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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.147, #1-3)
Separate and Concentrate Lactic Acid Using Combination of Nanofiltration and Reverse Osmosis Membranes
by Yebo Li; Abolghasem Shahbazi; Karen Williams; Caixia Wan (pp. 1-9).
The processes of lactic acid production include two key stages, which are (a) fermentation and (b) product recovery. In this study, free cell of Bifidobacterium longum was used to produce lactic acid from cheese whey. The produced lactic acid was then separated and purified from the fermentation broth using combination of nanofiltration and reverse osmosis membranes. Nanofiltration membrane with a molecular weight cutoff of 100–400 Da was used to separate lactic acid from lactose and cells in the cheese whey fermentation broth in the first step. The obtained permeate from the above nanofiltration is mainly composed of lactic acid and water, which was then concentrated with a reverse osmosis membrane in the second step. Among the tested nanofiltration membranes, HL membrane from GE Osmonics has the highest lactose retention (97 ± 1%). In the reverse osmosis process, the ADF membrane could retain 100% of lactic acid to obtain permeate with water only. The effect of membrane and pressure on permeate flux and retention of lactose/lactic acid was also reported in this paper.
Keywords: Cheese whey; Lactic acid; Membrane; Nanofiltration; Reverse osmosis
Parameter Estimation for Simultaneous Saccharification and Fermentation of Food Waste Into Ethanol Using Matlab Simulink
by Rebecca Anne Davis (pp. 11-21).
The increase in waste disposal and energy costs has provided an incentive to convert carbohydrate-rich food waste streams into fuel. For example, dining halls and restaurants discard foods that require tipping fees for removal. An effective use of food waste may be the enzymatic hydrolysis of the waste to simple sugars and fermentation of the sugars to ethanol. As these wastes have complex compositions which may change day-to-day, experiments were carried out to test fermentability of two different types of food waste at 27° C using Saccharomyces cerevisiae yeast (ATCC4124) and Genencor’s STARGEN™ enzyme in batch simultaneous saccharification and fermentation (SSF) experiments. A mathematical model of SSF based on experimentally matched rate equations for enzyme hydrolysis and yeast fermentation was developed in Matlab Simulink®. Using Simulink® parameter estimation 1.1.3, parameters for hydrolysis and fermentation were estimated through modified Michaelis–Menten and Monod-type equations with the aim of predicting changes in the levels of ethanol and glycerol from different initial concentrations of glucose, fructose, maltose, and starch. The model predictions and experimental observations agree reasonably well for the two food waste streams and a third validation dataset. The approach of using Simulink® as a dynamic visual model for SSF represents a simple method which can be applied to a variety of biological pathways and may be very useful for systems approaches in metabolic engineering in the future.
Keywords: Food waste; Hydrolysis; Ethanol; Simultaneous saccharification and fermentation; Matlab; Simulink®; Parameter estimation; Metabolic engineering
Lignin Peroxidase from Streptomyces viridosporus T7A: Enzyme Concentration Using Ultrafiltration
by Leda M. F. Gottschalk; Elba P. S. Bon; Ronaldo Nobrega (pp. 23-32).
It is well known that lignin degradation is a key step in the natural process of biomass decay whereby oxidative enzymes such as laccases and high redox potential ligninolytic peroxidases and oxidases play a central role. More recently, the importance of these enzymes has increased because of their prospective industrial use for the degradation of the biomass lignin to increase the accessibility of the cellulose and hemicellulose moieties to be used as renewable material for the production of fuels and chemicals. These biocatalysts also present potential application on environmental biocatalysis for the degradation of xenobiotics and recalcitrant pollutants. However, the cost for these enzymes production, separation, and concentration must be low to permit its industrial use. This work studied the concentration of lignin peroxidase (LiP), produced by Streptomyces viridosporus T7A, by ultrafiltration, in a laboratory-stirred cell, loaded with polysulfone (PS) or cellulose acetate (CA) membranes with molecular weight cutoffs (MWCO) of 10, 20, and 50 KDa. Experiments were carried out at 25 °C and pH 7.0 in accordance to the enzyme stability profile. The best process conditions and enzyme yield were obtained using a PS membrane with 10 KDa MWCO, whereby it was observed a tenfold LiP activity increase, reaching 1,000 U/L and 90% enzyme activity upholding.
Keywords: Streptomyces viridosporus ; Lignin peroxidase; Ultrafiltration; Enzyme concentration; Enzyme stability
Oxygen-controlled Biosurfactant Production in a Bench Scale Bioreactor
by Frederico de Araujo Kronemberger; Lidia Maria Melo Santa Anna; Ana Carolina Loureiro Brito Fernandes; Reginaldo Ramos de Menezes; Cristiano Piacsek Borges; Denise Maria Guimarães Freire (pp. 33-45).
Rhamnolipids have been pointed out as promising biosurfactants. The most studied microorganisms for the aerobic production of these molecules are the bacteria of the genus Pseudomonas. The aim of this work was to produce a rhamnolipid-type biosurfactant in a bench-scale bioreactor by one strain of Pseudomonas aeruginosa isolated from oil environments. To study the microorganism growth and production dependency on oxygen, a nondispersive oxygenation device was developed, and a programmable logic controller (PLC) was used to set the dissolved oxygen (DO) concentration. Using the data stored in a computer and the predetermined characteristics of the oxygenation device, it was possible to evaluate the oxygen uptake rate (OUR) and the specific OUR (SOUR) of this microorganism. These rates, obtained for some different DO concentrations, were then compared to the bacterial growth, to the carbon source consumption, and to the rhamnolipid and other virulence factors production. The SOUR presented an initial value of about 60.0 mgO2/gDW h. Then, when the exponential growth phase begins, there is a rise in this rate. After that, the SOUR reduces to about 20.0 mgO2/gDW h. The carbon source consumption is linear during the whole process.
Keywords: Pseudomonas aeruginosa ; Biosurfactant; Oxygenation; Rhamnolipid; Bioreactor
Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel
by Raquel L. C. Giordano; Joubert Trovati; Willibaldo Schmidell (pp. 47-61).
This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica–enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica–enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10−4 cm/s.
Keywords: Ethanol; Cassava starch; Saccharomices cerevisiae ; Glucoamylase; Packed-bed reactor; Simultaneous saccharification and fermentation
Lipase Production in Solid-State Fermentation Monitoring Biomass Growth of Aspergillus niger Using Digital Image Processing
by Júlio C. V. Dutra; Selma da C. Terzi; Juliana Vaz Bevilaqua; Mônica C. T. Damaso; Sônia Couri; Marta A. P. Langone; Lilian F. Senna (pp. 63-75).
The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.
Keywords: Lipase; Digital image processing; Aspergillus niger ; Solid-state fermentation; Glycosamine; Vegetable oils; Biodiesel
The Effects of Surfactants on the Estimation of Bacterial Density in Petroleum Samples
by Aderval Severino Luna; Antonio Carlos Augusto da Costa; Márcia Monteiro Machado Gonçalves; Kelly Yaeko Miyashiro de Almeida (pp. 77-84).
The effect of the surfactants polyoxyethylene monostearate (Tween 60), polyoxyethylene monooleate (Tween 80), cetyl trimethyl ammonium bromide (CTAB), and sodium dodecyl sulfate (SDS) on the estimation of bacterial density (sulfate-reducing bacteria [SRB] and general anaerobic bacteria [GAnB]) was examined in petroleum samples. Three different compositions of oil and water were selected to be representative of the real samples. The first one contained a high content of oil, the second one contained a medium content of oil, and the last one contained a low content of oil. The most probable number (MPN) was used to estimate the bacterial density. The results showed that the addition of surfactants did not improve the SRB quantification for the high or medium oil content in the petroleum samples. On other hand, Tween 60 and Tween 80 promoted a significant increase on the GAnB quantification at 0.01% or 0.03% m/v concentrations, respectively. CTAB increased SRB and GAnB estimation for the sample with a low oil content at 0.00005% and 0.0001% m/v, respectively.
Keywords: Sulphate-reducing bacteria (SRB); General anaerobic bacteria (GAnB); Petroleum; Surfactants; Most probable number (MPN)
An Alternative Application to the Portuguese Agro-Industrial Residue: Wheat Straw
by Denise S. Ruzene; Daniel P. Silva; António A. Vicente; Adilson R. Gonçalves; José A. Teixeira (pp. 85-96).
The effects of alkaline treatments of the wheat straw with sodium hydroxide were investigated. The optimal condition for extraction of hemicelluloses was found to be with 0.50 mol/l sodium hydroxide at 55 °C for 2 h. This resulted in the release of 17.3% of hemicellulose (% dry starting material), corresponding to the dissolution of 49.3% of the original hemicellulose. The yields were determined by gravimetric analysis and expressed as a proportion of the starting material. Chemical composition and physico-chemical properties of the samples of hemicelluloses were elucidated by a combination of sugar analyses, Fourier transform infrared (FTIR), and thermal analysis. The results showed that the treatments were very effective on the extraction of hemicelluloses from wheat straw and that the extraction intensity (expressed in terms of alkali concentration) had a great influence on the yield and chemical features of the hemicelluloses. The FTIR analysis revealed typical signal pattern for the hemicellulosic fraction in the 1,200–1,000 cm−1 region. Bands between 1,166 and 1,000 cm−1 are typical of xylans.
Keywords: Agro-industrial residue; Lignocellulosic materials; Wheat straw; Extraction; Hemicellulose; Papermaking
The Use of Seaweed and Sugarcane Bagasse for the Biological Treatment of Metal-contaminated Waters Under Sulfate-reducing Conditions
by Márcia Monteiro Machado Gonçalves; Luiz Antonio de Oliveira Mello; Antonio Carlos Augusto da Costa (pp. 97-105).
When wetlands reach maximum treatment capacity to remove heavy metals, removal can still take place through precipitation as sulfide because of the biological reduction of sulfate. To achieve this goal, anaerobic conditions must be attained, a sulfate source must exist, and an adequate substrate for sulfate-reducing bacteria (SRB) is also required. In the present work, two ligneous-cellulosic materials, a brown seaweed and sugarcane bagasse, have been selected as substrates for SRB growth. Experiments were simultaneously conducted in continuous operation in two columns (0.57 L each), one containing the ligneous-cellulosic material plus inoculum and another containing only the ligneous-cellulosic material. In this work, the removal of cadmium and zinc was studied because of their presence in effluents from mining/metallurgy operations. Results obtained indicated that the inoculated reactor was able to treat the effluent more efficiently than the noninoculated reactor considering the time course of the tests.
Keywords: Metal removal; Sulfate reduction; Sulfate-reducing bacteria (SRB); Ligneous-cellulosic materials; Wetlands
Development of Activity-based Cost Functions for Cellulase, Invertase, and Other Enzymes
by Chris C. Stowers; Elizabeth M. Ferguson; Robert D. Tanner (pp. 107-117).
As enzyme chemistry plays an increasingly important role in the chemical industry, cost analysis of these enzymes becomes a necessity. In this paper, we examine the aspects that affect the cost of enzymes based upon enzyme activity. The basis for this study stems from a previously developed objective function that quantifies the tradeoffs in enzyme purification via the foam fractionation process (Cherry et al., Braz J Chem Eng 17:233–238, 2000). A generalized cost function is developed from our results that could be used to aid in both industrial and lab scale chemical processing. The generalized cost function shows several nonobvious results that could lead to significant savings. Additionally, the parameters involved in the operation and scaling up of enzyme processing could be optimized to minimize costs. We show that there are typically three regimes in the enzyme cost analysis function: the low activity prelinear region, the moderate activity linear region, and high activity power-law region. The overall form of the cost analysis function appears to robustly fit the power law form.
Keywords: Cost function; Cellulase; Invertase; Foam fractionation; Separations cost
Reaction Kinetics of the Hydrothermal Treatment of Lignin
by Bo Zhang; Hua-Jiang Huang; Shri Ramaswamy (pp. 119-131).
Lignins derived from abundant and renewable resources are nontoxic and extremely versatile in performance, qualities that have made them increasingly important in many industrial applications. We have shown recently that liquefaction of lignin extracted from aspen wood resulted in a 90% yield of liquid. In this paper, the hydrothermal treatment of five types of lignin and biomass residues was studied: Kraft pine lignin provided by MeadWestvaco, Kraft pine lignin from Sigma-Aldrich, organosolv lignin extracted from oat hull, the residues of mixed southern hardwoods, and switchgrass after hydrolysis. The yields were found dependent on the composition or structure of the raw materials, which may result from different pretreatment processes. We propose a kinetic model to describe the hydrothermal treatment of Kraft pine lignin and compare it with another model from the literature. The kinetic parameters of the presented model were estimated, including the reaction constants, the pre-exponential factor, and the activation energy of the Arrhenius equations. Results show that the presented model is well in agreement with the experiments.
Keywords: Hydrothermal treatment; Degradation; Kinetic model; Kraft lignin; Biomass residue
Hydrodynamic Characterization of a Column-type Prototype Bioreactor
by Teodoro Espinosa-Solares; Marcos Morales-Contreras; Fabián Robles-Martínez; Melvin García-Nazariega; Consuelo Lobato-Calleros (pp. 133-142).
Agro-food industrial processes produce a large amount of residues, most of which are organic. One of the possible solutions for the treatment of these residues is anaerobic digestion in bioreactors. A novel 18-L bioreactor for treating waste water was designed based on pneumatic agitation and semispherical baffles. Flow patterns were visualized using the particle tracer technique. Circulation times were measured with the particle tracer and the thermal technique, while mixing times were measured using the thermal technique. Newtonian fluid and two non-Newtonian fluids were used to simulate the operational conditions. The results showed that the change from Newtonian to non-Newtonian properties reduces mixed zones and increases circulation and mixing times. Circulation time was similar when evaluated with the thermal and the tracer particle methods. It was possible to predict dimensionless mixing time (θ m) using an equivalent Froude number (Fr eq).
Keywords: Flow pattern; Circulation time; Dimensionless mixing time; Froude number; Non-Newtonian behavior; Pneumatic agitation
Thermal Effects on Hydrothermal Biomass Liquefaction
by Bo Zhang; Marc von Keitz; Kenneth Valentas (pp. 143-150).
Batch pressure vessels commonly used for hydrothermal liquefaction have typical heating times in the range of 30 to 60 min. Thermodynamically, the complex set of reactions are path dependent, so that the heating rate can possibly affect yields and the composition of the resultant liquid products. It is postulated that the mode of heat transfer becomes an uncontrolled variable in kinetic studies and can seriously impact scale-up. To confirm this hypothesis and minimize these heat-transfer-related artifacts, we designed a batch pressure vessel equipped with an induction heating system, which allows the reduction of heat-up times by about two orders of magnitude to several seconds, compared to tens of minutes with standard pressure reactors. This system was used to study the direct liquefaction of corn stover and aspen wood with a pretreatment. The heating rate was found to have no significant effect on the composition of the liquid products. However, the liquid yields are dependent on the heating rate. Varying the cooling rate does not show obvious effects. The results confirm that the heating rate, as governed by the mode of heat transfer, is an important factor that needs to be considered during scale-up.
Keywords: Liquefaction; Aspen; Corn stover; Heating rate; Cooling rate; Yield; Composition
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