Applied Biochemistry and Biotechnology (v.131, #1-3)

The development of cement and concrete additive by Byon-Wa Chun; Benita Dair; Patrick J. Macuch; Debbie Wiebe; Charloote Porteneuve; Ara Jeknavorian (645-658).
The present work attempted to utilize xylose by converting it to an aldonic acid. In the present study, xylose was converted to xyloni acid by using commercial glucose oxidase enzyme, palladium catalysis, and microbial bioconversion. The enzyme conversion was successfully done using a commercial glucose oxidase. The microbial conversion with Gluconobactor oxydans proceeded even with the presence of a large amount of lignosulfonate. Thus obtained xylonic acid products were evaluated as a cement dispersing agent in cement and concrete tests. It was found that xylonic acid is approximately twice as effective as lignosulfonate. Xylonic acid can be effectively utilized in concrete water reducer application.
Keywords: Xylose utilization; biochemical oxidation; xylonic acid; cement; concrete

Production of Bacillus sphaericus entomopathogenic biomass using brewery residues by Cristiane Darco Cruz Martins; Paula Fernandes De Aguiar; Eliana Flavia Camposese Sérvulo (659-667).
The use of brewery residues—yeast and trub—has been evaluated aiming to minimize the costs of the industrial production of Bacillus sphaericus-based bioinsecticide. Both brewery residues promoted growth and sporulation of the three B. sphaericus strains that were isolated from Brazilian soils (S1, S2, and S20). However, distinct growth and sporulation behaviors were observed in relation to the different nutritional conditions and strain used. The maximum sporulation percentage was obtained through the cultivation of S20 strain in brewery residual yeast. In general, the entomopathogenic biomasses produced showed good results for toxicity to Culex larvae. The minimum values of larvae population (LC50) were observed for the S20 strain grown on yeast brewery residue-containing media. After fermentation, a considerable decrease in the organic material of alternative media was verified, although the residual values were still higher than that considered appropriate for effluent discharge.
Keywords: Bacillus sphaericus ; bioinsecticide production; industrial residues; entomopathogenic biomass; spore/crystal toxins

Biodiesel fuel is an alternative and renewable energy source, which may help to reduce air pollution, as well as our dependence on petroleum for energy. Several processes have already been developed for the production of biodiesel. Alkali-catalyzed transesterification with short-chain alcohols, for example, generates high yields of methyl esters in short reaction times. In this study, we have evaluated the efficacy of batch (one- and two-stage) transesterification of rapeseed oil in the production of rapeseed methyl ester. The conversion of rapeseed oil exhibited similar reaction patterns and yields in 30- and 1-L reaction systems. Approximately 98% of the rapeseed oil was converted at 400 rpm within 20 min, under the following conditions: 1% (w/w) KOH, 1∶10 methanol molar ratio, and at 60°C. In the 30-L, two-stage transesterification process, approx 98.5% of the rapeseed oil was converted at a 1∶4.5 molar ratio and 1% (w/w) KOH at 60°C for 30 min (first reaction condition), and at a 1∶1 molar ratio and 0.2% (w/w) KOH at 60°C for 30 min (second reaction condition).
Keywords: Biodiesel fuel; transesterification; rapeseed oil; two stage

Optimization of distilled monoglycerides production by Leonardo Vasconcelos Fregolente; César Benedito Batistella; Rubens Maciel Filho; Maria Regina Wolf Maciel (680-693).
Monoglycerides (MG) are emulsifiers widely used in food and pharmaceutical industries. Current industrial processes for MG production consist of the interesterification of triglycerides with glycerol (GL), in the presence of inorganic catalysts at high temperatures (>200°C). This reaction is known as glycerolysis and produces a mixture of approx 50% of MG. This level of concentration is suitable for many applications, although, for some specific uses like margarine, shortening, icing, and cream filling, require distilled MGs, which are purified MG (min. 90%) obtained by the molecular distillation process. Therefore, in this work, a 23 factorial design was employed to evaluate the effects of reaction parameters in the MG content after the interesterification reaction of refined soybean oil with GL in the presence of sodium hydroxide as catalyst. After that, the MG content in the reaction product was enhanced through the molecular distillation process in order to obtain distilled MG.
Keywords: Glycerolysis; soybean oil; distilled monoglycerides; molecular distillation; short path distillation; factorial design

Production of lactic acid from cheese whey by batch and repeated batch cultures of Lactobacillus sp. RKY2 by Hyang-Ok Kim; Young-Jung Wee; Jin-Nam Kim; Jong-Sun Yun; Hwa-Won Ryu (694-704).
The fermentative production of lactic acid from cheese whey and corn steep liquor (CSL) as cheap raw materials was investigated by using Lactobacillus sp. RKY2 in order to develop a cost-effective fermentation medium. Lactic acid yields based on consumed lactose were obtained at more than 0.98 g/g from the medium containing whey lactose. Lactic acid productivities and yields obtained from whey lactose medium were slightly higher than those obtained from pure lactose medium. The lactic acid productivity gradually decreased with increase in substrate concentration owing to substrate and product inhibitions. The fermentation efficiencies were improved by the addition of more CSL to the medium. Moreover, through the cell-recycle repeated batch fermentation, lactic acid productivity was maximized to 6.34 g/L/h, which was 6.2 times higher than that of the batch fermentation.
Keywords: Corn steep liquor; lactic acid; Lactobacillus ; lactose; whey

Production of bacterial cellulose by Gluconacetobacter sp. RKY5 isolated from persimmon vinegar by Soo-Yeon Kim; Jin-Nam Kim; Young-Jung Wee; Don-Hee Park; Hwa-Won Ryu (705-715).
The optimum fermentation medium for the production of bacterial cellulose (BC) by a newly isolated Gluconacetobacter sp. RKY5 was investigated. The optimized medium composition for cellulose production was determined to be 15 g/L glycerol, 8 g/L yeast extract, 3 g/L K2HPO4, and 3 g/L acetic acid. Under these optimized culture medium, Gluconacetobacter sp. RKY5 produced 5.63 g/L of BC after 144 h of shaken culture, although 4.59 g/L of BC was produced after 144 h of static culture. The amount of BC produced by Gluconacetobacter sp. RKY5 was more than 2 times in the optimized medium found in this study than in a standard Hestrin and Shramm medium, which was generally used for the cultivation of BC-producing organisms.
Keywords: Bacterial cellulose; fermentation; Gluconacetobacter ; optimization; persimmon vinegar

Natural compounds obtained through centrifugal molecular distillation by Vanessa Mayumi Ito; Patricia Fazzio Martins; César Benedito Batistella; Rubens Maciel Filho; Maria Regina Wolf Maciel (716-726).
Soybean oil deodorized distillate (SODD) is a byproduct from refining edible soybean oil; however, the deodorization process removes unsaponifiable materials, such as sterols and tocopherols. Tocopherols are highly added value materials. Molecular distillation has large potential to be used in order to concentrate tocopherols, because it uses very low levels of temperatures because of the high vacuum and short operating time for separation and, also, it does not use solvents. However, nowadays, the conventional way to recover tocopherols is carrying out chemical reactions prior to molecular distillation, making the process not so suitable to deal with natural products. The purpose of this work is to use only molecular distillation in order to recover tocopherols from SODD. Experiments were performed in the range of 140–220°C. The feed flow rate varied from 5 to 15 g/min. The objective of this study was to remove the maximum amount of free fatty acids (FFA) and, so, to increase the tocopherol concentration without add any extra component to the system. The percentage of FFA in the distillate stream of the molecular still is large at low feed flow rates and low evaporator temperatures, avoiding thermal decomposition effects.
Keywords: Centrifugal distillation; molecular distillation; natural products; tocopherol

Biosurfactants production by Pseudomonas aeruginosa FR using palm oil by Fernando J. S. Oliveira; Leonardo Vazquez; Norberto P. De Campos; Francisca P. de França (727-737).
Biosurfactants production by a strain of Pseudomonas aeruginosa using palm oil as a sole carbon source was investigated. The experiments were carried out in 500-mL conical flasks containing 100 mL of mineral media supplemented with palm oil as the sole carbon source. The P. aeruginosa FR strain was able to reduce surface tension of three tested inorganic media. Rotation velocities from 100 to 150 rpm provided free-cell fermented media with the lowest surface tension of approx 33 mN/m. Emulsification index results of even 100% were achieved when diesel was used as oil phase. Eight surface-active compounds produced by the bacterium were identified by mass spectrometry.
Keywords: Biosurfactants production; rhamnolipids; Pseudomonas aeruginosa ; palm oil; Ellaus guineensis fruit; mass spectra

Novel approach of corn fiber utilization by G. Kálmán; K. Recseg; M. Gáspár; K. Réczey (738-750).
The corn wet milling process produces a 10% (w/w of the processed corn) byproduct called corn fiber, which is utilized worldwide as a low-value feedstock for cattle. The aim of this study was to find a higher value use of corn fiber. The main fractions of corn fiber are: 20% starch, 40% hemicellulose, 14% cellulose, and 14% protein. Extraction of the highly valuable, cholesterol-lowering corn fiber oil is not feasible owing to its low (2% w/w) concentration in the fiber. The developed technology is based on simple and inexpensive procedures, like washing with hot water, dilute acid hydrolysis at 120°C, enzymatic hydrolysis of cellulose, screening, drying, and extraction. The main fractions are sharply separated in the order of starch, hemicellulose, cellulose, lipoprotein, and lignin). The lipoprotein fraction adds up to 10% of the original dry corn fiber, and contains 45% corn fiber oil, thus yielding more oil than direct extraction of the fiber. It is concluded that the defined method makes the extraction of the corn fiber oil economically feasible. The fractionation process also significantly increases the yield of cholesterol-lowering substances (sterols and sterolesters). At the same time clear and utilizable fractions of monosaccharides, protein, and lignin are produced.
Keywords: Bioethanol; corn fiber hydrolysis; corn fiber oil; phytosterol

The production of nisin, a natural food preservative, by Lactococcus lactis subsp. lactis (ATCC 11454) is associated with the simultaneous formation of lactic acid during fermentation in a whey-based medium. As a result of the low concentration and high separation cost of lactic acid, recovering lactic acid as a product may not be economical, but its removal from the fermentation broth is important because the accumulation of lactic acid inhibits nisin biosynthesis. In this study, lactic acid removal was accomplished by biological means. A mixed culture of L. lactis and Saccharomyces cerevisiae was established in order to stimulate the production of nisin via the in situ consumption of lactic acid by the yeast strain, which is capable of utilizing lactic acid as carbon source. The S. cerevisiae in the mixed culture did not compete with the nisin-producing bacteria because the yeast does not utilize lactose, the major carbohydrate in whey for bacterial growth and nisin production. The results showed that lactic acid produced by the bacteria was almost totally utilized by the yeast and the pH of the mixed culture could be maintained at around 6.0. Nisin production by the mixed culture system reached 150.3 mg/L, which was 0.85 times higher than that by a pure culture of L. lactis.
Keywords: Nisin; whey; mixed culture; fermentation

Biochar as a precursor of activated carbon by R. Azargohar; A. K. Dalai (762-773).
Biochar was evaluated as a precursor of activated carbon. This product was produced by chemical activation using potassium hydroxide. The effects of operating conditions of activation process, such as temperature, activating agent to biochar mass ratio, and nitrogen flow rate, on the textural and chemical properties of the product were investigated. Activated carbon produced by this method has internal surface area at least 50 times than that of the precursor and is highly microporous, which is also confirmed by scanning electron microscopy analysis. Fourier-transform infrared spectroscopy analysis showed development of aromatization in the structure of activated carbon. X-ray diffraction data indicated the formation of small, two-dimensional graphite-like structure at high temperatures. Thermogravimetric study showed that when potassium hydroxide to biochar mass ratio was more than one, the weight loss decreased.
Keywords: Biochar; activated carbon; chemical activation; potassium hydroxide

Moisture sorption, transport, and hydrolytic degradation in polylactide by Richard A. Cairncross; Jeffrey G. Becker; Shri Ramaswamy; Ryan O'Connor (774-785).
Management of moisture penetration and hydrolytic degradation of polylactide (PLA) is extremely important during the manufacturing, shipping, storage, and end-use of PLA products. Moisture transport, crystallization, and degradation, in PLA have been measured through a variety of experimental techniques including size-exclusion chromatography, differential scanning calorimetry, and X-ray diffraction. Quartz crystal microbalance and dynamic vapor sorption experiments have also been used to measure moisture sorption isotherms in PLA films with varying crystallinity. A surprising result is that, within the accuracy of the experiments, crystalline and amorphous PLA films exhibit identical sorption isotherms.
Keywords: PLA; biodegradable polymers; bio-based polymers; polylactic acid; diffusion; hydrolysis

The conversion of glucose and fructose into gluconic acid (GA) and sorbitol (SOR) was conducted in a batch reactor with free (CTAB-treated or not) or immobilized cells of Zymomonas mobilis. High yields (more than 90%) of gluconic acid and sorbitol were attained at initial substrate concentration of 600 g/L (glucose plus fructose at 1:1 ratio), using cells with glucose-fructose-oxidoreductase activity of 75 U/L. The concentration of the products varied hyperbolically with time according to the equations (GA)=t(GA)max/(WGA +t), (SOR)=t (SOR)max/(WSor+t), vGA=[WGA (GA)max]/(WGA+t)2 and VSOR=[WSOR (SOR)max]/(WSOR+t)2. Taking the test carried out with free CTAB-treated cells as an example, the constant parameters were (GA)max= 541 g/L, (SOR)max=552 g/L, WGA=4.8h, WSOR=4.9h, υGA=112.7 g/L· and υSOR=112.7 g/L·.
Keywords: Zymomonas mobilis ; sorbitol; gluconic acid

Metabolic engineering of Saccharomyces cerevisiae for efficient production of pure l−(+)−lactic acid by Nobuhiro Ishida; Satoshi Saitoh; Toru Ohnishi; Kenro Tokuhiro; Eiji Nagamori; Katsuhiko Kitamoto; Haruo Takahashi (795-807).
We developed a metabolically engineered Saccharomyces cerevisiae, which produces optically pure l-lactic acid efficiently using cane juice-based medium. In this recombinant, the coding region of pyruvate decarboxylase (PDC)1 was completely deleted, and six copies of the bovine l-lactate dehydrogenase (l-LDH) genes were introduced on the genome under the control of the PDC1 promoter. To confirm optically pure lactate production in lowcost medium, cane juice-based medium was used in fermentation with neutralizing conditions. l-lactate production reached 122 g/L, with 61% of sugar being transformed into l-lactate finally. The optical purity of this l-lactate, that affects the physical characteristics of poly-l-lactic acid, was extremely high, 99.9% or over.
Keywords: Cane juice-based medium; l-lactic acid production; optical purity; Saccharomyces cerevisiae

A unicellular marine green alga, Chlamydomonas perigranulata, was demonstrated to synthesize starch through photosynthesis, store it in a cell, and ferment it under anaerobic conditions in the dark to produce ethanol, 2,3-butanediol (butanediol), acetic acid, and carbon dioxide (CO2). Previous fermentation data of an algal biomass cultivated outdoors in a 50-L tubular photo-bioreactor showed good carbon (C) recovery in the fermentation balance, with a higher ratio to alcohols and, therefore, lower ratio to CO2 in the C distribution of products than what would be expected from the embden-Myerhof-Parnas pathway. These findings led to a proposed concept for a CO2-ethanol conversion system (CDECS). The above data were evaluated in terms of hydrogen (H) recovery with the following results: C recovery at 105% was well balanced, although H recovery was as high as 139%, meaning an additional gain of H through fermentation. This finding was reproduced wholly in a set of experiments carried out in the same month of the following year, October, whereas another set of experiments was carried out in the following June provided ordinary fermentation results in terms of C and H recoveries with poor growth. Further analyses of these data revealed that butanediol is equal to ethanol as a product from a putative conversion system from CO2 to the detected fermentation products, leading to the revision of the CDECS concept to a CO2-alcohol conversion system (CDACS). The relevance of the CDACS will be discussed in relation to the cultivation conditions employed by chance.
Keywords: Marine green microalga; Chlamydomonas perigranulata ; endogenous starch fermentation; fuel alcohol; hydrogen recovery

Detailed analysis of modifications in lignin after treatment with cultures screened for lignin depolymerizing agents by Aarti Gidh; Dinesh Talreja; Todd B. Vinzant; Todd Clint Williford; Alfred Mikell (829-843).
Termites, beetles, and other arthropods can digest living and decaying wood plus other lignocellulosic plant litter. Microbial sources like other wood-eating insect guts and wastewater treatment sludge were screened for lignin depolymerization. Near infrared spectroscopy and atomic force microscopy (AFM) along with high-performance liquid chromatography (HPLC), were used to track changes in functional groups, size, shape, and molecular weight of lignin molecules during incubations. Odontotaenius disjunctus (Betsy beetle) guts dissected whole or separately as midgut, foregut, and hindgut, consumed corn stover but did not show lignin depolymerization. The sludge-treated lignin did show some reduction in molecular weight on the HPLC, particle size (350–650 nm initially to 135–220 nm by day 30) and particles per field on AFM. pH and the presence of nutrients had a substantial effect on the extent of depolymerization. Cultures in lignin and nutrients showed higher growth than cultures with lignin only. Colony characteristics within the beetle gut and the sludge were also evaluated.
Keywords: Lignin; beetles; NIR; HPLC; AFM; depolymerization

Lactic acid is used as a food additive for flavor and preservation and a precursor in the development of poly-lactic acid, a product used to make biodegradable plastics and textiles. Rhizopus oryzae NRRL 395 is known to be a strain that produces optically pure l-(+)-lactic acid. The morphology of Rhizopus cultures is complex, forming filamentous, clumps, and pellet mycelia. Different morphology growth has significant effects on lactic acid production. In bioreactors, the filamentous or clump mycelia increase the viscosity of the medium, wrap around impellers, and block the nutrient transportation, leading to a decrease in production efficiency and bioreactor performance. Growing fungi in pellet form can significantly improve these problems. In this study, factors that affect lactic acid production in pelletized flask cultures using R. oryzae NRRL 395 were investigated in detail. Completely randomized designs were used to determine the influence of culture temperature, time, concentration of glucose, and inoculum size. Lactic acid fermentation using clump and pellet morphologies were performed in a 5 L fermentor at the optimal values obtained from flask culture. Finally, fed-batch culture was used to enhance the lactate concentration in broth. The final lactate concentration of fed-batch culture reached 92 g/L. The data presented in the article can provide useful information on optimizing lactic acid production using alternative source materials.
Keywords: Rhizopus oryzae ; lactic acid; pellet morphology

Gram-positive bacteria have been explored to convert lignocellulosic biomass to biofuel and bioproducts. Our long-term goal is to create genetically engineered lactic acid bacteria (LAB) strains that convert agricultural biomass into ethanol and other value-added products. The immediate approaches toward this goal involve genetic manipulations by either introducing ethanol production pathway genes or inactivating pathways genes that lead to production of undesired byproducts. The widely studied species Lactobacillus plantarum is now considered a model for genetic manipulations of LAB. In this study, L. plantarum TF103 strain, in which two of the chromosomal L-ldh and D-ldh genes are inactivated, was used to introduce additional mutations on the chromosome to eliminate undesired fermentation products. We targeted the acetolactate synthase gene (als) that converts pyruvate to acetolactate, to eliminate the production of acetoin and 2,3-butanodial. A pBluescript derivative containing sections of the als coding region and an erythromycin resistance gene was directly introduced into L. plantarum TF103 cells to create mutations under selection pressure. The resulting erythromycin resistant (Emr) TF103 strain appears to have chromosomal mutations of both the als and the adjacent lysP genes as revealed by polymerase chain reaction and Southern blot analyses. Mutations were thus generated via targeted homologous recombination using a Gram-negative cloning vector, eliminating the use of a shuttle vector. This method should facilitate research in targeted inactivation of other genes in LAB.
Keywords: Lactic acid bacteria; als and lysP mutant; ethanol; acetoin; 2,3-butanodial; acetolactate synthase

Production of insoluble exopolysaccharide of Agrobacterium sp. (ATCC 31749 and IFO 13140) by Márcia Portilho; Graciette Matioli; Gisella Maria Zanin; Flávio Faria de Moraes; Adilma Regina Pippa Scamparini (864-869).
Agrobacterium isolated from soil samples produced two extracellular polysaccharides: succinoglycan, an acidic soluble polymer, and curdlan gum, a neutral, insoluble polymer. Maize glucose, cassava glucose, and maize maltose were used in fermentation medium to produce insoluble polysaccharide. Two Agrobacterium sp. strains which were used (ATCC 31749 and IFO 13140) in the production of insoluble exopolysaccharide presented equal or superior yields compared to the literature. The strain ATCC 31749 yielded better production when using maize maltose, whose yield was 85%, whereas strain IFO 13140 produced more when fed maize glucose, producing a yield of 50% (on reducing sugars).
Keywords: Microbial exopolysaccharides; Agrobacterium sp; microbial gums; curdlan; fermentation

Selective utilization of fructose to glucose by Candida magnoliae, an erythritol producer by Ji-Hee Yu; Dae-Hee Lee; Yong-Joo Oh; Ki-Cheol Han; Yeon-Woo Ryu; Jin-Ho Seo (870-879).
Candida magnoliae isolated from honeycomb is an industrially important yeast with high erythritol-producing ability. Erythritol has been used as functional sugar substitute for various foods. In order to analyze the physiological properties of C. magnoliae, a study on sugar utilization pattern was carried out. The fermentation kinetics of glucose and fructose revealed that C. magnoliae has the discrepancy in glucose and fructose utilization when it produces erythritol. In contrast to most yeasts, C. magnoliae showed preference for fructose to glucose as a carbon source, deserving the designation of fructophilic yeast. Such a peculiar pattern of sugar utilization in C. magnoliae seems to be related to the evolutionary environment.
Keywords: Candida magnoliae ; fructophilic yeast; fructose utilization; erythritol

Biosurfactant production by Rhodococcus erythropolis grown on glycerol as sole carbon source by Elisa M. P. Ciapina; Walber C. Melo; Lidia M. M. Santa Anna; Alexandre S. Santos; Denise M. G. Freire; Nei Pereira Jr. (880-886).
The production of biosurfactant by Rhodococcus erythropolis during the growth on glycerol was investigated. The process was carried out at 28°C in a 1.5-L bioreactor using glycerol as carbon source. The bioprocess was monitored through measurements of biosurfactant concentration and glycerol consumption. After 51 h of cultivation, 1.7 g/L of biosurfactant, surface, and interfacial tensions values (with n-hexadecane) of 43 and 15 mN/m, respectively, 67% of Emulsifying Index (E 24), and 94% of oil removal were obtained. The use of glycerol rather than what happens with hydrophobic carbon source allowed the release of the biosurfactant, originally associated to the cell wall.
Keywords: Biosurfactant; Rhodococcus ; oil removal

Methane production in a 100-L upflow bioreactor by anaerobic digestion of farm waste by Abhijeet P. Borole; K. Thomas Klasson; Whitney Ridenour; Justin Holland; Khursheed Karim; Muthanna H. Al-Dahhan (887-896).
Manure waste from dairy farms has been used for methane production for decades, however, problems such as digester failure are routine. The problem has been investigated in small scale (1–2 L) digesters in the laboratory; however, very little scale-up to intermediate scales are available. We report production of methane in a 100-L digester and the results of an investigation into the effect of partial mixing induced by gas upflow/recirculation in the digester. The digester was operated for a period of about 70 d (with 16-d hydraulic retention time) with and without the mixing induced by gas recirculation through an internal draft tube. The results show a clear effect of mixing on digester operation. Without any mixing, the digester performance deteriorated within 30–50 d, whereas with mixing continuous production of methane was observed. This study demonstrates the importance of mixing and its critical role in design of large scale anaerobic digesters.
Keywords: Anaerobic digestion; animal manure; gas recirculation; mixing; biogas

Biomodification of coal to remove mercury by K. Thomas Klasson; Abhijeet P. Borole; Catherine K. McKeown; Choo Y. Hamilton (897-908).
A biological process for removal of mercury from coal is under investigation. Iron and sulfur oxidizing bacteria have previously been used for desulfurization of coal and for mineral mining. We have shown that removal of mercury from coal is also possible via the same principles. Two pure cultures, Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans and four environmental consortium samples obtained from an acid mine drainage site were studied for mercury removal from coal. Four different coal samples were included in the study and the preliminary results have shown that up to 20% of the mercury can be removed in batch cultures compared to control. Additional parameters such as media composition and inoculum size were also studied. This is the first report demonstrating successful leaching of mercury from coal using biological treatment.
Keywords: Bioleaching; mercury; coal; ferrooxidans