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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.156, #1-3)


Optimization of Biodiesel Production from Castor Oil Using Response Surface Methodology by Gwi-Taek Jeong; Don-Hee Park (pp. 1-11).
The short supply of edible vegetable oils is the limiting factor in the progression of biodiesel technology; thus, in this study, we applied response surface methodology in order to optimize the reaction factors for biodiesel synthesis from inedible castor oil. Specifically, we evaluated the effects of multiple parameters and their reciprocal interactions using a five-level three-factor design. In a total of 20 individual experiments, we optimized the reaction temperature, oil-to-methanol molar ratio, and quantity of catalyst. Our model equation predicted that the following conditions would generate the maximum quantity of castor biodiesel (92 wt.%): a 40-min reaction at 35.5 °C, with an oil-to-methanol molar ratio of 1:8.24, and a catalyst concentration of 1.45% of KOH by weight of castor oil. Subsequent empirical analyses of the biodiesel generated under the predicted conditions showed that the model equation accurately predicted castor biodiesel yields within the tested ranges. The biodiesel produced from castor oil satisfied the relevant quality standards without regard to viscosity and cold filter plugging point.

Keywords: Castor oil; Biodiesel; Transesterification; Optimization; Response surface methodology; Viscosity; Alcohol solubility


Cell Immobilization with Polyurethane Foam for Retaining Trichoderma reesei Cells During Foam Fractionation for Cellulase Collection by Qin Zhang; Chi-Ming Lo; Lu-Kwang Ju (pp. 12-23).
In situ affinity foam fractionation is a potential powerful tool for continuous, selective removal of products from bioprocesses. When evaluating its applicability to cellulase production by Trichoderma reesei fermentation, we encountered the difficulty of significant removal of fungal mycelia along with the cellulase. To solve this problem, cell immobilization using cut pieces of hydrophilic polyurethane (PU) foam was evaluated. Five commercial PU foams with different pore sizes and porosities were tested. Two were found to support good cell growth, cellulase production, and cell loading (about 0.6 g dry cells per g PU). The PU-immobilized mycelia were successfully retained in the foaming process.

Keywords: Cell immobilization; Cellulase; Trichoderma reesei ; Affinity foam fractionation; Hydrophilic polyurethane


Biodiesel Production from Various Oils Under Supercritical Fluid Conditions by Candida antartica Lipase B Using a Stepwise Reaction Method by Jong Ho Lee; Cheong Hoon Kwon; Jeong Won Kang; Chulhwan Park; Bumseok Tae; Seung Wook Kim (pp. 24-34).
In this study, we evaluate the effects of various reaction factors, including pressure, temperature, agitation speed, enzyme concentration, and water content to increase biodiesel production. In addition, biodiesel was produced from various oils to establish the optimal enzymatic process of biodiesel production. Optimal conditions were determined to be as follows: pressure 130 bar, temperature 45 °C, agitation speed 200 rpm, enzyme concentration 20%, and water contents 10%. Among the various oils used for production, olive oil showed the highest yield (65.18%) upon transesterification. However, when biodiesel was produced using a batch system, biodiesel conversion yield was not increased over 65%; therefore, a stepwise reaction was conducted to increase biodiesel production. When a reaction medium with an initial concentration of methanol of 60 mmol was used and adjusted to maintain this concentration of methanol every 1.5 h during biodiesel production, the conversion yield of biodiesel was 98.92% at 6 h. Finally, reusability was evaluated using immobilized lipase to determine if this method was applicable for industrial biodiesel production. When biodiesel was produced repeatedly, the conversion rate was maintained at over 85% after eight reuses.

Keywords: Biodiesel; Initial reaction rate; Lipase activity; Optimization; Solubility; Supercritical fluid condition


Effect of Heating Strategy on Power Consumption and Performance of a Pilot Plant Anaerobic Digester by Teodoro Espinosa-Solares; Salvador Valle-Guadarrama; John Bombardiere; Max Domaschko; Michael Easter (pp. 35-44).
The effect of heating strategy on power consumption and performance of a pilot plant anaerobic digester treating chicken litter, under thermophilic conditions, has been studied. Heating strategy was evaluated using three different spans (0.2 °C, 0.6 °C, and 1.0 °C) for triggering the temperature control system from target temperature (56.7 °C). The hydraulic retention time in the pilot plant digester was in the range of 32 to 37 days, varying the total solids concentration fed from 5% to 6%. The results showed that under the experimental conditions, heating was the most energy-demanding process with 95.5% of the energy used. Increments up to 7.5% and 3.8%, respectively, on mechanical and heating power consumption, were observed as the span, for triggering the temperature control system from target temperature, was increased. Under the experimental conditions studied here, an increment of 30.6% on the global biodigester performance index was observed when a span of 1.0 °C was compared to the one of 0.2 °C.

Keywords: Temperature control; Chicken litter; Pneumatic agitation; Biogas mixing; Heat transfer coefficient


A Comparison Between Shaker and Bioreactor Performance Based on the Kinetic Parameters of Xanthan Gum Production by S. Faria; P. A. Vieira; M. M. Resende; F. P. França; V. L. Cardoso (pp. 45-58).
Xanthan gum production was studied using sugarcane broth as the raw material and batch fermentation by Xanthomonas campestris pv. campestris NRRL B-1459. The purpose of this study was to optimize the variables of sucrose, yeast extract, and ammonium nitrate concentrations and to determine the kinetic parameters of this bioreaction under optimized conditions. The effects of yeast extract and ammonium nitrate concentrations for a given sucrose concentration (12.1–37.8 g L−1) were evaluated by central composite design to maximize the conversion efficiency. In a bioreactor, the maximum conversion efficiency was achieved using 27.0 g L−1 sucrose, 2.7 g L−1 yeast extract, and 0.9 g L−1 NH4NO3. This point was assayed in a shaker and in a bioreactor to compare bioreaction parameters. These parameters were estimated by the unstructured kinetic model of Weiss and Ollis (Biotechnol Bioeng 22:859–873, 1980) to determinate the yields (Y P/S), the maximum growth specific rate (μ max), and the saturation cellular concentration (X*). The parameters of the model (μ max, X*, m, λ, α, and β) were obtained by nonlinear regression. For production of xanthan gum in a shaker, the values of μ max and Y P/S obtained were 0.119 h−1 and 0.34 g g−1, respectively, while in a bioreactor, they were 0.411 h−1 and 0.63 g g−1, respectively.

Keywords: Fermentation; Optimization; Xanthan gum; Kinetic; Sugarcane broth


Periodic Fermentor Yield and Enhanced Product Enrichment from Autonomous Oscillations by Chris C. Stowers; J. Brian Robertson; Hyunju Ban; Robert D. Tanner; Erik M. Boczko (pp. 59-75).
Four decades of work have clearly established the existence of autonomous oscillations in budding yeast culture across a range of operational parameters and in a few strains. Autonomous oscillations impact substrate conversion to biomass and products. Relatively little work has been done to quantify yield in this case. We have analyzed the yield of autonomously oscillating systems, grown under different conditions, and demonstrate that it too oscillates. Using experimental data and mathematical models of yeast growth and division, we demonstrate strategies to increase the efficient recovery of products. The analysis makes advantage of the population structure and synchrony of the system and our ability to target production within the cell cycle. While oscillatory phenomena in culture have generally been regarded with trepidation in the engineering art of bioprocess control, our results provide further evidence that autonomously oscillating systems can be a powerful tool, rather than an obstruction.

Keywords: Ambient biocomplexity; Protein purification; Enrichment ratio; Mitotic cell cycle


Improving the Performance of a Continuous Process for the Production of Ethanol from Starch by Joubert Trovati; Roberto C. Giordano; Raquel L. C. Giordano (pp. 76-90).
In a previous work, a continuous simultaneous saccharification and fermentation process to produce ethanol from cassava starch was studied, using a set of fixed-bed reactors. The biocatalyst consisted of glucoamylase immobilized in silica particles and co-immobilized with S. cerevisiae in pectin gel. Using 3.8 U mL−1 reactor and 0.05 gwet yeast mL−1 reactor at start-up, starch hydrolysis was the rate-limiting step. Maximum ethanol productivity was 5.8 gethanol L−1 h−1, with 94.0% conversion of total reducing sugars (TRS) and 83.0% of the ethanol theoretical yield. In this work, the molar mass of the substrate and the biocatalyst particle size were reduced in an attempt to improve the bioreactor performance. The diameters of silica and pectin gel particles were reduced from 100 μm and 3–4 mm, respectively, to 60 μm and 1–1.5 mm, and the degree of substrate prehydrolysis by α-amylase was increased. The bioreactor performance was assessed for different loads of immobilized glucoamylase (2.1, 2.8, and 3.8 U mL−1 reactor), for the same initial cell concentration (0.05 gwet yeast.mL−1 reactor). Feeding with 154.0 g L−1 of TRS and using 3.8 U mL−1 reactor, fermentation became the rate-limiting step. Productivity reached 11.7 g L−1 h−1, with 97.0% of TRS conversion and 92.0% of the ethanol theoretical yield. The reactor was operated during 275 h without any indication of destabilization.

Keywords: Ethanol; Cassava starch; Saccharomyces cerevisiae ; Glucoamylase; Packed-bed reactor; SSF; Mass transport effects


Carboxylate Platform: The MixAlco Process Part 1: Comparison of Three Biomass Conversion Platforms by Mark T. Holtzapple; Cesar B. Granda (pp. 95-106).
To convert biomass to liquid fuels, three platforms are compared: thermochemical, sugar, and carboxylate. To create a common basis, each platform is fed “ideal biomass,” which contains polysaccharides (68.3%) and lignin (31.7%). This ratio is typical of hardwood biomass and was selected so that when gasified and converted to hydrogen, the lignin has sufficient energy to produce ethanol from the carboxylic acids produced by the carboxylate platform. Using balanced chemical reactions, the theoretical yield and energy efficiency were determined for each platform. For all platforms, the ethanol yield can be increased by 71% to 107% by supplying external hydrogen produced from other sources (e.g., solar, wind, nuclear, fossil fuels). The alcohols can be converted to alkanes with a modest loss of energy efficiency (3 to 5 percentage points). Of the three platforms considered, the carboxylate platform has demonstrated the highest product yields.

Keywords: Carboxylate platform; Sugar platform; Thermochemical platform; Gasification; Energy efficiency; Yields; Alcohol; Alkanes


Carboxylate Platform: The MixAlco Process Part 2: Process Economics by Cesar B. Granda; Mark T. Holtzapple; Gary Luce; Katherine Searcy; Darryl L. Mamrosh (pp. 107-124).
The MixAlco process employs a mixed culture of acid-forming microorganisms to convert biomass to carboxylate salts, which are concentrated via vapor-compression evaporation and subsequently chemically converted to other chemical and fuel products. To make alcohols, hydrogen is required, which can be supplied from a number of processes, including gasifying biomass, separation from fermentor gases, methane reforming, or electrolysis. Using zeolite catalysts, the alcohols can be oligomerized into hydrocarbons, such as gasoline. A 40-tonne/h plant processing municipal solid waste ($45/tonne tipping fee) and using hydrogen from a pipeline or refinery ($2.00/kg H2) can sell alcohols for $1.13/gal or gasoline for $1.75/gal with a 15% return on investment ($0.61/gal of alcohol or $0.99/gal of gasoline for cash costs only). The capital cost is $1.95/annual gallon of mixed alcohols. An 800-tonne/h plant processing high-yield biomass ($60/tonne) and gasifying fermentation residues and waste biomass to hydrogen ($1.42/kg H2) can sell alcohols for $1.33/gal or gasoline for $2.04/gal with a 15% return on investment ($1.08/gal of alcohol or $1.68/gal of gasoline for cash costs only). The capital cost for the alcohol and gasification plants at 800 tonnes/h is $1.45/annual gallon of mixed alcohols.

Keywords: Carboxylate platform; MixAlco process; Economics; Alcohol; Hydrogen; Alkanes; Biogasoline


Determining Yields in High Solids Enzymatic Hydrolysis of Biomass by Jan B. Kristensen; Claus Felby; Henning Jørgensen (pp. 127-132).
As technologies for utilizing biomass for fuel and chemical production continue to improve, enzymatic hydrolysis can be run at still higher solids concentrations. For hydrolyses that initially contain little or no free water (10–40% total solids, w/w), the saccharification of insoluble polymers into soluble sugars involves changes of volume, density, and proportion of insoluble solids. This poses a new challenge when determining the degree of hydrolysis (conversion yield). Experiments have shown that calculating the yield from the resulting sugar concentration in the supernatant of the slurry and using the assumed initial volume leads to significant overestimations of the yield. By measuring the proportion of insoluble solids in the slurry as well as the sugar concentration and specific gravity of the aqueous phase, it is possible to precisely calculate the degree of conversion. The discrepancies between the different ways of calculating yields are demonstrated along with a nonlaborious method for approximating yields in high solids hydrolysis.

Keywords: Enzymatic hydrolysis; Biomass; Bioethanol; High solids; High dry matter; Yield


Separation and Immobilization of Lipase from Penicillium simplicissimum by Selective Adsorption on Hydrophobic Supports by Aline G. Cunha; Gloria Fernández-Lorente; Melissa L. E. Gutarra; Juliana V. Bevilaqua; Rodrigo V. Almeida; Lúcia M. C. Paiva; Roberto Fernández-Lafuente; Jose M. Guisán; Denise M. G. Freire (pp. 133-145).
Lipases are an enzyme class of a great importance as biocatalysts applied to organic chemistry. However, it is still necessary to search for new enzymes with special characteristics such as good stability towards high temperatures, organic solvents, and high stereoselectivity presence. The present work’s aim was to immobilize the lipases pool produced by Penicillium simplissicimum, a filamentous fungi strain isolated from Brazilian babassu cake residue. P. simplissicimum lipases were separated into three different fractions using selective adsorption method on different hydrophobic supports (butyl-, phenyl-, and octyl-agarose) at low ionic strength. After immobilization, it was observed that these fractions’ hyperactivation is in the range of 131% to 1133%. This phenomenon probably occurs due to enzyme open form stabilization when immobilized onto hydrophobic supports. Those fractions showed different thermal stability, specificity, and enantioselectivity towards some substrates. Enantiomeric ratio for the hydrolysis of (R,S) 2-O-butyryl-2-phenylacetic acid ranged from 1 to 7.9 for different immobilized P. simplissicimum lipase fractions. Asymmetry factor for diethyl 2-phenylmalonate hydrolysis ranged from 11.8 to 16.4 according to the immobilized P. simplissicimum lipase fractions. Those results showed that sequential adsorption methodology was an efficient strategy to obtain new biocatalysts with different enantioselectivity degrees, thermostability, and specificity prepared with a crude extract produced by a simple and low-cost technology.

Keywords: Fraction separation; Immobilization; Enantioselective hydrolysis
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