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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.132, #1-3)

Introduction to session 6 by Michael R. Ladisch (pp. 909-910).

Enzymatic conversion of waste cooking oils into alternative fuel—Biodiesel by Guanyi Chen; Ming Ying; Weizhung Li (pp. 911-921).
Production of biodiesel from pure oils through chemical conversion may not be applicable to waste oils/fats. Therefore, enzymatic conversion using immobilized lipase based on Rhizopus orzyae is considered in this article. This article studies this technological process, focusing on optimization of several process parameters, including the molar ratio of methanol to waste oils, biocatalyst load, and adding method, reaction temperature, and water content. The results indicate that methanol/oils ratio of 4, immobilized lipase/oils of 30 wt% and 40°C are suitable for waste oils under 1 atm. The irreversible inactivation of the lipase is presumed, and a stepwise addition of methanol to reduce inactivation of immobilized lipases is proposed. Under the optimum conditions the yield of methyl esters is around 88–90%.

Keywords: Biocatalyst; biodiesel; immobilized R. oryzae lipase; transesterification; waste oils


Inulin-containing biomass for ethanol production by Ma José Negro; Ignacio Ballesteros; Paloma Manzanares; José Miguel Oliva; Felicia Sáez; Mercedes Ballesteros (pp. 922-932).
The use of stalks instead of tubers as a source of carbohydrates for ethanol production has been investigated. The inulin present in the stalks of Jerusalem artichoke was extracted with water and the effect of solid-liquid ratio, temperature, and acid addition was studied and optimized in order to attain a high-fructose fermentable extract. The maximum extraction efficiency (corresponding to 35 g/L) of soluble sugars was obtained at 1/6 solidliquid ratio.Fermentations of hydrolyzed extracts by baker's yeast and direct fermentation by an inulinease activity yeast were also performed and the potential to use this feedstock for bioethanol production assessed. The results show that the carbohydrates derived from Jerusalem artichoke stalks can be converted efficiently to ethanol by acidic hydrolysis followed by fermentation with Saccharomyces cerevisiae or by direct fermentation of inulin using Kluyveromyces marxianus strains. In this last case about 30 h to complete fermentation was required in comparison with 8–9 h obtained in experiments with S. cerevisiae growth on acid extracted juices.

Keywords: Ethanol; fermentation; Jerusalem artichoke; sugar extraction


Production of medium-chain-length polyhydroxyalkanoates by pseudomonas aeruginosa with fatty acids and alternative carbon sources by Pui-Ling Chan; Vincent Yu; Lam Wai; Hoi-Fu Yu (pp. 933-941).
In this study, medium-chain-length polyhydroxyalkanoates (mcl-PHAs) were produced by Pseudomonas aeruginosa using different carbon sources. Decanoic acid induced the highest (9.71% [±0.7]) mcl-PHAs accumulation in bacterial cells at 47 h. The cells preferred to accumulate and degrade the poly-hydroxyoctanoate than polyhydroxydecanoate (PHD) during early stage and final stage, of the growth, respectively. The production cost of mcl-PHAs can be reduced by using edible oils as the carbon source. The bacteria accumulated 6% (±0.7) of mcl-PHAs in the presence of olive oil. Besides, reused oil was another potential carbon source for the reduction of the production cost of mcl-PHAs. Overall, PHD was the major constitutent in the accumulated mcl-PHAs.

Keywords: Edible oils; fatty acids; medium-chain-length polyhydro-xyalkanoates; Pseudomonas aeruginosa


Production and rheological characterization of biopolymer of Sphingomonas capsulata ATCC 14666 using conventional and industrial media by Ana Luiza Da Silva Berwanger; Natalia Molossi Domingues; Larissa Tonial Vanzo; Marco Di Luccio; Helen Treichel; Francine Ferreira Padilha; Adilma Regina Pippa Scamparini (pp. 942-950).
This work was aimed at the production and rheological characterization of biopolymer by Sphingomonas capsulata ATCC 14666, using conventional and industrial media. The productivity reached the maximum of 0.038 g/L·h, at 208 rpm and 4% (w/v) of sucrose. For this condition, different concentrations of industrial medium were tested (2.66, 4, 6, and 8%). The best productivity was obtained using pretreated molasses 8% (w/v) (0.296 g/L·h), residue of textured soybean protein 6% (wt/v) (0.244 g/L·h) and crude molasses 8% (w/v) (0.192 g/L·h), respectively. Apparent viscosity presented similar results when compared with those in the literature for other biopolymers.

Keywords: Agroindustry waste; biopolymers; experimental design; rheology; Sphingomonas capsulata


Inulinase production by Kluyveromyces marxianus NRRL Y-7571 using solid state fermentation by João Paulo Bender; Marcio Antônio Mazutti; Débora de Oliveira; Marco Di Luccio; Helen Treichel (pp. 951-958).
Inulinase is an enzyme relevant to fructose production by enzymatic hydrolysis of inulin. This enzyme is also applied in the production of fructo-oligosaccharides that may be used as a new food functional ingredient. Commercial inulinase is currently obtained using inulin as substrate, which is a relatively expensive raw material. In Brazil, the production of this enzyme using residues of sugarcane and corn industry (sugarcane bagasse, molasses, and corn steep liquor) is economically attractive, owing to the high amount and low cost of such residues. In this context, the aim of this work was the assessment of inulinase production by solid state fermentation using by Kluyveromyces marxianus NRRL Y-7571. The solid medium consisted of sugar cane bagasse supplemented with molasses and corn steep liquor. The production of inulinase was carried out using experimental design technique. The effect of temperature, moisture, and supplements content were investigated. The enzymatic activity reached a maximum of 445 units of inulinase per gram of dry substrate.

Keywords: Inulinase; solid state; Kluyveromyces marxianus


Macroscopic mass and energy balance of a pilot plant anaerobic bioreactor operated under thermophilic conditions by Teodoro Espinosa-Solares; John Bombardiere; Mark Chatfield; Max Domaschko; Michael Easter; David A. Stafford; Saul Castillo-Angeles; Nehemias Castellanos-Hernandez (pp. 959-968).
Intensive poultry production generates over 100,000 t of litter annually in West Virginia and 9×106 t nationwide. Current available technological alternatives based on thermophilic anaerobic digestion for residuals treatment are diverse. A modification of the typical continuous stirred tank reactor is a promising process being relatively stable and owing to its capability to manage considerable amounts of residuals at low operational cost. A 40-m3 pilot plant digester was used for performance evaluation considering energy input and methane production. Results suggest some changes to the pilot plant configuration are necessary to reduce power consumption although maximizing biodigester performance.

Keywords: Biogas; livestock residual; methane; thermophilic anaerobic digestion


Ethyl alcohol production optimization by coupling genetic algorithm and multilayer perceptron neural network by Elmer Ccopa Rivera; Aline C. da Costa; Maria Regina Wolf Maciel; Rubens Maciel Filho (pp. 969-984).
In this present article, genetic algorithms and multilayer perceptron neural network (MLPNN) have been integrated in order to reduce the complexity of an optimization problem. A data-driven identification method based on MLPNN and optimal design of experiments is described in detail. The nonlinear model of an extractive ethanol process, represented by a MLPNN, is optimized using real-coded and binary-coded genetic algorithms to determine the optimal operational conditions. In order to check the validity of the computational modeling, the results were compared with the optimization of a deterministic model, whose kinetic parameters were experimentally determined as functions of the temperature.

Keywords: Alcoholic fermentation process; artificial intelligence; design of experiments; modeling; penalty function


Lactic acid recovery from cheese whey fermentation broth using combined ultrafiltration and nanofiltration membranes by Yebo Li; Abolghasem Shahbazi (pp. 985-996).
The separation of lactic acid from lactose in the ultrafiltration permeate of cheese whey broth was studied using a cross-flow nanofiltration membrane unit. Experiments to test lactic acid recovery were conducted at three levels of pressure (1.4, 2.1, and 2.8 MPa), two levels of initial lactic acid concentration (18.6 and 27 g/L), and two types of nanofiltration membranes (DS-5DK and DS-5HL). Higher pressure caused significantly higher permeate flux and higher lactose and lactic acid retention (p<0.0001). Higher initial lactic acid concentrations also caused significantly higher permeate flux, but significantly lower lactose and lactic acid retention (p<0.0001). The two tested membranes demonstrated significant differences on the permeate flux and lactose and lactic acid retention. Membrane DS-5DK was found to retain 100% of lactose at an initial lactic acid concentration of 18.6 g/L for all the tested pressures, and had a retention level of 99.5% of lactose at initial lactic acid concentration of 27 g/L when the pressure reached 2.8 MPa. For all the test when lactose retention reached 99–100%, as much as 64% of the lactic acid could be recovered in the permeate.

Keywords: Cheese whey; fermentation; lactic acid; membrane; lactose; nanofiltration


Fermentation of rice straw/chicken manure to carboxylic acids using a mixed culture of marine mesophilic micoorganisms by Frank K. Agbogbo; Mark T. Holtzapple (pp. 997-1014).
Countercurrent fermentation of rice straw and chicken manure to carboxylic acids was performed using a mixed culture of marine mesophilic microorganisms. To increase the digestibility of the biomass, rice straw, and chicken manure were pretreated with 0.1 g Ca(OH)2/g biomass. Fermentation was performed for 80% rice straw and 20% chicken manure at various volatile solid loading rates (VSLR) and liquid residence times (LRT). The highest acid productivity of 1.69 g/(L·d) occurred at a total acid concentration of 32.4 g/L. The highest conversion (0.69 g VS digested/g VS fed) and yield (0.29 g total acids/g VS fed) were at a total acid concentration of 25 g/L. A Continuum Particle Distribution Model of the process predicted the experimental total acid concentration and conversion results with an average error of 6.41% and 6.15%, respectively. Results show how total acid concentrations, conversions, and yields vary with VSLR and LRT in the MixAlco process.

Keywords: Biomass; carboxylic acids; CPDM; digestion; fuels; mixed culture


Construction of recombinant Bacillus subtilis for production of polyhydroxyalkanoates by Yujie Wang; Lifang Ruan; Wai-Hung Lo; Hong Chua; Hoi-Fu Yu (pp. 1015-1022).
Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we constructed two recombinant plasmids, pBE2C1, and pBE2C1AB, containing one or two PHA synthse, genes, respectively. The two plasmids were inserted into Bacillus subtilis DB104 to generate modified strains, B. subtilis/pBE2C1 and B. subtilis/pBE2C1AB. The two recombinants strains were subjected to fermentation and showed PHA accumulation, the first reported example of mcl-PHA production in B. subtilis. Gas Chromatography analysis identified the compound produced by B. subtilis/pBE2C1 to be a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer whereas that produced by B. subtilis/pBE2C1AB was a hydroxybutyrate-co-hydroxyde-canoate-co-hydroxydodecanoate (HB-HD-HDD) polymer.

Keywords: Bacillus subtilis ; cloning and expression; P (HB-co-mclHA); PHA synthase gene; polyhydroxyalkanoates (PHAs)


Microorganism screening for limonene bioconversion and correlation with RAPD markers by Geciane Toniazzo; Lindomar Lerin; Débora De Oliveira; Cláudio Dariva; Rogério L. Cansian; Francine Ferreira Padilha; Octávio A. C. Antunes (pp. 1023-1033).
The use of microorganisms for biotransformations of monoterpenes has stimulated the biotechnological market. Aiming at the highest efficiency in the process of strains screening, the application of molecular biology techniques have been proposed. Based on these aspects, the objective of this work was to select different strains able to convert limonene using fermentative process and random amplified polymorphic DNA (RAPD) markers. The results obtained in the fermentative screening, from 17 strains tested, pointed out that four microorganisms were able to convert limonene into oxygenated derivatives. The RAPD study showed a polymorphism of 96.02% and a similarity from 16.02 to 51.51%. Based on this it was possible to observe a high genetic diversity, even among strains of same species, concluding that the RAPD was not able to correlate the genetic characteristics of the microorganism with the results obtained from the biotransformation process.

Keywords: Bioconversion; limonene; RAPD; screening


Use of different adsorbents for sorption and Bacillus polymyxa protease immobilization by Irem Kirkkopru; Cenk Alpaslan; Didem Omay; Yüksel Güvenilir (pp. 1034-1040).
Proteases constitute one of the most important groups of industrial enzymes, accounting for at least 25% of the total enzyme sales, with two-thirds of the proteases produced commercially being of microbial origin (1). Immobilized enzymes are currently the subject of considerable interest because of their advantages over soluble enzymes or alternative, technologies, and the steadily increasing number of applications for immobilized enzymes. The general application of immobilized proteins and enzymes has played a central role in the expansion of biotechnology and synthesis-related industries. Proteases have been immobilized on natural and synthetic supports (2,3).In the present work, a protease from Bacillus polymyxa was partially purified with 80% ammonium sulfate precipitation followed by dialysis and chromatography using a diethylaminoethyl (DEAE)-cellulose ion exchange column. Immobilization was evaluated by using different adsorbents (chitin, chitosan, alginate, synthetic zeolite, and raw zeolite) and the storage stability and recycle of the immobilized protease determined. Immobilization yields were estimated to be 96% and 7.5%, by using alginate and chitosan, respectively, after, 24 h. The yield of the immobilization was 17% for alginate at 16h and the enzyme did not adsorb on the chitin, chitosan, synthetic zeolite, and raw zeolite.

Keywords: Alginate; Bacillus polymyxa ; chitin; immobilization; protease


Simulation and optimization of a supercritical extraction process for recovering provitamin A by Elenise Bannwart de Moraes; Mario Eusebio Torres Alvarez; Maria Regina Wolf Maciel; Rubens Maciel Filho (pp. 1041-1050).
In this work, a simulation procedure of a supercritical extraction process was developed through the use of the commercial simulator HYSYSTM (Hyprotech Ltd.), adapting the existing units to the operating conditions typical of the supercritical extraction process. The objective is to recover provitamin A (β-carotene) from palm oil (esterified) using carbon dioxide/ethanol as the supercritical mixed solvent. This example characterizes the problem for recovering high added value product from natural sources, as the palm oil, which is desired by the market. Owing to the fact that esterified palm oil is a complex mixture, made by several components, in order to characterize this system in the simulator, it was necessary to create hypothetical components using the UNIFAC (universal function-group activity coefficients model) group contribution, because they are not present in a conventional database and, then, their physical properties must be estimated and/or predicted before the simulation. The optimization was carried out in each simulation for each equipment, in terms of operating conditions (temperature and pressure), in order to obtain the maximum recovery of carotenes. According to the results, it was possible to concentrate carotenes through two cycles of supercritical extraction with high yield. Furthermore, ethyl esters (biodiesel) were also obtained, as a byproduct of the proposed process, which can also be used as an alternative fuel, with the important characteristic that it is renewable.

Keywords: Biodiesel; carotenes; palm oil; supercritical extraction


Affinity foam fractionation of Trichoderma cellulase by Qin Zhang; Chi-Ming Lo; Lu-Kwang Ju (pp. 1051-1065).
Cellulase could not be selectively collected from fermentation broth by simple foam fractionation, because of the presence of other more surface-active compounds. A new approach of affinity foam fractionation was investigated for improvement. A hardwood hydrolysate (containing cellulose oligomers, substrates to cellulase) and two substrate analogs, i.e., carboxymethyl cellulose (CMC) and xylan hydrolysate, were added before the foaming process. The substrates and substrate analogs were indeed found to bind the cellulase selectively and form more hydrophobic complexes that partition more readily onto bubble surfaces. In this study, the effects of the type and concentration of substrate/analog as well as the presence of cells at different growth stages were examined. The foam fractionation properties evaluated included foaming speed, foam stability, foamate volume, and enrichment of filter paper unit (FPU) and individual cellulase components (i.e., endoglucanases, exoglucanases, and β-glucosidases). Depending on the broth and substrate/analog employed, the foamate FPU could be more than fourfold higher than the starting broth FPU. Addition of substrate/analog also deterred the enrichment of other extracellular proteins, resulting in the desired cellulase purification in the foamate. The value of E/P (enzyme activity-FPU/g/L of proteins) in the foamate reached as high as 18, from a lactose-based fermentation broth with original E/P of 5.6. Among cellulase components, exoglucanases were enriched the most and β-glucosidases the least. The study with CMC of different molecular weights (MW) and degrees of substitution (DS) indicated that the CMC with low DS and high MW performed better in cellulase foam fractionation.

Keywords: Affinity foam fractionation; carboxymethyl cellulose; cellulose; cellulose hydrolysate; xylan hydrolysate


Molecular distillation by Elenise Bannwart de Moraes; Patricia Fazzio Martins; César Benedito Batistella; Mario Eusebio Torres Alvarez; Rubens Maciel Filho; Maria Regina Wolf Maciel (pp. 1066-1076).
Molecular distillation was studied for the separation of tocopherols from soya sludge, both experimentally and by simulation, under different operating conditions, with good agreement. Evaporator temperatures varied from 100°C to 160°C and feed flow rates ranged from 0.1 to 0.8 kg/h. The process pressure was maintained at 10−6 bar, the feed temperature at 50°C, the condenser temperature at 60°C, and the stirring at 350 rpm. For each process condition, samples of both streams (distillate and residue) were collected and stored at −18°C before tocopherols analyses. Owing to the differences between molecular weights and vapor pressures of free fatty acids and tocopherols, tocopherols preferentially remained in the residue at evaporator temperatures of 100°C and 120°C, whereas for higher temperatures (140°C and 160°C) and lower feed flow rate, tocopherols tended to migrate to the distillate stream.

Keywords: Free fatty acids; molecular distillation; soya sludge; tocopherols


Application of two-stage biofilter system for the removal of odorous compounds by Gwi-Taek Jeong; Donn-Hee Park; Gwang-Yeon Lee; Jin-Myeong Cha (pp. 1077-1088).
Biofiltration is a biological process which is considered to be one of the more successful examples of biotechnological applications to environmental engineering, and is most commonly used in the removal of odoriferous compounds. In this study, we have attempted to assess the efficiency with which both single and complex odoriferous compounds could be removed, using one- or two-stage biofiltration systems. The tested single odor gases, limonene, α-pinene, and iso-butyl alcohol, were separately evaluated in the biofilters. Both limonene and α-pinene were removed by 90% or more EC (elimination capacity), 364 g/m3/h and 321 g/m3/h, respectively, at an input concentration of 50 ppm and a retention time of 30 s. The iso-butyl alcohol was maintained with an effective removal yield of more than 90% (EC 375 g/m3/h) at an input concentration of 100 ppm. The complex gas removal scheme was applied with a 200 ppm inlet concentration of ethanol, 70 ppm of acetaldehyde, and 70 ppm of toluene with residence time of 45 s in a one- or two-stage biofiltration system. The removal yield of toluene was determined to be lower than that of the other gases in the one-stage biofilter. Otherwise, the complex gases were sufficiently eliminated by the two-stage biofiltration system.

Keywords: Biofilm; biofilter; complex odor; two-stages; VOCs
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