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.     

Lactic acid production from agricultural resources as cheap raw materials

Agricultural resources such as barley, wheat, and corn were hydrolyzed by commercial amylolytic enzymes and fermented into lactic acid by Enterococcus faecalis RKY1. Although no additional nutrients were supplemented to those resources, lactic acid productivities were obtained at >0.8 g/l h from barley and wheat. When 200 g/l of whole wheat flour was hydrolyzed by amylolytic enzymes after the pre-treatment with 0.3% (v/v) sulfuric acid and sterilized by filtration, E. faecalis RKY1 efficiently produced lactic acid with 2.6 g/l h of lactic acid productivity and 5.90 g/l of maximal dry cell weight without additional nutrients. Lactic acid productivity and cell growth could be enhanced to 31% and 12% higher values than those of non-adapted RKY1, by adaptation of E. faecalis RKY1 to CSL-based medium. When the medium contained 200 g/l of whole wheat flour hydrolyzate, 15 g/l of corn steep liquor, and 1.5 g/l of yeast extract, lactic acid productivity and maximal dry cell weight were obtained at 5.36 g/l h and 14.08 g/l, respectively. This result represented an improvement of up to 106% of lactic acid productivity and 138% of maximal dry cell weight in comparison to the fermentation from whole wheat flour hydrolyzate only.     

The effect of whey protein hydrolyzates on the lactic acid fermentation

Summary The batch fermentation of whey permeate to lactic acid was improved markedly by the addition of enzymehydrolyzed whey protein. Acid concentrations greater than 90 g/l were achieved at a productivity of 4.3 g/l per h and a 98% substrate use. Cell mass concentration reached 6 g/l. The acid productivity achieved is somewhat higher than that typical for fermentation of whole whey. The process economics, based on in-house hydrolyzate preparation, look promising. Presented in this paper are the experimental results showing the effects of hydrolyzate concentration on acid and cell mass production.     

Use of response surface methodology for selection of nutrient levels for culturing Paecilomyces variotii in eucalyptus hemicellulosic hydrolyzate

Eucalyptus hemicellulose was hydrolyzed by treating eucalyptus wood chips with sulfuric acid. The hydrolyzate was used as the substrate to produce single-cell protein by growing Paecilomyces variotii IOC-3764 for 72 or 96 h. The influences of rice bran, ammonium sulfate and fermentation time were verified by a 23 full-factorial central composite design. At the optimum process conditions, the cell concentration was 12.06 g/l, which was obtained when the microorganisms were cultivated for 89 h in a medium composed of 10 g/l rice bran, 2.0 g/l nitrogen and 1.1 g/l sodium phosphate. The mathematical model Y = 10.65 + 2.40X2 + 2.36X3 + 1.16X2X3 - 2.10X2(2) - 1.06X3(2) describes biomass production by P. variotii in eucalyptus hemicellulosic hydrolyzate with a determination coefficient of R2 = 0.9561, where X2 and X3 are ammonium sulfate and fermentation time, respectively.     

Fed-batch Cultivation of Mucor indicus in Dilute-acid Lignocellulosic Hydrolyzate for Ethanol Production

Mucor indicus fermented dilute-acid lignocellulosic hydrolyzates to ethanol in fed-batch cultivation with complete hexose utilization and partial uptake of xylose. The fungus was tolerant to the inhibitors present in the hydrolyzates. It grew in media containing furfural (1 g/l), hydroxymethylfurfural (1 g/l), vanillin (1 g/l), or acetic acid (7 g/l), but did not germinate directly in the hydrolyzate. However, with fed-batch methodology, after initial growth of M. indicus in 500 ml enzymatic wheat hydrolyzate, lignocellulosic hydrolyzate was fermented with feeding rates 55 and 100 ml/h. The fungus consumed more than 46% of the initial xylose, while less than half of this xylose was excreted in the form of xylitol. The ethanol yield was 0.43 g/g total consumed sugar, and reached the maximum concentration of 19.6 g ethanol/l at the end of feeding phase. Filamentous growth, which is regarded as the main obstacle to large-scale cultivation of M. indicus, was avoided in the fed-batch experiments.     

Evaluation of plastic-composite supports in repeated fed-batch biofilm lactic acid fermentation by Lactobacillus casei

A customized stirred-tank biofilm reactor was designed for plastic-composite supports (PCS). In repeated-batch studies, the PCS-biofilm reactors outperformed the suspended-cell reactors by demonstrating higher lactic acid productivities (2.45 g l(-1) h(-1) vs 1.75 g l(-1) h(-1)) and greater glucose consumption rates (3.27 g l(-1) h(-1) vs 2.09 g l(-1) h(-1)). In the repeated fed-batch studies, reactors were spiked periodically with concentrated glucose (75%) to maintain a concentration of approximately 80 g of glucose l(-1) in the bioreactor. In suspended-cell fermentations with 10 g of yeast extract (YE) l(-1) and zero, one, two, and three glucose spikes, the lactic acid productivities were 2.64, 1.58, 0.80, and 0.62 g l(-1) h(-1), respectively. In comparison, biofilm reactors with 7 g of YE l(-1) and zero, one, two, and three glucose spikes achieved lactic acid productivities of 4.20, 2.78, 0.66, and 0.94 g l(-1) h(-1), respectively. The use of nystatin (30 U ml(-1)) subdued the contaminating yeast population with no effect on the lactic acid productivity of the biofilm reactors, but it did affect productivity in the suspended-cell bioreactor. Overall, in repeated fed-batch fermentations, the biofilm reactors consistently outperformed the suspended-cell bioreactors, required less YE, and produced up to 146 g of lactic acid l(-1) with 7 g of YE l(-1), whereas the suspended-cell reactor produced 132 g l(-1) with 10 g of YE l(-1).     

Optimisation of media and cultivation conditions for L(+)(S)-lactic acid production by Lactobacillus casei NRRL B-441

Process variables and concentration of carbon in media were optimised for lactic acid production by Lactobacillus casei NRRL B-441. Lactic acid yield was inversely proportional to initial glucose concentration within the experimental area (80-160 g l(-1)). The highest lactic acid concentration in batch fermentation, 118.6 g l(-1), was obtained with 160 g 1(-1) glucose. The maximum volumetric productivity, 4.4 g 1(-1) h(-1) at 15 h, was achieved at an initial glucose concentration of 100 g l(-1). Similar lactic acid concentrations were reached with a fedbatch approach using growing cells, in which case the fermentation time was much shorter. Statistical experimental design and response surface methodology were used for optimising the process variables. The temperature and pH optima for lactic acid production were 35 degrees C, pH 6.3. Malt sprout extract supplemented with yeast extract (4 g l(-1)) appeared to be an economical alternative to yeast extract alone (22 g l(-1)) although the fermentation time was a little longer. The results demonstrated both the separation of the growth and lactic acid production phases and lactic acid production by non-growing cells without any nutrient supplements. Resting L. casei cells converted 120 g l(-1) glucose to lactic acid with 100% yield and a maximum volumetric productivity of 3.5 g l(-1) h(-1).     

Fermentative production of dl-lactic acid from amylase-treated rice and wheat brans hydrolyzate by a novel lactic acid bacterium, Lactobacillus sp.

Rice and wheat brans, without additional nutrients and hydrolyzed by alpha-amylase and amyloglucosidase, were fermented to DL-lactic acid using a newly isolated strain of Lactobacillus sp. RKY2. In batch fermentations at 36 degrees C and pH 6, the amount of lactic acid in fermentation broth reached 129 g l(-1) by supplementation of rice bran with whole rice flour. The maximum productivity was 3.1 g lactic acid l(-1) h(-1) in rice bran medium supplemented with whole rice flour or whole wheat flour.     

A continuous lactic acid production system using an immobilized packed bed of Lactobacillus helveticus

A 5 l packed bed bioreactor was used to study the effect of initial lactose concentration and hydraulic retention time (HRT) on cell growth, lactose utilization and lactic acid production. Up to 95% of the initial lactose concentration was utilized at longer HRTs (30-36 h). The study showed that lactic acid production increased with increases in HRT (12-36 h) and initial lactose concentrations. The highest lactic acid production rate (3.90 g l(-1) h(-1)) was obtained with an initial lactose concentration of 100 g/l and an HRT of 18 h, whereas the lowest lactic acid production rate (1.35 g l(-1) h(-1)) was obtained with an initial lactose concentration of 50 g/l and an HRT of 36 h. This suggested that optimal lactic acid production can be achieved at an HRT of 18 h and initial lactose concentration of 100 g/l.     

利用固定化米根霉在三相流化床中发酵生成L-乳酸

用聚氨酯泡沫吸附固定米根霉菌丝,在三相流化床中对葡萄糖、木糖以及木糖渣的纤维素酶解液等不同碳源进行Ｌ－乳酸发酵研究,并对游离菌丝和固定化菌丝发酵Ｌ－乳酸进行了比较。结果表明,聚氨酯泡沫是米根霉的良好载体,具有经济、高效等特点。实验条件下,不同碳源的乳酸转化率分别为：葡萄糖,８２．５％;木糖,５３．８％;木糖渣酶水解液,７１．９％。三相流化床中固定化米根霉产酸速率（对葡萄糖）为１９．１ｇ．ｈ＾－１．     

Production of 10-hydroxystearic acid from oleic acid and olive oil hydrolyzate by an oleate hydratase from Lysinibacillus fusiformis

A recombinant enzyme from Lysinibacillus fusiformis was expressed, purified, and identified as an oleate hydratase because the hydration activity of the enzyme was the highest for oleic acid (with a k (cat) of 850?min(-1) and a K (m) of 540?μM), followed by palmitoleic acid, γ-linolenic acid, linoleic acid, myristoleic acid, and α-linolenic acid. The optimal reaction conditions for the enzymatic production of 10-hydroxystearic acid were pH 6.5, 35?°C, 4% (v/v) ethanol, 2,500?U ml(-1) (8.3?mg?ml(-1)) of enzyme, and 40?g l(-1) oleic acid. Under these conditions, 40?g l(-1) (142?mM) oleic acid was converted into 40?g l(-1) (133?mM) 10-hydroxystearic acid for 150?min, with a molar yield of 94% and a productivity of 16?g l(-1)?h(-1), and olive oil hydrolyzate containing 40?g l(-1) oleic acid was converted into 40?g l(-1) 10-hydroxystearic acid for 300?min, with a productivity of 8?g l(-1)?h(-1).     

Production of mannitol and lactic acid by fermentation with Lactobacillus intermedius NRRL B-3693

Lactobacillus intermedius B-3693 was selected as a good producer of mannitol from fructose after screening 72 bacterial strains. The bacterium produced mannitol, lactic acid, and acetic acid from fructose in pH-controlled batch fermentation. Typical yields of mannitol, lactic acid, and acetic acid from 250 g/L fructose were 0.70, 0.16, and 0.12 g, respectively per g of fructose. The fermentation time was greatly dependent on fructose concentration but the product yields were not dependent on fructose level. Fed-batch fermentation decreased the time of maximum mannitol production from fructose (300 g/L) from 136 to 92 h. One-third of fructose could be replaced with glucose, maltose, galactose, mannose, raffinose, or starch with glucoamylase (simultaneous saccharification and fermentation), and two-thirds of fructose could be replaced with sucrose. L. intermedius B-3693 did not co-utilize lactose, cellobiose, glycerol, or xylose with fructose. It produced lactic acid and ethanol but no acetic acid from glucose. The bacterium produced 21.3 +/- 0.6 g lactic acid, 10.5 +/- 0.3 g acetic acid, and 4.7 +/- 0.0 g ethanol per L of fermentation broth from dilute acid (15% solids, 0.5% H(2)SO(4), 121 degrees C, 1 h) pretreated enzyme (cellulase, beta-glucosidase) saccharified corn fiber hydrolyzate.     

Application of bipolar electrodialysis on recovery of free lactic acid after simultaneous saccharification and fermentation of cassava starch

The efficiency of bipolar electrodialysis (BED) for the recovery of lactic acid from fermentation broth was evaluated. Three systems of BED (bipolar-anion, bipolar-cation and bipolar-anion-cation) at fixed voltage (20 V) were compared using a model solution of ammonium lactate (100 g l(-1)). Results showed that bipolar-anion (BED-anion) was the most beneficial in terms of lactate flux, current efficiency, energy consumption and recovery ratio. Consequently, BED-anion was used to purify lactic acid from fermentation broth which had been pre-treated with mono-polar electrodialysis (MED). The final lactic acid concentration and lactate flux obtained were 144 g l(-1) and 393 g m(-2) h(-1), respectively. Using the two-step process (MED and BED-anion) the concentration of fermentation broth was increased by 33% and the total energy consumption was 2.76 kW h kg(-1).     

Lantana camara for fuel ethanol production using thermotolerant yeast

AIM: Evaluation of Lantana camara's use as feedstock for fuel ethanol production. METHODS AND RESULTS: Lantana camara plant material was hydrolysed with 1% sulfuric acid for 18 h at room temperature, followed by heat treatment of 121 degrees C for 20 min. Hemicellulosic hydrolyzate was separated and used for detoxification by ethyl acetate and overliming. Cellulosic fraction was hydrolysed with Aspergillus niger crude cellulase enzyme for 18 h at 55 degrees C. Using 15% (dw/v) substrate 73 g l(-1) total reducing sugars were obtained to give 78.7% hydrolysis of carbohydrate content. Acid and enzyme hydrolyzates were mixed equally and used for fermentation with thermotolerant Saccharomyces cerevisiae (VS(3)). Yeast fermented L. camara hydrolyzate well with a fermentation efficiency of 83.7% to give an ethanol yield of 0.431 +/- 0.018 g ethanol pre g sugar and productivity of 0.5 +/- 0.021 g l(-1) h(-1). CONCLUSIONS: Even though inhibitors were present in L. camara hydrolyzate, maximum sugars were utilized by thermotolerant yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of L. camara for fuel ethanol production with improved strains and detoxification can be recommended.     

Co-culturing of Lactobacillus paracasei subsp. paracasei with a Lactobacillus delbrueckii subsp. delbrueckii Mutant to Make High Cell Density for Increased Lactate Productivity from Cassava Bagasse Hydrolysate

To increase the productivity of lactic acid, a co-culture of lactobacilli was made by mixing 1:1 ratio of Lactobacillus paracasei subsp. paracasei and a fast growing L. delbrueckii subsp. delbrueckii mutant. The culture was embedded on to polyurethane foam (PUF) cubes as a biofilm and used for fermentation. In order to prevent the cell leakage, the PUF cubes were further entrapped in calcium cross-linked alginate. The maximum lactic acid production using a high cell density free culture was >38 g l(-1) from ~40 g l(-1) of reducing sugar within 12 h of fermentation. Using PUF biofilms, the same yield of lactic acid attained after 24 h. When the cubes were further coated with alginate it took 36 h for the maximum yield. Even though, the productivity is slightly lesser with the alginate coating, cell leakage was decreased and cubes were reused without much decrease in production in repeated batches. Using a conventional control inoculum (3%, w/v), it took 120 h to yield same amount of lactic acid.     

Anaerobic fermentative production of lactic acid using cheese whey and corn steep liquor

Cheese whey was the most suitable substrate for production of lactic acid under anaerobic conditions by Entercoccus flavescens which, on supplementating with corn steep liquor (5% v/v) and 10 mM CaCO₃ at pH 5.5, 37°C, yielded 12.6 g lactic acid/l in 36 h. Production was scaled up to a 10 l bioreactor under controlled pH and continuous CO₂ supply and gave 28 g lactic acid/l in 30 h resulting in a net 8.7-fold increase in production as compared to unoptimized conditions.     

Neutralization/recovery of lactic acid from Lactococcus lactis: effects on biomass,lactic acid,and nisin production

Besides lactic acid, many lactic acid bacteria also produce proteinaceous metabolites (bacteriocins) such as nisin. As catabolite repression and end-product inhibition limit production of both products, we have investigated the use of alternative methods of supplying substrate and neutralizing or extracting lactic acid to increase yields. Fed-batch fermentation trials using a stillage-based medium with pH control by NH4OH resulted in improved lactic acid (83.4 g/l, 3.18 g/l/h, 95% yield) and nisin (1,260 IU/ml, 84,000 IU/l/h, 14,900 IU/g) production. Removing particulate matter from the stillage-based medium increased nisin production (1,590 IU/ml, 33,700 IU/g), but decreased lactic acid production (58.5 g/l, 1.40 g/l/h, 96% yield). Removing lactic acid by ion exchange resins stimulated higher lactic acid concentrations (60 to 65 g/l) and productivities (2.0 to 2.6 g/l/h) in the filtered stillage medium at the expense of nisin production (1,500 IU/ml, 25,800 IU/g).     

Production of lactic acid and fructose from media with cane sugar using mutant of Lactobacillus delbrueckii NCIM 2365

AIMS: To examine the potential of Lactobacillus delbrueckii mutant, Uc-3 to produce lactic acid and fructose from sucrose-based media. METHODS AND RESULTS: The mutant of L. delbrueckii NCIM 2365 was cultivated in shake flask containing hydrolysed cane sugar (sucrose)-based medium. The lactic acid yield and volumetric productivity with hydrolysed cane concentration up to 200 g l(-1) were in the range of 92-97% of the theoretical value and between 2.7 and 3.8 g l(-1) h(-1), respectively. The fructose fraction of the syrup produced was more than 95% when the total initial sugar concentration in the medium was higher (150-200 g l(-1)). There are no unwanted byproducts detected in the fermentation broth. CONCLUSIONS: We demonstrated that L. delbrueckii mutant Uc-3 was able to utilize glucose preferentially to produce lactic acid and fructose from hydrolysed cane sugar in batch fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings will be useful in the production of lactic acid and high fructose syrups using media with high concentrations of sucrose-based raw materials. This approach can lead to modification of the traditional fermentation processes to obtain value-added byproducts, attaining better process economics.     

Thermophilic production of lactic acid using integrated membrane bioreactor systems coupled with monopolar electrodialysis

The thermophilic Bacillus strain BS119 was selected for this study to demonstrate the long term performance of lactic acid production and simultaneous pre-purification. Integrated continuous cell recycle cultivation using ultra-filtration membrane bioreactor (MBR) systems was investigated. The permeate from the MBR was routed to an on-line electrodialysis (ED) to recover, pre-purify and concentrate lactate. The cultivation and ED was operated at 60 degrees C for more than 1,000 h at a pH of 6.5. At lower dilution rate (0.02 h(-1)), lactate concentration reached a maximum of 55 g l(-1) with clearly lower residual glucose levels. At 0.04 h(-1), lactate concentration was significantly lower at 35 g l(-1). Maximal volumetric productivities of 1.38 g l(-1) h(-1) were achieved. Under stable conditions, lactic acid yield on consumed glucose appeared stable at around 80%. It could be demonstrated that the addition of supplements like yeast extract and peptone severely influences product formation. Integration of mono-polar ED with the MBR systems yields lactate solutions with concentrations of up to 115 g l(-1). Because of the low substrate feed concentrations (less than 50 g l(-1)), lactate flux was rather poor, reaching a low maximum of 140 g m(-2) h(-1); nevertheless, stack energy consumption was positive with an average of 0.49 kWh kg(-1) lactate.     

Lactic acid production by batch fermentation of whey permeate: A mathematical model

Summary The batch fermentation of whey permeate to lactic acid was improved by supplementing the broth with enzyme-hydrolyzed whey protein. A mathematical model based on laboratory results predicts to a 99% confidence limit the kinetics of this fermentation. Cell growth, acid production and protein and sugar use rates are defined in quantifiable terms related to the state of cell metabolism. The model shows that the constants of the Leudeking-Piret model are not true constants, but must vary with the medium composition, and especially the peptide average molecular weight. The kinetic mechanism on which the model is based also is presented.Nomenclature K _i lactic acid inhibition constant (g/l) - K _pr protein saturation constant during cell growth (g/l) - K _pr protein saturation constant during maintenance (g/l) - K _s lactose saturation constant (g/l) - [LA] lactic acid concentration (g/l) - [PR] protein concentration (g/l) - [S] lactose concentration (g/l) - t time (h) - [X] cell mass concentration (g/l) - , fermentation constants of Leudeking and Piret - specific growth rate (l/h) - Y _{g, LA/S} acid yield during cell growth (g acid/g sugar) - Y _{m, LA/S} acid yield during maintenance (g acid/g sugar) - Y _x/pr yield (g cells/g protein) - specific sugar use rate during cell growth (g sugar/h·g cell) - specific sugar use rate during maintenance (g sugar/h·cell)