Biochemical Engineering Journal (v.49, #2)

BEJ Keywords (II).

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation in the genome. Scanning a genome for SNPs can help identify millions of potentially informative biomarkers. SNPs have been extensively used as molecular markers in human disease genetics, pharmacogenetics, and breeding, but SNPs have not been widely used in the bioprocess community. In biotechnology applications such as bioprocess development, SNPs may serve as genetic markers for phenotypes of interest such as those related to cell growth and viability, specific productivity, or stability. Furthermore, SNPs that relate to particular phenotypes may be targets for metabolic and cellular engineering. This review introduces study designs that have been used to link SNPs and phenotypes. The review then focuses on the downstream effects of the SNPs at DNA, RNA and protein levels. Finally, this review discusses specific examples to apply SNPs for breeding, strain evolution, and biomolecule production. Large scale SNP studies represent an opportunity to apply new genome-scale technologies to address current limitations and questions relevant to the biotechnology community such as cell line generation and selection.
Keywords: Single nucleotide polymorphism; Genotyping; Genetic markers; Functional genomics; Strain development; Genetic engineering;

Magnetic polymeric nanospheres as an immobilized metal affinity chromatography (IMAC) support for catalase by M. Emin Çorman; Nevra Öztürk; Nalan Tüzmen; Sinan Akgöl; Adil Denizli (159-164).
Novel magnetic nanospheres with an average size of 118 nm utilizing N-methacryloyl-(l)-cysteine methyl ester (MAC) as a monomer were prepared by surfactant free emulsion polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAC conducted in an aqueous dispersion medium. MAC was synthesized using methacryloyl chloride and l-cysteine methyl ester. l-Cysteine groups of the mag-poly(HEMA–MAC) nanospheres were chelated with Fe3+ ions. Specific surface area of the nonporous nanospheres was found to be 2452 m2/g. Mag-poly(HEMA–MAC)-Fe3+ nanospheres contained 0.81 mmol sulphur/g polymer were used in the adsorption of catalase in batch system. Using an optimized adsorption protocol, a very high loading of 820 mg catalase/g nanosphere was obtained. The adsorption phenomena appeared to follow a typical Langmuir isotherm. The immobilized catalase has more resistence to temperature inactivation than that of their free form. The optimum pH value of catalase was not affected by the immobilization reaction, but the pH profile was broadened for the immobilized enzyme. Kinetic parameters were determined for immobilized catalase as well as for the free enzyme. The values of the Michaelis constant K m of catalase were significantly smaller (ca. 2.5 times) upon immobilization, indicating increased affinity of the enzyme for its substrate, whereas V max value of free catalase was higher than that of the immobilized enzyme. It was also observed that enzyme could be repeatedly adsorbed and desorbed on the mag-poly(HEMA–MAC)-Fe3+ nanospheres without loss of adsorption capacity or enzymatic activity.
Keywords: Catalase; Nanospheres; Support; IMAC; Cysteine;

Simplified dynamic pressure method for k L a measurement in aerated bioreactors by F. Scargiali; A. Busciglio; F. Grisafi; A. Brucato (165-172).
A simplified version of the dynamic pressure method for measuring mass-transfer coefficients in gas–liquid systems is proposed. With this method oxygen concentration in the liquid phase is monitored after a sudden change of total pressure is applied to the system. With respect to the original technique introduced by Linek the simplified version here proposed greatly simplifies the data treatment, yet resulting in good accuracy for most practical purposes. In practice, with the help of a simple mathematical model, it is found that the dynamic oxygen concentration response, when plotted as residual driving force versus time in a semi-log diagram, should be expected to finally settle on a straight line. From the slope of this last k L a can be immediately computed. Experimental data obtained on a lab-size stirred tank reactor confirm all model predictions, including the feature that the adoption of large pressure changes may lead to better accuracy. Mass transfer coefficient data obtained by means of the simplified dynamic pressure method (SDPM) here proposed are compared with the relevant data obtained on the same system by the most accurate physical technique (pure oxygen absorption in a pre-evacuated liquid), as well as with the literature data, resulting in SDPM full validation. All k L a data obtained are finally organized by a conventional power law correlation.
Keywords: Dynamic modelling; Gas–liquid mass transfer; Multiphase bioreactors; Oxygen transfer; k L a ; Stirred vessels;

Modelling aerobic granular SBR at variable COD/N ratios including accurate description of total solids concentration by José Ramón Vázquez-Padín; Anuska Mosquera-Corral; José Luis Campos; Ramón Méndez; Julián Carrera; Julio Pérez (173-184).
The operation of a sequencing batch reactor (SBR) with aerobic granular biomass was successfully simulated using a one-dimensional biofilm model. The biological processes considered were described based on the activated sludge model (ASM) platform with two main modifications: (i) simultaneous growth and storage of organic substrates by heterotrophic bacteria; and (ii) inclusion of nitrite as intermediate compound in the nitrification and denitrification processes. Three different operational conditions were evaluated, characterized by different chemical oxygen demand to nitrogen (COD/N) ratios in the influent of: 0, 1.25 and 5.5 kg kg−1, representing a purely autotrophic media and two heterotrophic media, respectively. An accurate description of the experimental concentrations of COD, ammonium, nitrite, nitrate, dissolved oxygen (DO) and alkalinity along the cycles was obtained. Total solids concentration inside the reactor (5.0, 2.0 and 1.0 kg VSS m−3 for (COD/N) ratio of 5.5, 1.25 and 0 kg kg−1, respectively) and biofilm density ( 23 kg m granule − 3 ) were correctly described with the model. To obtain an accurate description of both solids concentration and biofilm density different densities were defined for the particulate compounds and a porosity profile along the granule was imposed. Oxygen penetration depths obtained with the model were 0.35 × 10−3, 0.30 × 10−3 and 0.12 × 10−3  m for (COD/N) ratio of 5.5, 1.25 and 0 kg kg−1, respectively. The values were in agreement with those used in the description of the porosity profiles.
Keywords: Aerobic granulation; Mass transfer; Microbial kinetics; Biomass density; Solids concentration; Wastewater treatment;

Biosurfactant production by Pseudomonas aeruginosa SP4 using sequencing batch reactors: Effect of oil-to-glucose ratio by Sira Pansiripat; Orathai Pornsunthorntawee; Ratana Rujiravanit; Boonyarach Kitiyanan; Pastra Somboonthanate; Sumaeth Chavadej (185-191).
Sequencing batch reactors were used for biosurfactant production from Pseudomonas aeruginosa SP4. The lab-scale aerobic sequencing batch reactor units were operated at an oil loading rate of 2 kg/m3  day, a cycle time of 2 days/cycle, and a temperature of 37 °C. A mineral medium with palm oil was used as the culture medium. Glucose, a supplemental carbon source, was added for enhancing the microbial growth, which, in turn, gave a better process stability. The optimum oil-to-glucose ratio for the biosurfactant production was 40:1, providing a surface tension reduction of 58.5%, a chemical oxygen demand removal of 85.1%, and an oil removal of 77.7%. The maximum biosurfactant concentration in the bioreactors was about 1.1 times the critical micelle concentration. The biosurfactant product was predominantly composed of Rha-Rha-C8-C10 and Rha-Rha-C10-C8, and its critical micelle concentration was 150 mg/l.
Keywords: Biosurfactants; Pseudomonas aeruginosa; Sequencing batch reactor (SBR); Glycolipids; Rhamnolipids;

Study of hydrodynamics, mass transfer, energy consumption, and biomass production from natural gas in a forced-liquid vertical tubular loop bioreactor by F. Yazdian; M. Pesaran Hajiabbas; S.A. Shojaosadati; M. Nosrati; E. Vasheghani-Farahani; M.R. Mehrnia (192-200).
A forced-liquid vertical tubular loop bioreactor (VTLB) has been used for the production of biomass from natural gas. Hydrodynamic characteristics and mass-transfer coefficients have been determined as functions of design and operational parameters. Energy consumption for different gas and liquid flow rates has been studied. Liquid flow rate was found to have a remarkable effect on gas hold-up and k L a due to its influence on mixing time. The values of k L a for gases have been determined for different geometrical and operational factors. New correlations for mixing time, gas hold-up, and k L a were obtained. A gas mixture of 40 vol% methane and 60 vol% air proved to be optimal for biomass production.
Keywords: Vertical tubular loop bioreactor; Hydrodynamics; Mixing; Gas hold-up; Mass transfer; Biomass;

A salt tolerant Enterobacter cloacae mutant for bioaugmentation of petroleum- and salt-contaminated soil by Xiufu Hua; Jun Wang; Zuojun Wu; Hongxing Zhang; Heping Li; Xinghui Xing; Zheng Liu (201-206).
A NaCl-tolerant Enterobacter cloacae variant (MU-1) was obtained by mutagenesis using atmospheric pressure glow discharge (APGD) plasmas. The variant exhibited regular growth behavior in slurry cultivation and reached a cell density of 5.72 × 108 and 6.44 × 108  colony-forming units (CFU/mL) in the presence and absence of 7.5% NaCl, respectively, when crude oil was used as the sole carbon source (crude oil/soil = 1.5%). The total petroleum hydrocarbon (TPH) degradation percentage was 7.94% with mutant MU-1 in the presence of 7.5% NaCl whereas that of the wild-type strain was 3.17%. When cultivated in saline medium, MU-1 showed a slight change in membrane permeability but significant increases in both the K+ concentration inside the cell membrane (from 234.24 to 1422.88 ppm/g dry cell weight in the first 2 h) and the exopolysaccharide (EPS) level outside the membrane (from 1350 to 1825 mg/g dry cell weight). The rapid increase in K+ inside the cell and the simultaneous accumulation of EPS outside the cell may be responsible for maintaining the osmotic balance during saline cultivation, and this could facilitate the microbial growth and TPH degradation of MU-1.
Keywords: Bioremediation; Microbial growth; Biodegradation; Bioprocess design; Enterobacter cloacae; Salinity tolerance mutagenesis;

Two-step lipase catalysis for production of biodiesel by Md. Mahabubur Rahman Talukder; Jin Chuan Wu; Ng Mei Fen; Yeo Li Shi Melissa (207-212).
Lipase-catalyzed methanolysis of vegetable oils has attracted considerable interests for the production of biodiesel (BD). However, the activity of lipase such as Novozym 435 (immobilized Candida antarctica lipase B) is negatively affected by methanol. To minimize this problem, two-step lipase catalysis was investigated. Crude palm oil (CPO), which is relatively cheaper because of avoiding refining cost, was used as the source of BD. CPO was first hydrolysed to fatty acids (FA), which was then esterified to BD. Candida rugosa and Novozym 435 lipases were used as biocatalysts for the hydrolysis of CPO and the esterification of FA, respectively. The complete conversion of CPO to FA was achieved under an optimal condition of buffer to CPO ratio 1:1 (v/v), buffer pH 7.0, lipase 0.1 wt.% of CPO, isooctane to CPO ratio 1:1 (v/v), temperature 30 °C, shaking speed 250 rpm and time 4 h. The methyl esterification of FA with 1.2-fold stoichiometric excess of methanol reached the equilibrium after 2 h at which BD yield was 98%. C. rugosa and Novozym 435 lipases were repeatedly used for 10 and 50 cycles, respectively without significant loss of their activities. The developed two-step process is very promising because of its feedstock flexibility: it can be used for production of BD and FA from crude, refined and waste oils.
Keywords: Biodiesel; Lipase; Two-step catalysis; Crude palm oil; Methanolysis;

The effect of the total volumetric power input under different aeration and agitation intensities on pellet morphology and glucoamylase production by Aspergillus niger AB1.13 was investigated in a 28 L stirred tank reactor. The pellet macro-morphology including pellet size and concentration was determined by the laser diffraction technique and digital image analysis. The pellet micro-morphology including pellet internal and surface structure was characterized by microscopic image analysis of pellet slices and sedimentation velocity measurements. The total volumetric power input, which comprises the volumetric power input by aeration and agitation, was kept constant for all cultivations, while the volumetric power input by aeration increased by the same degree as the volumetric power input by agitation decreased. Results revealed a notable variation in morphological data between pellets grown at different ratios of aeration to agitation, by which glucoamylase production yields were correspondingly altered. An increasing ratio of the volumetric power input by aeration resulted in a significant decrease in pellet size, a considerable increase in pellet concentration, a less compact surface structure of the pellets and a favorable production of glucoamylase.
Keywords: Aspergillus niger; Aeration; Agitation; Pellet morphology; Product formation; Glucoamylase;

A simple lumped kinetic model was used to simulate the single- and two-component breakthrough curves for the cation-exchange adsorption of pure α-lactalbumin (ALA) and β-lactoglobulin (BLG) onto a 5-ml SP Sepharose FF column at pH 3.7 and flow rate of 2 ml min−1. When compared to equivalent experimental results, the model accurately predicted the single-component BLG adsorption profiles using Langmuir isotherm parameters (q m, maximum binding capacity of the adsorbent; K d, dissociation constant for the protein–adsorbent interaction) obtained in batch experiments. The breakthrough curve for ALA, however, was well predicted only after increasing q m which indicates the occurrence of additional adsorption processes in the packed-bed relative to the batch system. An overshoot of the concentration of BLG in the bed exit stream observed experimentally in the two-component system, was only predicted after correcting the two isotherm parameters in order to account for the unexpected finding that the weakly bound ALA was able to displace the strongly bound BLG. A fitting mechanism was proposed for this situation. The correction factors employed for the pure binary mixture were used to simulate the breakthrough curves of the two proteins in experiments conducted with whey concentrate in each of the two stages of a novel separation process, and there was agreement between the experimental and theoretical results. These considerations should be helpful in developing a model compatible with the proposed mechanisms of adsorption for these two proteins.
Keywords: Simulation; Simple kinetic model; α-Lactalbumin; β-Lactoglobulin; Whey concentrate; Packed-bed adsorption;

The potential of oxic-settling-anoxic (OSA) process with addition of 3,3′,4′,5-tetrachlorosalicylanilide (TCS) to reduce excess sludge production was investigated. TCS was dosed into aeration tank with 0.05, 0.10 and 0.15 g every other day in three lab-scale OSA processes, respectively to form the TCS and OSA combined processes. The OSA and TCS combined processes reduced sludge yield by 21–56% under the same sludge retention time (6.75 h) in sludge anoxic holding tank. Substrate removal capability, effluent NH3-N concentrations and total phosphorus removal rates were not adversely affected by the presence of TCS or insertion of sludge anoxic holding tank, but total nitrogen removal rates only decreased significantly in the system with addition of 0.15 g TCS during the 60-day continuous operation. The settleability of sludge in four systems was qualitatively comparable and not significantly different. Microscopic examination and the banding patterns of DGGE profiles demonstrated that microbial population changed after TCS addition and insertion of anoxic sludge holding tank. The results suggest that TCS and OSA combined process is effective in reducing sludge yield, and process performance as well as sludge settleability are not significantly affected by introduction of the chemical uncoupler. The results imply that reduction of excess sludge production is due to uncoupled metabolism at low TCS dosage, but microbial death at high TCS dosage.
Keywords: Activated sludge; Waste treatment; Oxic-settling-anoxic (OSA) process; 3,3′,4′,5-Tetrachlorosalicylanilide (TCS); Metabolic engineering; Biomass;

Influence of zinc on ferrous iron bio-oxidation: Biological or physical nature? by Alfonso Mazuelos; Nieves Iglesias; Rafael Romero; Miguel Ángel Mejías; Francisco Carranza (235-240).
Ferrous iron bio-oxidation is negatively affected by the presence of heavy metals. Although the available information relates this phenomenon to purely biological aspects, it is contradictory with respect to tolerance levels, mechanisms and kinetics. This dispersion of results may be due to the empirical nature of the approaches which are based on batch cultures and fail to consider the conditions of aeration of the biomass.In the present work, the influence of Zn2+ in the range of 0–40 g/L is tested in continuous packed-bed bioreactors, by studying oxygen partial pressure and aeration flow rate as variables. Results show that when oxygen is the limiting reagent under identical aeration conditions, the bio-oxidation rate decreases by 0.8% per gram per litre of Zn2+. The cause of this result is purely thermodynamic; the solubility of oxygen in the medium decreases in equivalent proportions of the bio-oxidation rate owing to the salting-out effect.This finding leads to redesign of reactors for continuous ferrous iron bio-oxidation with the presence of Zn2+, whereby special attention is paid to the aeration system and its control during the operation.
Keywords: Bio-oxidation; Ferrous iron; Bioreactor system; Oxygen solubility; Zinc tolerance; Immobilized cells; Mass transfer; Biofilm;

Exopolysaccharides (EPSs) from Cordyceps taii have potent pharmacological effects and present an unparalleled resource for drug discovery, and optimization of EPS yield through systematic analysis and refinement of fermentation parameters was therefore sought. A one-variable-at-a-time method identified xylose (NH4)2SO4, and vitamins A and D as the optimum source of carbon, nitrogen, and growth-stimulating factor, respectively. Ammonium salts led to acidification of the culture medium, and the combination of (NH4)2SO4 with soybean steep liquor, the best organic nitrogen source, was therefore selected for subsequent optimization. Further experiments identified only xylose, soybean steep liquor and (NH4)2SO4 as having statistically significant effects on EPS production by a Plackett–Burman design. Re-optimization of these factors employed a path of steepest ascent approach followed by a central composite design modeling the response surface as a function of the experimental conditions. This predicted that optimum production of EPS would be achieved using a combination of xylose 31.27 g/l, soybean steep liquor 4.85 g/l, and (NH4)2SO4 0.15 g/l. Experimental validation revealed that the actual yield of EPS (43.87 ± 0.28 g/l) was accurately predicted by the model. This level of production represented an increase of over 8-fold vs. the highest yield (5.21 ± 0.14 g/l) obtained in preliminary experiments.
Keywords: Cordyceps; Biomass; Submerged; Shake-flask; Optimisation; Combinatorial experimental design;

The transesterification of methyl butyrate, ethyl butyrate and butyl butyrate to geranyl butyrate was investigated in supercritical carbon dioxide. The effect of chain length of the butyrate on the rate of transesterification was investigated. The initial rates followed the trend: ethyl butyrate < butyl butyrate < methyl butyrate. The transesterification of butyl butyrate to geranyl butyrate in various supercritical fluids such as ethylene, methane, ethane was also examined. The initial rate of transesterification of butyl butyrate in different supercritical fluids followed the order: ScCO2  < ScC2H6  < ScC2H4  < ScCH4. The highest initial rate was obtained in supercritical methane and the reasons for this observation were proposed. The Ping-Pong Bi–Bi model with inhibition by both acid and alcohol was used to model the experimental data and determine the kinetics of the reaction.
Keywords: Supercritical carbon dioxide; Supercritical methane; Novozym 435; Transesterification; Ping-Pong Bi–Bi mechanism; Kinetics;

Effect of particle size distribution on the simulation of immobilized enzyme reactor performance by Pedro Valencia; Sebastián Flores; Lorena Wilson; Andrés Illanes (256-263).
A mathematical model that describes the heterogeneous reaction–diffusion process involved in a batch reactor with immobilized enzyme is presented. The model is based on equations considering reaction and diffusion components including biocatalyst particle size distribution. The reaction system includes the bulk liquid phase containing the dissolved substrate (and products) and the solid biocatalyst phase represented by spherical porous particles carrying the enzyme. The model developed is illustrated for the case of penicillin G hydrolysis with immobilized penicillin acylase, which is a complex reaction system in which both products of reaction and the substrate itself are inhibitors. Significant differences in batch reactor performance simulation are observed when considering biocatalyst particles of a single radius and particle size distribution. The magnitude of these differences is proportional to the dispersion (standard deviation) considered in that size distribution function.
Keywords: Immobilized enzyme; Enzyme kinetics; Penicillin acylase; Penicillin hydrolysis; Diffusional restrictions;

Preparation and characterization of a novel macroporous immobilized micro-organism carrier by Xue Bai; Zhengfang Ye; Yanfeng Li; Liuqing Yang; Yanzhi Qu; Xiaozhe Yang (264-270).
The traditional PVA–boric acid method was modified using calcium carbonate as a pore-forming agent to form the macroporous structure and formulated macroporous carrier (MPC) post-crosslinked with glutaraldehyde. The pore volumes, pore structure, porosity, and swelling behavior of MPCs were evaluated. The crosslinking density of MPCs with four different crosslinker dosages was calculated from their swelling properties using the modified Flory equation. MPCs demonstrated high swelling capacity, large specific surface area, high diffusion coefficient, as well as chemical and mechanical strength. The high crosslinking degree MPCs resulted in high biomass densities and low activity yield and vice versa. The characterizations of MPC suggest significant potential of its use for microbial immobilization and provide a scientific basis for immobilized carrier design and optimization.
Keywords: Macroporous carrier; Polyvinyl alcohol; Adsorption; Activated sludge; Biomass; Waste treatment;

Various forms of polymers are involved and play different roles in enhanced biological phosphorus removal (EBPR), among which, total carbohydrate has been often measured to represent glycogen (intracellular carbohydrate) in previous EBPR studies. In this study, intracellular carbohydrate was tested after extracellular carbohydrate extraction, and its dynamics and function in activated sludge performing EBPR at 20 °C were examined: (i) during a reactor phase, (ii) when excess organic carbon was available under anaerobic conditions, and (iii) during anaerobic and aerobic starvation conditions. The proportion of intracellular carbohydrate to the total carbohydrate was only 62% in activated sludge taken at the end of the aerobic phase. Intracellular carbohydrate alone, or combined with polyphosphate, was used as an energy source for organic carbon uptake under the anaerobic condition. Intracellular carbohydrates were degraded for maintenance during starvation, with the first-order degradation coefficient of 0.41 1/d under anaerobic starvation and 0.19 1/d under aerobic starvation. Intracellular carbohydrate rather than total carbohydrate should be tested in EBPR studies so as to better understand, operate, control and model the EBPR process.
Keywords: Enhanced biological phosphorus removal; Carbohydrate; Intracellular; Polyphosphate; Starvation;