Batch production of L(+) lactic acid from whey by Lactobacillus casei (NRRL B‐441)

The effects of temperature, pH, and medium composition on lactic acid production by Lactobacillus casei were investigated. The highest lactic acid productivity values were obtained at 37 °C and pH 5.5. The productivity was 1.87 g dm^?3 h^?1 at 37 °C in shake flasks. In the fermenter, a productivity of 3.97 g dm^?3 h^?1 was obtained at pH 5.5. The most appropriate yeast extract concentration was 5.0 g dm^?3. Whey yielded a higher productivity value than the analytical lactose and glucose. Initial whey lactose concentration did not affect lactic acid productivity. MnSO₄ ·H₂O was necessary for lactic acid production by L casei from whey. Product yields were approximately 0.93 g lactic acid g lactose^?1. Copyright © 2004 Society of Chemical Industry     

Optimization of lactic acid production from whey by L casei NRRL B‐441 immobilized in chitosan stabilized Ca‐alginate beads

The production of lactic acid from whey by Lactobacillus casei NRRL B‐441 immobilized in chitosan‐stabilized Ca‐alginate beads was investigated. Higher lactic acid production and lower cell leakage were observed with alginate–chitosan beads compared with Ca‐alginate beads. The highest lactic acid concentration (131.2 g dm^?3) was obtained with cells entrapped in 1.3–1.7 mm alginate–chitosan beads prepared from 2% (w/v) Na‐alginate. The gel beads produced lactic acid for five consecutive batch fermentations without marked activity loss and deformation. Response surface methodology was used to investigate the effects of three fermentation parameters (initial sugar, yeast extract and calcium carbonate concentrations) on the concentration of lactic acid. Results of the statistical analysis showed that the fit of the model was good in all cases. Initial sugar, yeast extract and calcium carbonate concentrations had a strong linear effect on lactic acid production. The maximum lactic acid concentration of 136.3 g dm^?3 was obtained at the optimum concentrations of process variables (initial sugar 147.35 g dm^?3, yeast extract 28.81 g dm^?3, CaCO₃ 97.55 g dm^?3). These values were obtained by fitting of the experimental data to the model equation. The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in lactic acid production using alginate–chitosan‐immobilized cells. Copyright © 2005 Society of Chemical Industry     

Strategies to improve the bioconversion of processed wood into lactic acid by simultaneous saccharification and fermentation

In the one‐step conversion of wood into lactic acid by Simultaneous Saccharification and Fermentation (SSF), inhibition effects caused by hydrolysis‐ and fermentation‐derived compounds on both enzymatic activity and fermentative ability of microorganisms appear when the operation is carried out under conditions leading to high productivities. The main effects inhibiting SSF have been assessed, and the results obtained in fed‐batch experiments allowed the definition of strategies for improving the overall bioconversion process. As cellobiose caused significant inhibition of cellulases, the supplementation of media with β‐glucosidase resulted in improved kinetics and yields. The inhibition of both enzymatic activity and microbial metabolism by lactic acid was confirmed. Intermittent removal of lactic acid by passing the fermentation media through an anion‐exchange resin column resulted in increased productivities and yields. Improved conversion of pretreated wood into lactic acid (67% conversion of cellulose into lactic acid, with maximum lactic acid concentration of 108 g dm^?3 and a productivity of 0.94 g dm^?3 h^?1) was achieved combining multiple substrate addition, supplementation with fresh nutrients and enzymes and removal of lactic acid. © 2001 Society of Chemical Industry     

Biotechnological production of lactic acid integrated with potato wastewater treatment by Rhizopus arrhizus

This paper describes a feasibility study of a for lactic acid production integrated with are treatment of wastewater from an industrial starch plant. Rhizopus oryzae two strains, Rhizopus arrhizus and Rhizopus oligosporus were tested with respect to their capability to carry out simultaneous saccharification and fermentation to lactic acid using potato wastewater. Rhizopus arrhizus DAR 36017 was identified as a suitable strain that demonstrated a high capacity for starch saccharification and lactic acid synthesis. The optimal conditions, in terms of pH, temperature and starch concentration, for lactic acid production were determined. The selected fungal strain grew well in a pH range from 3.0 to 7.0. The addition of CaCO₃10 g dm^?3 maintained the pH at 5.0–6.0 and significantly enhanced lactic acid production. Kinetic study revealed that almost complete starch saccharification and a lactic acid yield of 450g kg^?1 could be achieved in 20 h and 28 h cultivation, respectively. The maximum lactic acid production 21 g dm^?3 and mycelial biomass (1.7 g dm^?3) were obtained at 30 °C. Besides the multiple bioproducts, total removal of suspended solids and 90% reduction of COD were achieved in a single no‐aseptic operation. Copyright © 2003 Society of Chemical Industry     

Protein hydrolysis under anaerobic,saline conditions in presence of acetic acid

The hydrolysis of soluble proteins in an anaerobic, saline (24 g dm^?3 NaCl) and mesophilic (37 °C) environment was studied. The inhibitory effect of a volatile fatty acid, acetic acid (HAc), on the hydrolysis rate and hydrolytic biomass activity for a model saline wastewater with a high protein load (total organic carbon, 1153 mg dm^?3 and 1572 mg dm^?3 proteins) was studied. Initial inhibitor concentrations were tested in the range of 0–2000 mg dm^?3 HAc. The microbiological characterization was performed using a total microorganism count by epifluorescence, and hydrolytic bacterial activity was determined by plate count. The protein hydrolysis was modeled according to first order kinetics. The effect of biomass on hydrolysis was analyzed by varying its concentration in the range of 42–210 mg dm^?3 volatile suspended solids. The following apparent hydrolysis kinetic constants (K_h) for proteins at 37 °C were obtained: 1.3, 0.8, 0.6, 0.2 and 0.1 d^?1 for initial concentrations of 250, 500, 750, 880, and 1000 mg dm^?3 HAc, respectively. At concentrations of HAc greater than 1000 mg dm^?3, total inhibition of hydrolysis was observed. The intrinsic hydrolysis constant ( ) at 37 °C, without inhibition, was 2.3 d^?1. The hydrolysis kinetic constant was not affected by the biomass concentration. The hydrolysis kinetics constant was filted to three models: Luong, Levenspiel and non‐competitive inhibition. The model that best represented the experimental data was Luong, obtaining an inhibition constant (K_I) of 1087 mg dm^?3 of HAc and the exponent γ = 0.54. The hydrolysis was inhibited by the presence of HAc, which corresponds to an intermediate compound of the anaerobic process. Copyright © 2004 Society of Chemical Industry     

An alternative for pre‐treatment of high‐strength raw whey wastewaters: submerged membrane bioreactors

The direct treatment of whey wastewater at various sludge ages (10–75 days) and high biomass concentration (above 50 g mixed liquor suspended solid (MLSS) dm^?3) in a submerged membrane bioreactor (sMBR) is described. The chemical oxygen demand (COD) of raw whey varied in the range of 60 and 90 g dm^?3. After feeding the sMBR with raw whey, effluent COD reduced to about 20 g dm^?3. The effluent was free of suspended solids and total coliform bacteria. Total phosphorus (TP) and orthophosphate (Ortho‐P) in the influent varied between 204 and 880 mg dm^?3 and between 180 and 620 mg dm^?3, and effluent TP and Ortho‐P reduced to 113 and 109 mg dm^?3, respectively. The ammonium and nitrate concentrations in the influent were in the ranges of 3.4 and 120 mg dm^?3 and 10 and 503 mg dm^?3, respectively. The effluent ammonium concentration varied between 17.6 and 198 mg dm^?3 and nitrate concentrations varied between 0.9 and 69 mg dm^?3. Effluent turbidity varied between 23 and 111 FAU (Formazin Attenuation Unit). The results show that sMBR is an effective pre‐treatment system for high‐strength agro‐wastewaters because of its ability to reduce the pollution load. Copyright © 2004 Society of Chemical Industry     

Kinetics of lactic acid fermentation on glucose and corn by Lactobacillus amylophilus

The biosynthesis of ammonium lactate, a product of lactic acid fermentation was studied from corn and glucose at five different pH values of 5.4 to 7.8. In the glucose fermentations, a 100% conversion of substrate was obtained resulting in a maximum lactic acid production yield of 93.2%. The optimum pH for the maximum volumetric rate of lactic acid biosynthesis (1.56 g dm^?3 h^?1) was between 6.0 and 6.5. The corn fermentations were slower than the glucose fermentations with a resulting lactic acid yield of 67.5%. Hydrolysis of corn by enzymatic or chemical methods as well as the use of ammonium hydroxide for pH control increased both the final concentration and the rates of lactic acid production. An enhanced yield of more than 90% was finally obtained in the corn fermentations. A logistic model adequately described the kinetics of biomass growth, lactic acid production and sugar utilization in the glucose fermentations at different pH values. The dynamics of lactic acid formation in the corn fermentations were also successfully described by the developed model. The dependence of the model parameters on pH was investigated.     

Kinetics of syntrophic acetogenesis in a saline medium

BACKGROUND: The anaerobic degradation kinetics of volatile fatty acids (VFA) in a saline (24 g NaCl dm^?3) and mesophilic (37 °C) medium was studied under batch test conditions. The acetate production kinetics without inhibition by propionic, butyric and valeric acids was determined. The inhibition of acetate production during syntrophic acetogenesis by VFA and pH was studied. The acetogenesis without inhibition was modelled using a Monod equation. The pH inhibition was represented by a Michaelis pH function, while the inhibition by acetic acid (HAc) was represented by a non‐competitive model. RESULTS: The specific maximum degradation rate and saturation constant (k_{max, VFA}, K_{S, max}) values were (5.89, 15.95), (7.97, 25.99) and (7.75 g VFA g^?1 volatile suspended solids day^?1, 11.52 mg VFA dm^?3) for propionic, butyric and valeric acids respectively, with maximum velocity at pH 7. The inhibition constants (K_{I, HAc}) were 1295, 671 and 572 mg HAc dm^?3 for propionic, butyric and valeric acids respectively. CONCLUSION: VFA and pH can be inhibitory for acetogenesis under these conditions. Copyright © 2008 Society of Chemical Industry     

Effect of sucrose on nisin production in batch and fed‐batch culture by Lactococcus lactis

The effects of sucrose on cell growth and nisin production by Lactococcus lactis were investigated in batch and pH feed‐back controlled fed‐batch cultures. In batch cultures, nisin titer reached its maximum, 2658 IU cm^?3, at the initial sucrose concentration of 30 g dm^?3. With sucrose concentrations higher than 30 g dm^?3, nisin production decreased while the biomass was not influenced significantly. By using the pH feed‐back controlled method, residual sucrose concentration could be controlled well in fed‐batch cultures and three conditions (sucrose maintained at 2, 16, 20 g dm^?3, respectively) were evaluated. Maintaining a low sucrose concentration at 2 g dm^?3 during feeding favored nisin biosynthesis, and the maximum nisin titer obtained was 4961 IU cm^?3 compared with 3370 IU cm^?3 (16 g sucrose dm^?3)and 3498 IU cm^?3 (20 g sucrose dm^?3), respectively. Copyright © 2005 Society of Chemical Industry     

D‐Lactic acid production from waste cardboard

The effects caused by alkaline treatment on the susceptibility of waste cardboard to enzymatic hydrolysis have been studied. Optimised conditions leading to extensive saccharification of both cellulose (870 g kg^?1 conversion) and hemicelluloses (845 g kg^?1 conversion) were identified. Samples treated under selected operational conditions were employed for producing D ‐lactic acid by simultaneous saccharification and fermentation (SSF) in media containing cellulases, β‐glucosidase and Lactobacillus coryniformis ssp torquens cells. SSF fed‐batch experiments led to D ‐lactic acid concentrations up to 23.4 g dm^?3 at a product yield of 514 g lactic acid kg^?1 of potential glucose and a volumetric productivity of 0.48 g dm^?3 h^?1. Copyright © 2004 Society of Chemical Industry     

Effects of polyacrylamide‐co‐acrylic acid on cellulose production by Acetobacter xylinum

Polyacrylamide‐co‐acrylic acid (PA) added to shake flask cultures of Acetobacter xylinum at concentrations up to 3 g dm^?3 resulted in increased production of bacterial cellulose. For PA concentrations of 0–3 g dm^?3, 7‐day cellulose production rose monotonically from 2.7 ± 0.8 to 6.5 ± 0.5 g dm^?3 at a shaker speed of 175 rpm, and from 1.7 ± 0.01 to 3.7 ± 0.5 g dm^?3 at shaker speed of 375 rpm. Addition of PA also changed the morphology of the biomass from amorphous/stringy forms to spheroidal particles with diameters ≤2 mm. Similarly, bioreactor cultures grown in the absence of PA formed long fibrous masses which deposited on the internals, while those grown in the presence of 1–2 g dm^?3 PA formed small discrete particles with diameters ≤0.1 mm. Tests performed with 1 and 2 g dm^?3 PA, and stirrer speeds from 500 to 900 rpm, appeared to give the highest cellulose concentration of 5.3 ± 0.7 g dm^?3 in 64–68.5 h in the presence of 2 g dm^?3 PA at 700 rpm, although this value was statistically indistinguishable from that obtained at 1 g dm^?3 PA and 900 rpm. A qualitative model is proposed to describe the mechanisms by which PA affects biomass morphology, resulting in its advantageous formation as small, dispersed, spheroidal pellets. Quantitative analysis of the results gave inverse correlations between both the fraction of fructose carbon going to cellulose synthesis and the specific fructose consumption rate, and the maximum cellulose concentration and the fraction of fructose carbon going to by‐product formation. Since cellulose yield was almost universally improved by higher polyacrylamide concentration, it appears likely that increased viscosity reduces fructose uptake rate by limiting mass transfer. Copyright © 2003 Society of Chemical Industry     

In‐line mixing for production of citric acid by Candida lipolytica grown on n‐paraffins

This study reports on the effects of internal fermenter and external in‐line agitation and fed‐batch mode of operation on citric acid production from Candida lipolytica using n‐paraffin as the carbon source. An optimum range of fermenter agitation speeds in the range 800–1000 rpm corresponding to Reynolds numbers of 50433–62947 (based on initial batch conditions) seemed to give the best balance between substrate utilization for biomass growth and citric acid production. Proof of concept evidence is presented that indicates that an external in‐line agitator could be used in place of high speed internal agitation to increase citric acid production. However, more work is required to optimize the external agitator concept. Application of multiple fed‐batch feedings can be used to extend the batch fermentation and increase final citric acid concentrations and product yield. Experiments were conducted implementing a three‐cycle fed‐batch process which increased overall citric acid yields to 0.8–1.0 g citric acid g^?1 n‐paraffin, approximately 200% improvement from those found in the normal batch process. The three‐cycle fed‐batch mode of operation also increased the final citric acid concentration to 42 g dm^?3 from about 6 g dm^?3 for normal batch operation. Increased citric acid concentrations in three‐cycle fed‐batch mode was achieved at longer fermentation times. Copyright © 2004 Society of Chemical Industry     

Fermentation optimization for the production of lovastatin by Aspergillus terreus: use of response surface methodology

A Box–Behnken experimental design was used to investigate the effects of five factors—ie oxygen content in the gas phase; concentrations of C, N and P; and fermentation time—on the concentrations of biomass and lovastatin produced in batch cultures of Aspergillus terreus. The values of the various factors in the experiment ranged widely, as follows: 20–80% (v/v) oxygen in the aeration gas; 8–48 g dm^?3 C‐concentration; 0.2–0.6 g dm^?3 N‐concentration; 0.5–2.5 g dm^?3 phosphate‐concentration; and 7–11 days fermentation time. No previous work has used statistical analysis in documenting the interactions between oxygen supply and nutrient concentrations in lovastatin production. The Box–Behnken design identified the oxygen content in the gas phase as the principal factor influencing the production of lovastatin. Both a limitation and excess of oxygen reduced lovastatin titers. A medium containing 48 g dm^?3 C supplied as lactose, 0.46 g dm^?3 N supplied as soybean meal, and 0.79 g dm^?3 phosphate supplied as KH₂PO₄, was shown to support high titers (～230 mg dm^?3) of lovastatin in a 7‐day fermentation in oxygen‐rich conditions (80% v/v oxygen in the aeration gas). Under these conditions, the culture medium had excess carbon but limiting amounts of nitrogen. The optimized fermentation conditions raised the lovastatin titer by four‐fold compared with the worst‐case scenario within the range of factors investigated. Copyright © 2004 Society of Chemical Industry     

Production of fructose and ethanol from media with high sucrose concentrations by a mutant of Saccharomyces cerevisiae

The production of enriched fructose syrups and ethanol from a synthetic medium with high sucrose concentrations was studied in a batch process using Saccharomyces cerevisiae ATCC 36858. The results showed that the fructose yield was above 92% of theoretical values in synthetic media with sucrose concentrations between 180 g dm^?3 and 726 g dm^?3. Ethanol yield was about 82% in media with sucrose concentrations up to 451 g dm^?3. A product containing 178 g dm^?3 fructose, which represents 97% of the total sugar content, and 79 g dm^?3 ethanol was obtained using a medium with 360 g dm^?3 sucrose. The fructose fraction in the carbohydrates content in the produced syrups decreased with increases in the initial sucrose concentration. In a medium with initial sucrose concentration of 574 g dm^?3, the fructose content in the produced broth was 59% of the total carbohydrates. Glycerol and fructo‐oligosaccharides were also produced in this process. The produced fructo‐oligosaccharides started to be consumed when the concentration of sucrose in the media was less than 30% of its initial value. Complete hydrolysis of these sugars was noticed in media with sucrose concentrations below 451 g dm^?3. These findings will be useful in the production of ethanol and high fructose syrups using sucrose‐based raw materials with high concentrations of this carbohydrate. © 2001 Society of Chemical Industry     

Cell recycle and broth reuse fermentation with cross-flow filtration and ion-exchange resin

A novel integrated fermentation system in which cross-flow filtration was coupled to an anion-exchange resin column was developed to achieve biomass recycle and broth reuse for lactic acid fermentation. An anion-exchange resin column was employed to recover lactic acid from the spent broth. The effluent was diluted with fresh medium, supplemented with glucose and nutrients. Spent broth was reused for three consecutive biomass recycle fermentations with no significant decrease in fermentation performance. The fermentation system enabled simultaneously high productivity of lactic acid (average value 7·75 g dm⁻³ h⁻¹ and total amount of lactic acid produced 85·21 g dm⁻³ after 11 h fermentation), high productivity of cells (average value 2·00 g dm⁻³ h⁻¹) and efficient utilization of medium (about 75% of the spent broth was reutilized). The system described may be applied to other organic fermentations subject to end-product inhibition.     

The effect of raw glycerol concentration on the production of 1,3‐propanediol by Clostridium butyricum

Raw glycerol, the main by‐product of the bio‐diesel production process, was converted to 1,3‐propanediol by Clostridium butyricum F2b. In batch cultures, 47.1 g dm^?3 of 1,3‐propanediol were produced. Continuous cultures were conducted at a constant dilution rate (= 0.04 h^?1) and various inlet glycerol concentrations with 1,3‐propanediol produced at levels up to 44.0 g dm^?3. At increasing glycerol concentrations in the inlet medium, biomass yield decreased. This decrease was attributed to the microbial metabolism being directed towards the biosynthesis of organic acids (and hence carbon losses as CO₂) instead of biochemical anabolic reactions. An autonomous analytical model was developed, and quantified the effect of inlet glycerol concentration on the production of biomass and metabolites. Indeed, high inlet substrate concentrations positively affected the biosynthesis, principally of butyric acid and to a lesser extent that of acetic acid. In contrast, at increased glycerol concentrations, the relative increase of 1,3‐propanediol production per unit of substrate consumed was lower as compared with that of acetic and, mainly, butyric acid. This could be explained by the fact that the butyric acid pathway represents an alternative and competitive one to that of 1,3‐propanediol for re‐generation of NADH₂ equivalents in the microbial cell. Copyright © 2004 Society of Chemical Industry     

Experimentation of a new mode of batch culture for lactic acid bacteria: cell reuse with an initial period of cell reactivation at acidic pH

Cell reuse was compared with conventional batch culture for lactic acid fermentation, the objective was to simplify the batch process and to alleviate the need for added nitrogen. At high levels of nitrogen supplementation to the culture medium (20 g dm^?3 yeast extract and 5 g dm^?3 each of tryptic and pancreatic casein peptones), similar mean production rates were obtained with partial cell reuse and the conventional batch process, without any additional gain when cells were initially reactivated at acidic pH. On the other hand, cell reuse with an initial period without pH control appeared particularly effective for low levels of nitrogen supplementation (5 g dm^?3 yeast extract): a 57% increase in the mean production rate with regard to the conventional batch process was obtained. © 2001 Society of Chemical Industry     

Plasmid stability in a recombinant S cerevisiae strain secreting a bifunctional fusion protein

The recombinant Saccharomyces cerevisiae strain, YPB‐G, producing and secreting Bacillus subtilis α‐amylase and Aspergillus awamori glucoamylase as a fusion protein yielded efficient utilisation of starch. A segregated population balance model has been used to determine the probability of plasmid loss and plasmid copy number. The kinetics of cell growth and product (fusion protein) formation were based on a genetically structured model. The predictions were compared with the experimental observations obtained for the unstable recombinant S cerevisiae cells in a 1.5 dm^?3 batch bioreactor with 30 g dm³ initial starch under non‐aerated conditions. The main advantage of the present model is that three different genetic classes were defined on the basis of the existence of plasmid and of the expression of the enzymes, ie cells containing plasmids and expressing the gene product, x₁; cells containing plasmids and but not expressing the gene product, x₂; and cells without plasmids, x₃. It is confirmed by this model that the cells without plasmids outgrow and dominate in the fermentation medium (2.27 g dm^?3 vs 0.51 g dm^?3) as more and more glucose becomes available by the degradation of starch. © 2001 Society of Chemical Industry     

Volatile fatty acid production during anaerobic mesophilic digestion of solid potato waste

The production of volatile fatty acids by anaerobic digestion of solid potato waste was investigated using a batch solid waste reactor with a working capacity of 2 dm^?3 at 37°C. Solid potato waste was packed into the digester and the organic content of the waste was released by microbial activity by circulating water over the bed, using batch loads of 500 g or 1000 g potato waste. The sequence of appearance of the volatile fatty acids was (acetic, propionic); (n‐butyric); (n‐valeric, iso‐valeric, caproic); (iso‐butyric). After 300 h digestion of potato waste on a small scale, the fermentation products were chiefly (mg g^?1 total VFAs): acetic acid (420), butyric acid (310), propionic acid (140) and caproic acid (90), with insignificant amounts of iso‐butyric acid, n‐valeric and iso‐valeric acids. When the load of potato solids was increased, the volatile fatty acid content was similar, but butyric acid constituted 110 mg g^?1 and lactic acid 400 mg g^?1 of the total volatile fatty acids. The maximum soluble chemical oxygen demand (COD) achieved under the experimental conditions used was 27 and 37 g COD dm^?3 at low and high loadings of potato solids, respectively. The total volatile fatty acids reached 19 g dm^?3 of leachate at both loads of potato solid waste. Gas production was negligible, indicating that methanogenic activity was effectively inhibited. Copyright © 2004 Society of Chemical Industry     

Stoichiometric analysis of Kluyveromyces fragilis growth on lactose

This paper describes results of the application of simple metabolic model principles to aerobic growth of Kluyveromyces fragilis during the utilisation of a semi‐synthetic medium simulating dairy waste (whey). The theoretical yield coefficients (maximum, true and real) of biomass on lactose and oxygen, available electrons and ATP were determined and compared with experimental data. The experimental runs were performed in a B Brown fermenter (15 dm³ of operating capacity). The yeast K fragilis was cultured in semi‐synthetic medium containing lactose (40 g dm⁻³) as a main carbon and energy source and ammonia salts as a main nitrogen source (C/N = 8.74). The yeast growth is closely related to the concentration of oxygen in the media. The experimental yields of biomass on lactose and oxygen reached 94% and 88% of real maximum theoretical values, respectively. Calculated true biomass yield coefficients were closely correlated with the values resulting from the balance analysis of stoichiometric equations. The specific maintenance requirement (m_ATP) was 14.7 mmole ATP(g DM h)^‐1. Determination of the elemental composition of the biomass during the course of fermentation resulted in observations of the cellular C/N ratio changes. The conversion of lactose carbon to biomass and CO₂ was 74.3%–81.2%. The recovery of ATP reached 79%, and the recovery of oxygen 96.2%. © 2000 Society of Chemical Industry