byLactobacillus helveticus

Summary The effect of pH on growth and lactic acid production ofLactobacillus helveticus was investigated in a continuous culture using supplemented whey ultrafiltrate. Maximum lactate productivity of 5 gl^–1h^–1 occurred at pH 5.5. Whey permeates concentrated up to four times were fermented using batch cultures. Maximum lactic acid concentration of 95 gl^–1 was attained, but residual sugars indicated a possible limitation in growth factors.Nomenclature D Dilution rate [h^–1] - X Biomass [gl^–1] - Glu Glucose consentration [gl^–1] - Gal Galactose consentration [gl^–1] - S Substrate, Lactose consentration [gl^–1] - P Product, Lactate consentration [gl^–1] - Y_p/s Yield, defined as P/S [gg^–1] - r_i Rate of synthesis or consumption of i [gl^–1h^–1]     

Analysis of growth and production coupling for batch cultures of Lactobacillus helveticus with the help of an unstructured model

An unstructured model has been developed by assuming a sigmoidal variation with time of specific growth rate and introducing an additional term in Luedeking and Piret production kinetics. The model fitted growth and production kinetic data. The model was helpful for examining the influence of initial yeast extract concentration on growth and production coupling. In richly supplemented media, the growth associated production mechanism prevailed, while in the case of poor nitrogen supplementation, most lactic acid production was non-growth-associated. Both contributions might be calculated from model parameters; this was especially interesting when the forms of growth and production kinetics did not allow a clear conclusion. The additional term in the production rate expression was a convenient way to characterize ‘usable nitrogen’ concentration in the medium. From the model a clear criterion has been derived for optimizing the nitrogen supplementation from an economic point of view.     

Xanthan production from olive-mill wastewaters

Olive mill wastewaters (OMW) are a by-product from olive oil manufacture that cause environmental pollution. These wastes have been used as substrate for the production of the extracellular polysaccharide xanthan by Xanthomonas campestris NRRL B1459-S4L41. Growth and xanthan production on dilute OMW as a sole source of nutrients were obtained at OMW concentrations below 60%, yielding a maximal xanthan production of 4.4gl⁻¹ at 30–40% OMW concentration. Addition of nitrogen and/or salts led to significantly increased xanthan yields with a maximum of 7.7gl⁻¹. The N/salts supplements also allowed an increase in the optimal OMW concentration. Inocula pre-grown on OMW can be used. Results suggest that an improved xanthan yield could be obtained with adequate balance between waste concentration and nitrogen or salt supplementation. OMW is proposed as a low-cost substrate for xanthan production with the additional environmental benefit of this use.     

Variations of alcohol dehydrogenase activity and fermentative pyruvate,ethanol production of F. equiseti and F. acuminatum depend on the yeast extract and urea concentrations

In this study, pyruvate production of Fusarium equiseti was significantly increased when the yeast extract concentration was raised from 5 to 25 g/L while it was increased to only up to 10 g/L yeast extract in F. acuminatum. Upon supplementation with urea as an alternative nitrogen source, production of pyruvate for both of the Fusarium species were decreased with respect to increase in urea concentration in medium. On the other hand, ethanol production and alcohol dehydrogenase activity of F. equiseti were decreased approximately 1.9- and 1.6-fold with an increase in yeast concentration from 5 to 25 whereas the levels of F. acuminatum were increased 2.3- and 1.8-fold, respectively. In addition, ethanol productions and ADH activities in F. equiseti and F. acuminatum significantly increased on the 12th day up to 15 and 25 g/L urea concentrations, respectively. However, they were significantly decreased under these conditions at higher nitrogen sources. In addition, ethanol production and alcohol dehydrogenase activity in urea supplemented medium were higher than yeast extract supplemented. The results may suggest that the pyruvate, ethanol production and ADH enzyme activity variations and balance between aerobic and anaerobic respiration in F. equiseti and F. acuminatum were effected from yeast extract and urea concentrations in the nutrient medium.     

Kinetics of growth and medium de-acidification for Geotrichum candidum and Penicillium camemberti cultivated on complex liquid media

Growth kinetics of Geotrichum candidum and Penicillium camemberti in submerged cultures under conditions of low aeration rate and uncontrolled pH were continuously recorded turbidimetrically. In these conditions the exponential growth phase was short, and ceased at a total biomass concentration of about 0.5 gl^–1 for G. candidum and 0.8 gl^–1 for P. candidum. The succeeding linear growth phase was also short, and its end corresponded to a biomass of 1.5 and 2.5 gl^–1 for G. candidum and P. camemberti respectively. A fair growth was recorded for P. camemberti on peptone-lactate medium, but G. candidum required addition of trace elements. For neither of these species growth was stimulated by growth factors of yeast extract. In peptone-lactate medium, the final pH did not depend on supplementation: 8–8.4 was recorded for G. candidum and 8.7–8.8 for P. camemberti.     

Influence of growth factor supplements on butyric acid production from sucrose byClostridium butyricum

During batch fermentation of sucrose to butyric acid byClostridium butyricum the effect of growth factor supplementation was determined: addition of yeast extract (5 g/L) stimulated most. Using biotin as the sole growth factor, average productivity was definitely lower. Beet molasses as a combined source of carbon and growth factor were effective only at a high concentration (150 g/L). The optimal butyric acid production (45 g/L, yield 45%) was achieved with sucrose concentration of 100 g/L in a medium supplemented with yeast extract (5 g/L). It represents an average productivity of 0.90 gL⁻¹ h⁻¹ and relative butyric acid concentration of 91%.     

Concomitant wastewater treatment with lipid and carotenoid production by the oleaginous yeast Rhodosporidium toruloides grown on brewery effluent enriched with sugarcane molasses and urea

In this study, secondary brewery wastewater (SBWW) supplemented with sugarcane molasses (SCM) was used for SBWW treatment with concomitant lipid and carotenoid production by the yeast Rhodosporidium toruloides NCYC 921. In order to improve the biomass production, ammonium sulfate, yeast extract and urea were tested as nitrogen sources. Urea was chosen as the best low-cost nitrogen source. A fed-batch cultivation was carried out with SBWW supplemented with 10 g L⁻¹ of sugarcane molasses as carbon source, and 2 g L⁻¹ of urea as nitrogen source. A maximum biomass concentration of 42.5 g L⁻¹ was obtained at t = 126.5 h and the maximum biomass productivity was 0.55 g L⁻¹ h⁻¹ at t = 48.25 h. The maximum lipid content was 29.9 % w/w (DCW) at t = 94 h of cultivation and the maximum carotenoid content was 0.23 mg g⁻¹ at 120 h of cultivation. Relatively to the SBWW treatment, after the batch phase, 45.8 % of total Kjeldahl nitrogen removal, 81.7 % of COD removal and 100 % of sugar consumption were observed. Flow cytometry analysis revealed that 27.27 % of the cells had injured membrane after the inoculation. This proportion was reduced to 10.37 % at the end of the cultivation, indicating that cells adapted to the growth conditions.     

Production of lactic acid by Lactobacillus rhamnosus with vitamin-supplemented soybean hydrolysate

Batch fermentation studies were performed to evaluate the potentials of a complex nitrogen source, soybean, as an alternative to yeast extract for the economical production of lactic acid by Lactobacillus rhamnosus. An enzyme-hydrolysate of soybean meal, Soytone, with an adequate supplementation of vitamins was found to be highly effective in supporting lactic acid production from glucose and lactose. The effects of seven selected vitamins: d-biotin, pyridoxine, p-aminobenzoic acid, nicotinic acid, thiamine, pantothenic acid, and riboflavin, on cell growth and lactic acid production were investigated to provide the basis for the optimization of vitamin supplementation to minimize the cost. Pantothenic acid was the most required compound while the other six vitamins were also essential for high lactic acid productivity. As a result of the optimization, 15 g/l yeast extract could be successfully replaced with 19.3 g/l Soytone supplemented with the vitamins, resulting in a production of 125 g/l lactic acid from 150 g/l glucose. The volumetric productivity and lactate yield were 2.27 g/l/h and 92%, respectively, which were higher than those with 15 g/l yeast extract. The raw material cost was estimated to be 21.4 cent/kg lactic acid, which was only approximately 41% of that with yeast extract.     

Effect of temperature and various nitrogen sources on L(+)-lactic acid production by Lactobacillus casei

 Two homofermentative strains, Lactobacillus casei NRRL B-441 and Lactobacillus casei subsp. rhamnosus NRRL B-445 were selected for further study from 17 lactic acid bacterial strains screened for lactic acid production. The effect of temperature on lactic acid production with the selected strains was investigated by adapting both strains to four different temperatures. The production of L(+)-lactic acid by both strains was most efficient at 37°C, although with L. casei the highest lactic acid concentration was obtained at 41°C. The maximal volumetric productivity with L. casei was 4.1 g l^-1 h^-1 and with L. casei subsp. rhamnosus 3.5 g l^-1 h^-1. The composition of the medium was studied in order to replace the costly yeast extract with less expensive sources of nitrogen and amino acids. From 11 different nitrogen sources investigated at 37°C, barley malt sprouts (88 g l^-1 lactic acid in 66 h) and grass extract (74 g l^-1 lactic acid in 73 h) were the best economic alternatives. The effect of different combinations of yeast extract, peptone and malt sprouts was further studied by using statistical experimental design, and an empirical second-order polynomial model was constructed on the basis of the results. With the right combination most of the yeast extract could be substituted by barley malt sprouts for efficient lactic acid production. A method for extraction of nutrients and growth factors from malt sprouts is also described. Received: 25 September 1995/Accepted: 24 October 1995     

Joint effect of nitrogen sources and B vitamin supplementation of date juice on lactic acid production by Lactobacillus casei subsp. rhamnosus

The use of date juice as a substrate for lactic acid production was investigated. Various nitrogen sources were compared with yeast extract for efficient lactic acid production by Lactobacillus casei subsp. rhamnosus. Among different nitrogen sources added to date juice (yeast extract, ammonium sulfate, tryptic soy, urea, peptone, and casein hydrolysate), yeast extract was the most efficient. The effect of yeast extract could have been due to its B vitamin content. The addition of five B vitamins at less than 25 mg/l to date juice with any nitrogen source enhanced lactic acid production to some extent, except for date juice with yeast extract or urea or peptone. The most significant increase was obtained with ammonium sulfate. Half of the yeast extract content (10 g/l) in a supplemented date juice could be replaced by a mixture of B vitamins at less than 25 mg/l, and ammonium sulfate at 2.6 g/l with no significant decrease in lactic acid production.     

Cauliflower waste incorporation into cane molasses improves ethanol production using <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> MTCC 178

Diluted cane molasses having total sugar and reducing sugar content of 9.60 and 3.80% (w/v) respectively was subjected to ethanol production by Saccharomyces cerevisiae MTCC 178. Incorporation of dried Cauliflower Waste (CW) in molasses at the level of 15 % increased ethanol production by nearly 36 % compared to molasses alone. Addition of 0.2 % yeast extract improved ethanol production by nearly 49 % as compared to molasses alone. When the medium containing diluted molasses and 0.2 % yeast extract was supplemented with 15 % CW, 29 % more ethanol was produced compared to molasses with 0.2 % yeast extract. Cell biomass, ethanol production, final ethanol concentration and fermentation efficiency of 2.65 mg mL⁻¹, 41.2 gL⁻¹, 0.358 gg⁻¹ and 70.11 % respectively were found to be best at 15% CW supplementation level besides reduction in fermentation time but further increase in CW level resulted in decline on account of all the above parameters. This is probably the first report to our knowledge, in which CW was used in enhancing ethanol production significantly using a small quantity of yeast extract.     

Channelling of glucose by methanol for citric acid production from Aspergillus niger

Citric acid produced by Aspergillus niger was increased from 4.6g l^-1 to 7.8gl^-1 by supplementing basal medium with methanol (30mll^-1). While stimulating citric acid production, methanol did not improve membrane permeability of the fungus for citric acid. Methanol inhibited the germination of Aspergillus spores. An increase in glucose concentration from 50gl^-1 to 100gl^-1 in the presence of methanol (30mll^-1) improved citric acid production (1.6-fold) while at higher levels of glucose concentration methanol had no effect on citic acid production.     

戊糖乳杆菌发酵培养基氮源条件的优化

对戊糖乳杆菌发酵培养基的氮源条件进行了优化。通过单因素实验及响应面分析优化利用木糖高产乳酸的戊糖乳杆菌发酵培养基的不同氮源组合。优化得到的牛肉膏与柠檬酸氢二铵复合的最佳组成为牛肉膏17.72 g/L,柠檬酸氢二铵1.91 g/L,得到乳酸实际最大产量42.37 g/L。添加玉米浆与酵母粉和无机氮源复合的最佳组成为玉米浆46.54 g/L,酵母粉21.95 g/L,柠檬酸氢二铵9.95 g/L,可得到乳酸最大产量41.06 g/L。通过响应面优化减少了有机氮源的种类。牛肉膏与柠檬酸氢二铵的复合得到了更高的乳酸产量,且减少了有机氮源用量,节约了成本。玉米浆与酵母粉的复合解决了单一玉米浆造成的木糖利用速率过低的问题,同样得到较高浓度的乳酸。     

Study of docosahexaenoic acid production by the heterotrophic microalga <Emphasis Type="Italic">Crypthecodinium cohnii</Emphasis> CCMP 316 using carob pulp as a promising carbon source

In this work, carob pulp syrup was used as carbon source in C. cohnii fermentations for docosahexaenoic acid production. In preliminary experiments different carob pulp dilutions supplemented with sea salt were tested. The highest biomass productivity (4 mg/lh) and specific growth rate (0.04/h) were observed at the highest carob pulp dilution (1:10.5 (v/v), corresponding to 8.8 g/l glucose). Ammonium chloride and yeast extract were tested as nitrogen sources using different carob pulp syrup dilutions, supplemented with sea salt as growth medium. The best results were observed for yeast extract as nitrogen source. A C. cohnii fed-batch fermentation was carried out using diluted carob pulp syrup (1:10.5 v/v) supplemented with yeast extract and sea salt. The biomass productivity was 420 mg/lh, and the specific growth rate 0.05/h. Under these conditions the DHA concentration and DHA production volumetric rate attained 1.9 g/l and 18.5 mg/lh respectively after 100.4 h. The easy, clean and safe handling of carob pulp syrup makes this feedstock a promising carbon source for large-scale DHA production from C. cohnii. In this way, this carob industry by-product could be usefully disposed of through microbial production of a high value fermentation product.