Applicability of pectate-entrapped <Emphasis Type="Italic">Lactobacillus casei</Emphasis> cells for <Emphasis Type="SmallCaps">l</Emphasis>(+) lactic acid production from whey

Lactic acid is a versatile organic acid, which finds major application in the food, pharmaceuticals, and chemical industries. Microbial fermentation has the advantage that by choosing a strain of lactic acid bacteria producing only one of the isomers, an optically pure product can be obtained. The production of l(+) lactic acid is of significant importance from nutritional viewpoint and finds greater use in food industry. In view of economic significance of immobilization technology over the free-cell system, immobilized preparation of Lactobacillus casei was employed in the present investigation to produce l(+) lactic acid from whey medium. The process conditions for the immobilization of this bacterium using calcium pectate gel were optimized, and the developed cell system was found stable during whey fermentation to lactic acid. A high lactose conversion (94.37%) to lactic acid (32.95 g/l) was achieved with the developed immobilized system. The long-term viability of the pectate-entrapped bacterial cells was tested by reusing the immobilized bacterial biomass, and the entrapped bacterial cells showed no decrease in lactose conversion to lactic acid up to 16 batches, which proved its high stability and potential for commercial application.     

Production of l (+) lactic acid by Lactobacillus delbrueckii immobilized in functionalized alginate matrices

The role of functionalized alginate gels as immobilized matrices in production of l (+) lactic acid by Lactobacillus delbrueckii was studied. L. delbrueckii cells immobilized in functionalized alginate beads showed enhanced bead stability and selectivity towards production of optically pure l (+) lactic acid in higher yields (1.74Yp/s) compared to natural alginate. Palmitoylated alginate beads revealed 99% enantiomeric selectivity (ee) in production of l (+) lactic acid. Metabolite analysis during fermentation indicated low by-product (acetic acid, propionic acid and ethanol) formation on repeated batch fermentation with functionalized immobilized microbial cells. The scanning electron microscopic studies showed dense entrapped microbial cell biomass in modified immobilized beads compared to native alginate. Thus the methodology has great importance in large-scale production of optically pure lactic acid.     

Lactic acid production from cellulosic waste by immobilized cells of Lactobacillus delbrueckii

Industrial waste corn cob residue (from xylose manufacturing) without pretreatment was hydrolyzed by cellulase and cellobiase. The cellulosic hydrolysate contained 52.4 g l⁻¹ of glucose and was used as carbon source for lactic acid fermentation by cells of Lactobacillus delbrueckii ZU-S2 immobilized in calcium alginate gel beads. The final concentration of lactic acid and the yield of lactic acid from glucose were 48.7 g l⁻¹ and 95.2%, respectively, which were comparative to the results of pure glucose fermentation. The immobilized cells were quite stable and reusable, and the average yield of lactic acid from glucose in the hydrolysate was 95.0% in 12 repeated batches of fermentation. The suitable dilution rate of continuous fermentation process was 0.13 h⁻¹, and the yield of lactic acid from glucose and the productivity were 92.4% and 5.746 g l⁻¹ h⁻¹, respectively. The production of lactic acid by simultaneous saccharification and fermentation (SSF) process was carried out in a coupling bioreactor, the final concentration of lactic acid was 55.6 g l⁻¹, the conversion efficiency of lactic acid from cellulose was 91.3% and the productivity was 0.927 g l⁻¹ h⁻¹. By using fed-batch technique in the SSF process, the final concentration of lactic acid and the productivity increased to 107.6 g l⁻¹ and 1.345 g l⁻¹ h⁻¹, respectively, while the dosage of cellulase per gram substrate decreased greatly. This research work should advance the bioconversion of renewable cellulosic resources and reduce environmental pollution.     

Immobilized growing lactic acid bacteria with κ-carrageenan — locust bean gum gel

Summary A cell entrapment process using -carrageenan — locust bean gum gel is presented. Streptococcus thermophilus, Lactobacillus bulgaricus and S. lactis were immobilized in small gel beads (0.5–1.0 mm and 1.0–2.0 mm diameter) and fermentations in bench bioreactors were conducted. Viability of entrapped cells, lactose utilization, lactic acid production and cell release rates were measured during fermentation. The procedure was effective for S. thermophilus, L. bulgaricus and S. lactis, and the viability of these bacteria remained very high throughout entrapment steps and subsequent storage. Bead diameter influenced the fermentation rate: smaller beads (0.5–1.0 mm) permitted an increase in release rates, lactose utilization and acid production by entrapped cells, approximating values attained with free cells.     

Improved immobilization of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> ATCC 7469 in polyacrylamide gel,preventing cell leakage during lactic acid fermentation

A new procedure for improved immobilization of Lactobacillus rhamnosus ATCC 7469, producing solely l(+)-lactic acid, in polyacrylamide was developed. A series of gels with varied ingredients concentrations and order of addition was prepared and were tested in batch and repeat-batch processes. Our results revealed that the crucial step for successful immobilization was the initial incubation of the cells in pure 10% AA that leads to improved entrapment in the polyacrylamide gel. In contrast, all gels derived from previously prepared stock AA/MBAA released high amount of cells and free biomass was formed. The most efficient immobilization was achieved using gel, containing L. rhamnosus, incubated in 10% AA (acrylamide) and with 1% MBAA (N,N′-methylene-bis-acrylamide) added. This gel possessed optimal permeation characteristics and at the same time, the cells were completely retained in the polymer lattice (0.03 g free biomass/l at 48 h of the batch process). In addition, it yielded highly concentrated lactic acid: the conversion ratio was about 85% without pH-control for initial lactose concentrations of up to 30 g/l. A series of additional immobilization experiments showed the potential of physicochemical interactions between the monomers of acrylamide and the cell surface of L. rhamnosus.     

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.     

Lactic acid production from whey permeate by immobilized Lactobacillus casei

Two matrices have been assessed for their ability to immobilize Lactobacillus casei cells for lactic acid fermentation in whey permeate medium. Agar at 2% concentration was found to be a better gel than polyacrylamide in its effectiveness to entrap the bacterial cells to carry out batch fermentation up to three repeat runs. Of the various physiological parameters studied, temperature and pH were observed to have no significant influence on the fermentation ability of the immobilized organism. A temperature range of 40–50°C and a pH range of 4.5–6.0 rather than specific values, were found to be optimum when fermentation was carried out under stationary conditions. In batch fermentation ～90% conversion of the substrate (lactose) was achieved in 48 h using immobilized cell gel cubes of 4 × 2 × 2 mm size, containing 400 mg dry bacterial cells per flask and 4.5% w/v (initial) whey lactose content as substrate. However, further increase in substrate levels tested (>4.5% w/v) did not improve the process efficiency. Supplementation of Mg²⁺ (1 mM) and agricultural by-products (mustard oil cake, 6%) in the whey permeate medium further improved the acid production ability of the immobilized cells under study.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Repeated fed-batch lactic acid production in a packed bed-stirred fermentor system using a pH feedback feeding method

Lactic acid production by repeated fed-batch fermentation using free and immobilized cells of Lactobacillus lactis-11 in a packed bed-stirred fermentor (PBSF) system filled with different support materials including ceramic beads, macro-activated carbon cylinders and glass fiber balls was investigated. The results showed that the optimal support materials were the ceramic beads with diameters of 1–2 mm. Compared with the free cell fermentation system, lactic acid production and volumetric productivity in the PBSF system increased by 16.6 and 12.5%, respectively. Though the concentration of free cells decreased sharply, lactic acid production remained stable in five consecutive fed-batch runs using the PBSF system. pH gradients, immobilized cell concentration and mass diffusion in the packed bed were all affected by the recirculation rate of the culture broth. Maximum lactic acid production, productivity and yield occurred at a recirculation rate of 50 mL min⁻¹.     

Sugar Utilization and Acid Production by Free and Entrapped Cells of Streptococcus salivarius subsp. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactococcus lactis subsp. lactis in a Whey Permeate Medium

Cells of Streptococcus salivarius subsp. thermophilus and Lactococcus lactis subsp. lactis entrapped in k-carrageenan-locust bean gum gel performed similarly to free cells in the conversion of lactose to lactic acid. Bead diameter influenced the fermentation rate. Cells entrapped in smaller beads (0.5 to 1.0 mm) showed higher release rates, higher lactose, glucose, and formic acid utilization, higher galactose accumulation, and higher lactic acid production than did cells entrapped in larger beads (1.0 to 2.0 mm). Values for smaller beads were comparable with those for free cells. Immobilization affected the fermentation rate of lactic acid bacteria, especially Lactobacillus delbrueckii subsp. bulgaricus. Entrapped cells of L. delbrueckii subsp. bulgaricus demonstrated a lower lactic acid production than did free cells in batch fermentation. The kinetics of the production of formic and pyruvic acids by L. lactis subsp. lactis and S. salivarius subsp. thermophilus are presented.     

L-Lactic acid production from raw cassava starch in a circulating loop bioreactor with cells immobilized in loofa (Luffa cylindrica)

l-Lactic acid was produced from raw cassava starch, by simultaneous enzyme production, starch saccharification and fermentation in a circulating loop bioreactor with Aspergillus awamori and Lactococcus lactis spp. lactis immobilized in loofa sponge. A. awamori was immobilized directly in cylindrical loofa sponge while the L. lactis was immobilized in a loofa sponge alginate gel cube. In the loofa sponge alginate gel cube, the sponge serves as skeletal support for the gel with the cells. The alginate gel formed a hard outer layer covering the soft porous gel inside. By controlling the rate and frequency of broth circulation between the riser and downcomer columns, the riser could be maintained under aerobic condition while the downcomer was under anaerobic condition. Repeated fed-batch l-lactic acid production was performed for more than 400 h and the average lactic acid yield and productivity from raw cassava starch were 0.76 g lactic acid g^–1 starch and 1.6 g lactic acid l^–1 h^–1, respectively.     

Removal of skim milk lactose by fermentation using free and immobilized Kluyveromyces marxianus cells

Kluyveromyces marxianus CBS 6164 cells, free or immobilized in Ca-alginate (2%) beads, are able to consume more than 99% of the skim milk lactose in anaerobic conditions. In batches at 30 °C, the lactose consumption after 3.5 h of skim milk fermentation by 30 and 50 g free K. marxianus cells per liter was around 99 and 99.6% respectively, with an approximate conversion of lactose to ethanol and CO₂ of 80%. The immobilized cells, easy to handle and showing a faster and easier separation from the fermented medium compared to the free ones, were used in more than 23 batches (cycles of re-use) without losing their activity.     

Lactic acid production from hemicellulosic hydrolyzate by cells of <Emphasis Type="Italic">Lactobacillus bifermentans</Emphasis> immobilized in Ca-alginate using response surface methodology

Consumption of hexoses/pentoses and production of lactic acid by Lactobacillus bifermentans were investigated in optimized culture medium and hemicellulosic hydrolyzates. The hydrolyzate used had the following composition (expressed in gL⁻¹): xylose 50 ± 5 gL⁻¹; glucose 18 ± 3 gL⁻¹; arabinose 29 ± 5 gL⁻¹. The immobilization experiments were conducted with microbial cells entrapped in calcium alginate beads. The results indicate that maximum concentrations of lactic acid were produced after 54 h of fermentation. All glucose and arabinose in wheat bran hydrolyzate were consumed during fermentation. Only xylose was not completely consumed. The substrate consumption rate was 3.2 gh⁻¹, 1.9 gh⁻¹, 1.6 gh⁻¹ respectively for glucose, arabinose, and xylose. The optimized culture condition gave a lactic acid concentration and metabolic yield of 62.77 gL⁻¹ and 0.83 gg⁻¹. These parameters improved to 41.3 gL⁻¹ and 0.47 gg⁻¹ respectively, when cell free was used.     

Determination of the intrinsic fermentation kinetics of Saccharomyces cerevisiae cells immobilized in Ca-alginate beads and observations on their growth

Continuous fermentation experiments in a well-stirred fermentor with Saccharomyces cerevisiae cells immobilized in Ca-alginate beads of small diameter (approx. 1 mm) have been performed in order to discover their intrinsic fermentation kinetics, and compare them to the fermentation kinetics for free cells, by fitting both sets of results to the same model. The results show similar kinetic parameters for free and immobilized cells. The changes in cell concentration inside the beads and microscopical observations of transverse sections throughout the experiments, allowed discernment of two different scenarios of cell growth inside the beads: low cell density and fully developed growth. Correspondence to: F. Gòdia     

Uptake of lactose and continuous lactic acid fermentation by entrapped non-growing Lactobacillus helveticus in whey permeate

 Continuous production of lactic acid from lactose has been carried out in a stirred-tank reactor with non-growing Lactobacillus helveticus entrapped in calcium alginate beads. A considerably longer operation half-life was obtained in a continuously operated reactor than in a batch-operated reactor. It is possible to simulate the action of entrapped non-growing cells on the basis of information from diffusion and kinetic experiments with suspended free cells. The simulation fit the experimental data over a broad range of substrate concentrations if the specific lactic acid production rate, q _P, was used as a variable parameter in the model. The dynamic mathematical model used is divided into three parts: the reactor model, which describes the mass balance in a continuously operated stirred-tank reactor with immobilized biomass, the mass-transfer model including both external diffusion and internal mass transfer, and the kinetic model for uptake of substrate on the basis of a Michaelis-Menten-type mechanism. From kinetic data obtained for free biomass experiments it was found, with the use of non-linear parameter estimation techniques, that the conversion rate of lactose by L. helveticus followed a Michaelis-Menten-type mechanism with K _S at half-saturation=0.22±0.01 g/l. The maximum specific lactose uptake rate for growing cells, q _S,max, varied between 4.32±0.02 g lactose g cells^-1 h^-1 and 4.89 ±0.02 g lactose g cells^-1 h^-1. The initial specific lactose uptake rate for non-growing cells, q _S,0, was found to be approximately 40% of the maximum specific lactose uptake rate for growing cells. Received: 4 October 1995/Received last revision: 23 April 1996/Accepted: 29 April 1996     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.     

Immobilization of Oenococcus oeni in lentikats® to develop malolactic fermentation in wines

Entrapment of Oenococcus oeni into a polymeric matrix based on polyvinyl alcohol (PVA) (Lentikats®) was successfully used to get a better development of malolactic fermentation (MLF) in wine. The incubation of immobilized cells in a nutrient medium before starting the MLF, did not improve the degradation of malic acid. In only one day, 100% of conversion of malic acid was achieved using a high concentration of immobilized cells (0.35 g gel/ml of wine with a cell‐loading of 0.25 mg cells/mg of gel). While a low concentration of 0.21 g gel/ml of wine (cell‐loading of 0.25 mg cells/mg of gel) needed 3 days to get a reduction of 40%. The entrapped cells could be reused through six cycles (runs of 3 days), retaining 75% of efficacy for the conversion of malic acid into lactic acid. The immobilized cells in PVA hydrogels gave better performance than free cells because of the increase of the alcohol toleration. Consequently, the inhibitory effect of ethanol for developing MLF could be reduced using immobilized cells into PVA hydrogels. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Lactic acid fermentation by Lactobacillus casei in free cell form and immobilised on gluten pellets

A comparative study of the fermentation of a range of carbohydrate substrates, at various temperatures, was carried out using a commercial Lactobacillus casei strain in a free cell form and immobilised on gluten pellets. This strain required yeast extract, l-cysteine HCl and Mn²⁺ at 5, 0.5 and 0.1 g l^–1, respectively, for maximum growth and lactic acid production. Sugar fermentation using free cells showed preference in the order glucose, sucrose, fructose while lactose was poorly utilised. Optimum temperature for growth and lactic acid production over (18–30 h) was 43 °C. L. casei was successfully immobilised on gluten pellets and fermented glucose and sucrose in a shorter time (18 h) with increased lactic acid production (42 and 41 g l^–1 on glucose and sucrose, respectively).     

Optimal conditions for production of lactic acid from cheese whey permeate by Ca-alginate-entrapped Lactobacillus helveticus

Whole cells of Lactobacillus helveticus were immobilized in calcium-alginate beads to produce lactic acid from cheese whey ultrafiltrate. Ca-alginate-entrapped cells were characterized by higher fermentation rates and optimum pH than free cells. No difference could be observed in the profile of cell activity against temperature for either type of cells. After a heat treatment, cell activity was higher for free cells than for immobilized cells. Continuous lactic acid fermentation using a packed bed reactor was investigated.     

拉曼光谱分析技术在资源微生物发酵性能评价中的应用

微生物发酵过程是细胞新陈代谢进行物质转化的过程,为了提高目标产物的转化率,需要对微生物发酵动态特性进行实时分析,以便实时优化发酵过程。拉曼光谱(Raman spectroscopy)量化测试作为一种有应用前景的在线过程分析技术,可以在避免微生物污染的条件下,实现精准监测,进而用于优化控制微生物发酵过程。【目的】以运动发酵单胞菌(Zymomonas mobilis)为例,建立微生物发酵过程中葡萄糖、木糖、乙醇和乳酸浓度拉曼光谱预测模型,并进行准确性验证。【方法】采用浸入式在线拉曼探头,收集运动发酵单胞菌发酵过程中多个组分的拉曼光谱,采用偏最小二乘法对光谱信号进行预处理和多元数据分析,结合离线色谱分析数据,对拉曼光谱进行建模分析和浓度预测。【结果】针对运动发酵单胞菌,首先实现拉曼分析仪对单一产品乙醇发酵过程的精准检测,其次基于多元变量分析,建立葡萄糖、乙醇和乳酸浓度变化的预测模型,实现对发酵过程中各成分浓度变化的准确有效分析。【结论】成功建立了一种评价资源微生物尤其是工业菌株发酵液多种组分的拉曼光谱分析方法。该方法为运动发酵单胞菌等工业菌株利用多组分底物工业化生产不同产物的实时检测,以及其他微生物尤其工业菌株的选育和过程优化提供了新方法。