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.     

Optimum conditions for the biological production of lactic acid by a newly isolated lactic acid bacterium,Lactobacillus sp. RKY2

Lactic acid is a green chemical that can be used as a raw material for biodegradable polymer. To produce lactic acid through microbial fermentation, we previously screened a novel lactic acid bacterium. In this work, we optimized lactic acid fermentation using a newly isolated and homofermentative lactic acid bacterium. The optimum medium components were found to be glucose, yeast extract, (NH₄)₂HPO₄, and MnSO₄. The optimum pH and temperature for a batch culture ofLactobacillus sp. RKY2 was found to be 6.0 and 36°C, respectively. Under the optimized culture conditions, the maximum lactic acid concentration (153.9 g/L) was obtained from 200 g/L of glucose and 15 g/L of yeast extract, and maximum lactic acid productivity (6.21 gL⁻¹h⁻¹) was obtained from 100 g/L of glucose and 20 g/L of yeast extract. In all cases, the lactic acid yields were found to be above 0.91 g/g. This article provides the optimized conditions for a batch culture ofLactobacillus sp. RKY2, which resulted in highest productivity of lactic acid.     

Ethanol and xylitol production from glucose and xylose at high temperature by <Emphasis Type="Italic">Kluyveromyces</Emphasis> sp. IIPE453

A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45°C to 50°C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 ± 0.5 g l⁻¹ (10.4% v/v) on initial glucose concentration of 200 g l⁻¹, and ethanol concentration of 1.75 ± 0.05 g l⁻¹ as well as xylitol concentration of 11.5 ± 0.4 g l⁻¹ on initial xylose concentration of 20 g l⁻¹ at 50°C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹, achieving maximum ethanol concentration of 38 ± 0.5 g l⁻¹ and xylitol concentration of 14.5 ± 0.2 g l⁻¹ in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹ by recycling the cells, achieving maximum ethanol concentration of 30.8 ± 6.2 g l⁻¹ and xylitol concentration of 7.35 ± 3.3 g l⁻¹ with ethanol productivity of 3.1 ± 0.6 g l⁻¹ h⁻¹ and xylitol productivity of 0.75 ± 0.35 g l⁻¹ h⁻¹, respectively.     

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.     

Lactic acid production from xylose by the fungus Rhizopus oryzae

Lignocellulosic biomass is considered nowadays to be an economically attractive carbohydrate feedstock for large-scale fermentation of bulk chemicals such as lactic acid. The filamentous fungus Rhizopus oryzae is able to grow in mineral medium with glucose as sole carbon source and to produce optically pure l(+)-lactic acid. Less is known about the conversion by R. oryzae of pentose sugars such as xylose, which is abundantly present in lignocellulosic hydrolysates. This paper describes the conversion of xylose in synthetic media into lactic acid by ten R. oryzae strains resulting in yields between 0.41 and 0.71 g g⁻¹. By-products were fungal biomass, xylitol, glycerol, ethanol and carbon dioxide. The growth of R. oryzae CBS 112.07 in media with initial xylose concentrations above 40 g l⁻¹ showed inhibition of substrate consumption and lactic acid production rates. In case of mixed substrates, diauxic growth was observed where consumption of glucose and xylose occurred subsequently. Sugar consumption rate and lactic acid production rate were significantly higher during glucose consumption phase compared to xylose consumption phase. Available xylose (10.3 g l⁻¹) and glucose (19.2 g l⁻¹) present in a mild-temperature alkaline treated wheat straw hydrolysate was converted subsequently by R. oryzae with rates of 2.2 g glucose l⁻¹ h⁻¹ and 0.5 g xylose l⁻¹ h⁻¹. This resulted mainly into the product lactic acid (6.8 g l⁻¹) and ethanol (5.7 g l⁻¹).     

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.     

Fermentative production of<Emphasis Type="SmallCaps"> l</Emphasis>-(+)-lactic acid by an alkaliphilic marine microorganism

Of six strains of lactic acid-producing alkaliphilic microorganisms, Halolactibacillus halophilus was most efficient. It produced the highest concentration and yield of lactic acid, with minimal amounts of acetic and formic acid when sucrose and glucose were used as substrate. Mannose and xylose were poorly utilized. In batch fermentation at 30°C, pH 9 with 4 and 8% (w/v) sucrose, lactic acid was produced at 37.7 and 65.8 g l⁻¹, with yields of 95 and 83%, respectively. Likewise, when 4 and 8% (w/v) glucose were used, 33.4 and 59.6 g lactic acid l⁻¹ were produced with 85 and 76% yields, respectively. l-(+)-lactic acid had an optical purity of 98.8% (from sucrose) and 98.3% (from glucose).     

Brewer’s spent grain as raw material for lactic acid production by<Emphasis Type="Italic"> Lactobacillus delbrueckii</Emphasis>

Chemically pre-treated brewer’s spent grain was saccharified with cellulase producing a hydrolysate with approx. 50 g glucose l⁻¹. This hydrolysate was used as a fermentation medium without any nutrient supplementation by Lactobacillus delbrueckii, which produced L-lactic acid (5.4 g l⁻¹) at 0.73 g g⁻¹ glucose consumed (73% efficiency). An inoculum of 1 g dry cells l⁻¹ gave the best yield of the process, but the pH decrease affected the microorganism capacity to consume glucose and convert it into lactic acid.     

Production of xylitol by Candida peltata

Candida peltata NRRL Y-6888 to ferment xylose to xylitol was evaluated under different fermentation conditions such as pH, temperature, aeration, substrate concentration and in the presence of glucose, arabinose, ethanol, methanol and organic acids. Maximum xylitol yield of 0.56 g g⁻¹ xylose was obtained when the yeast was cultivated at pH 6.0, 28°C and 200 rpm on 50 g L⁻¹ xylose. The yeast produced ethanol (0.41 g g⁻¹ in 40 h) from glucose (50 g L⁻¹) and arabitol (0.55 g g⁻¹ in 87 h) from arabinose (50 g L⁻¹). It preferentially utilized glucose > xylose > arabinose from mixed substrates. Glucose (10 g L⁻¹), ethanol (7.5 g L⁻¹) and acetate (5 g L⁻¹) inhibited xylitol production by 61, 84 and 68%, respectively. Arabinose (10 g L⁻¹) had no inhibitory effect on xylitol production. Received 24 December 1998/ Accepted in revised form 18 March 1999     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

H<Subscript>2</Subscript> synthesis from pentoses and biomass in <Emphasis Type="Italic">Thermotoga</Emphasis> spp.

We have investigated H₂ production on glucose, xylose, arabinose, and glycerol in Thermotoga maritima and T. neapolitana. Both species metabolised all sugars with hydrogen yields of 2.7–3.8 mol mol⁻¹ sugar. Both pentoses were at least comparable to glucose with respect to their qualities as substrates for hydrogen production, while glycerol was not metabolised by either species. Glycerol was also not metabolised by T. elfii. We also demonstrated that T. neapolitana can use wet oxidised wheat straws, in which most sugars are stored in glycoside polymers, for growth and efficient hydrogen production, while glucose, xylose and arabinose are consumed in parallel.     

Diacetyl fermentation coupled with pervaporation using oleyl alcohol supported liquid membrane

Pervaporation using oleyl alcohol supported liquid membrane was successfully applied to diacetyl fermentation by immobilized lactic acid bacteria. Diacetyl productivity was about 10 g·m⁻³·h⁻¹, while productivity during batch fermentation was about 6 g·m⁻³·h⁻¹. Diacetyl yield from consumed glucose was about 0.04 g·g⁻¹ which was 4 times as large as that of batch fermentation. The pervaporation functioned favorably on actual fermentation broth. The flux of the permeate and the diacetyl separation factor for the pervaporation were about 9 g·m⁻²·h⁻¹ and 36, respectively, and these values were maintained at almost constant levels during fermentation. Diacetyl concentration in the permeate was about 2 kg·m⁻³, which is sufficiently high for commercial use.     

Simultaneous bioconversion of glucose and xylose to ethanol by Saccharomyces cerevisiae in the presence of xylose isomerase

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of a glucose/xylose mixture was carried out by Saccharomyces cerevisiae in the presence of xylose isomerase. The SIF of 50 g l⁻¹ xylose gave an ethanol concentration and metabolic yield of 7.5 g l⁻¹ and 0.36 g (g xylose consumed)⁻¹. These parameters improved to 13.4 g l⁻¹ and 0.40 respectively, when borate was added to the medium. The SICF of a mixture of 50 g l⁻¹ glucose and 50 g l⁻¹ xylose gave an ethanol concentration and metabolic yield of 29.8 g l⁻¹ and 0.42 respectively, in the presence of borate. Temperature modulation from 30 °C to 35 °C during fermentation further enhanced the above parameters to 39 g l⁻¹ and 0.45 respectively. The approach was extended to the bioconversion of sugars present in a real lignocellulose hydrolysate (peanut-shell hydrolysate) to ethanol, with a fairly good yield. Received: 14 May 1999 / Received revision: 27 September 1999 / Accepted: 2 October 1999     

Production of cephamycin C by <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> NT4 using solid-state fermentation

Cephamycin C is an extracellular broad spectrum β-lactam antibiotic produced by Streptomyces clavuligerus, S. cattleya and Nocardia lactamdurans. In the present study, different substrates for solid-state fermentation were screened for maximum cephamycin C production by S. clavuligerus NT4. The fermentation parameters such as substrate concentration, moisture content, potassium dihydrogen phosphate, inoculum size and ammonium oxalate were optimized by response surface methodology (RSM). The optimized conditions yielded 21.68 ± 0.76 mg gds⁻¹ of cephamycin C as compared to 10.50 ± 1.04 mg gds⁻¹ before optimization. Effect of various amino acids on cephamycin C production was further studied by using RSM, which resulted in increased yield of 27.41 ± 0.65 mg gds⁻¹.     

<Emphasis Type="SmallCaps">l</Emphasis>(+)-Lactic acid production from non-food carbohydrates by thermotolerant <Emphasis Type="Italic">Bacillus coagulans</Emphasis>

Lactic acid is used as an additive in foods, pharmaceuticals, and cosmetics, and is also an industrial chemical. Optically pure lactic acid is increasingly used as a renewable bio-based product to replace petroleum-based plastics. However, current production of lactic acid depends on carbohydrate feedstocks that have alternate uses as foods. The use of non-food feedstocks by current commercial biocatalysts is limited by inefficient pathways for pentose utilization. B. coagulans strain 36D1 is a thermotolerant bacterium that can grow and efficiently ferment pentoses using the pentose-phosphate pathway and all other sugar constituents of lignocellulosic biomass at 50°C and pH 5.0, conditions that also favor simultaneous enzymatic saccharification and fermentation (SSF) of cellulose. Using this bacterial biocatalyst, high levels (150–180 g l⁻¹) of lactic acid were produced from xylose and glucose with minimal by-products in mineral salts medium. In a fed-batch SSF of crystalline cellulose with fungal enzymes and B. coagulans, lactic acid titer was 80 g l⁻¹ and the yield was close to 80%. These results demonstrate that B. coagulans can effectively ferment non-food carbohydrates from lignocellulose to l(+)-lactic acid at sufficient concentrations for commercial application. The high temperature fermentation of pentoses and hexoses to lactic acid by B. coagulans has these additional advantages: reduction in cellulase loading in SSF of cellulose with a decrease in enzyme cost in the process and a reduction in contamination of large-scale fermentations.     

Fermentation of sugar mixtures using Escherichia coli catabolite repression mutants engineered for production of L-lactic acid

Conversion of lignocellulose to lactic acid requires strains capable of fermenting sugar mixtures of glucose and xylose. Recombinant Escherichia coli strains were engineered to selectively produce L-lactic acid and then used to ferment sugar mixtures. Three of these strains were catabolite repression mutants (ptsG ⁻) that have the ability to simultaneously ferment glucose and xylose. The best results were obtained for ptsG ⁻ strain FBR19. FBR19 cultures had a yield of 0.77 (g lactic acid/g added sugar) when used to ferment a 100 g/l total equal mixture of glucose and xylose. The strain also consumed 75% of the xylose. In comparison, the ptsG ⁺ strains had yields of 0.47–0.48 g/g and consumed 18–22% of the xylose. FBR19 was subsequently used to ferment a variety of glucose (0–40 g/l) and xylose (40 g/l) mixtures. The lactic acid yields ranged from 0.74 to 1.00 g/g. Further experiments were conducted to discover the mechanism leading to the poor yields for ptsG ⁺ strains. Xylose isomerase (XI) activity, a marker for induction of xylose metabolism, was monitored for FBR19 and a ptsG ⁺ control during fermentations of a sugar mixture. Crude protein extracts prepared from FBR19 had 10–12 times the specific XI activity of comparable samples from ptsG ⁺ strains. Therefore, higher expression of xylose metabolic genes in the ptsG ⁻ strain may be responsible for superior conversion of xylose to product compared to the ptsG ⁺ fermentations. Received 14 December 2000/ Accepted in revised form 28 June 2002     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from sugarcane molasses,sugarcane juice and sugar beet juice by <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40°C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l⁻¹) and sugarcane juice (133 g total sugar l⁻¹) was 107 g l⁻¹ and 120 g l⁻¹, respectively. With 10% (w/v) sugar beet juice (105 g total sugar l⁻¹), 84 g lactic acid l⁻¹ was produced. The optical purities of d-lactic acid from the feedstocks ranged from 97.2 to 98.3%.     

Efficient production of lactic acid from sucrose and corncob hydrolysate by a newly isolated <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> GY18

The aim of this study is to investigate production of l-lactic acid from sucrose and corncob hydrolysate by the newly isolated R. oryzae GY18. R. oryzae GY18 was capable of utilizing sucrose as a sole source, producing 97.5 g l⁻¹ l-lactic acid from 120 g l⁻¹ sucrose. In addition, the strain was also efficiently able to utilize glucose and/or xylose to produce high yields of l-lactic acid. It was capable of producing up to 115 and 54.2 g l⁻¹ lactic acid with yields of up to 0.81 g g⁻¹ glucose and 0.90 g g⁻¹ xylose, respectively. Corncob hydrolysates obtained by dilute acid hydrolysis and enzymatic hydrolysis of the cellulose-enriched residue were used for lactic acid production by R. oryzae GY18. A yield of 355 g lactic acid per kg corncobs was obtained after 72 h incubation. Therefore, sucrose and corncobs could serve as potential sources of raw materials for efficient production of lactic acid by R. oryzae GY18.     

Butanol production from wheat straw hydrolysate using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In these studies, butanol (acetone butanol ethanol or ABE) was produced from wheat straw hydrolysate (WSH) in batch cultures using Clostridium beijerinckii P260. In control fermentation 48.9 g L⁻¹ glucose (initial sugar 62.0 g L⁻¹) was used to produce 20.1 g L⁻¹ ABE with a productivity and yield of 0.28 g L⁻¹ h⁻¹ and 0.41, respectively. In a similar experiment where WSH (60.2 g L⁻¹ total sugars obtained from hydrolysis of 86 g L⁻¹ wheat straw) was used, the culture produced 25.0 g L⁻¹ ABE with a productivity and yield of 0.60 g L⁻¹ h⁻¹ and 0.42, respectively. These results are superior to the control experiment and productivity was improved by 214%. When WSH was supplemented with 35 g L⁻¹ glucose, a reactor productivity was improved to 0.63 g L⁻¹ h⁻¹ with a yield of 0.42. In this case, ABE concentration in the broth was 28.2 g L⁻¹. When WSH was supplemented with 60 g L⁻¹ glucose, the resultant medium containing 128.3 g L⁻¹ sugars was successfully fermented (due to product removal) to produce 47.6 g L⁻¹ ABE, and the culture utilized all the sugars (glucose, xylose, arabinose, galactose, and mannose). These results demonstrate that C. beijerinckii P260 has excellent capacity to convert biomass derived sugars to solvents and can produce over 28 g L⁻¹ (in one case 41.7 g L⁻¹ from glucose) ABE from WSH. Medium containing 250 g L⁻¹ glucose resulted in no growth and no ABE production. Mixtures containing WSH + 140 g L⁻¹ glucose (total sugar approximately 200 g L⁻¹) showed poor growth and poor ABE production. Mention of trade names or commercial products in this article is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

Cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025

The potential of cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025 was evaluated in this work. This strain was preliminarily cultivated in a synthetic medium containing glucose and xylose and was able to produce ethanol and xylitol at pH 4.5. Next, cashew apple bagasse hydrolysate (CABH) was prepared by a diluted sulfuric acid pretreatment and used as fermentation media. This hydrolysate is rich in glucose, xylose, and arabinose and contains traces of formic acid and acetic acid. In batch fermentations of CABH at pH 4.5, the strain produced only ethanol. The effects of temperature on the kinetic parameters of ethanol fermentation by K. marxianus CE025 using CABH were also evaluated. Maximum specific growth rate (μ_max), overall yields of ethanol based on glucose consumption Y_{P \mathord}