Biochemical Engineering Journal (v.35, #2)

BEJ Keywords (II).

In the course of the production of rhamnolipids with surface-active properties, Pseudomonas aeruginosa 47T2, when cultivated in mineral medium with waste cooking, accumulates polyhydroxyalkanoates (PHA) up to 36% of its cell dry weight (CDW) with the following composition (%): 9.4 C8:0; 47.8 C10:0; 15.3 C12:0; 7.8 C12:1; 1.0 C14:0; 11.4 C14:1; 7.0 C14:2. At the same time, average molecular weights, which ranged between 38.8 and 37.1 kDa were determined making use of the Mark–Houwink relationship and the intrinsic viscosities determined for tetrahydrofuran (THF) at 40 °C as solvent. The resulting polymers varied in the nature of the monomers and number. The highest production rate was at 37 °C, 8.2 mg of PHA/(g of residual biomass h) and the amounts of saturated and unsaturated compounds were strongly temperature dependent. Finally, the effect of the incubation temperature, carbon and nitrogen sources and the concentration of potassium phosphate in relation to the polymer composition are evaluated. PHA monomer compositions were determined by GC–MS and corroborated by NMR spectra analysis.
Keywords: Polyhydroxyalkanoates; Pseudomonas; Waste oily substrate; Monomer characterization;

Production of carotenoids by the isolated yeast of Rhodotorula glutinis by Zümriye Aksu; Ayşe Tuğba Eren (107-113).
The production of carotenoids by the strain of Rhodotorula glutinis, a red soil yeast isolated from IPRAŞ refinery wastewater, was investigated in a batch system as a function of initial pH, temperature, aeration rate, initial sugar (glucose, molasses sucrose and whey lactose) and ammonium sulphate concentrations and activator (cotton seed oil and Tween 80) addition. Optimum pH and temperature for total carotenoids production were determined as 6 and 30 °C, respectively. Total carotenoids concentration and carotenoids production yield significantly enhanced with increasing aeration rate up to 2.4 vvm. An initial ammonium sulphate concentration of 2 g l−1 gave the maximum carotenoids production. Only the cotton seed oil improved the carotenoids productivity of the yeast considerably at 5 g l−1 initial glucose concentration. In general, the increase in initial glucose and molasses sucrose concentrations extended the growth of yeast and total carotenoids production while increase in whey lactose concentration did not show the same effect. The highest carotenoid concentration (125.0 mg total carotenoids per liter of fermentation broth) was obtained when 20 g l−1 molasses sucrose was used as the carbon source while the highest product yield based on the maximum cell concentration (35.5 mg total carotenoids per gram of dry cells) was achieved when 13.2 g l−1 whey lactose was the carbon source in the broth.
Keywords: Rhodotorula glutinis; Carotenoids; Fermentation; Glucose; Molasses sucrose; Whey lactose;

The goal of the present paper is to propose a mathematical model, based on experimental data and suitable for description and prediction of the process of microbial synthesis of CGTase by immobilised bacterial cells.The model consists of a set of ordinary differential equations taking into account the microbial growth, the substrate inhibition, the decay of the microbial culture with time and the product degradation. The possible cell leakage and the resulting growth of a free culture are taken into account. The model is very sensitive towards the yield coefficients of bacterial growth and product formation. The model is suitable for estimation of the specific growth rates and the yield coefficients for free and immobilised cell growth.The model is used to evaluate kinetic constants from experimental data for the microbial production of CGTase by cells of the strain Bacillus circulans ATCC 21783 attached to poly-sulfonate membranes in a fluidized bed bioreactor operating in a batch mode.The attached cells showed higher yield than the free ones and the estimated leakage rates are pretty low. It was concluded therefore that the contribution of the immobilised cells for the CGTase formation in the considered case is dominant.
Keywords: Cyclodextrin-glucano-transferase; Enzyme production; Immobilised cells; Kinetic parameters; Modelling;

Studies were carried out on decolorization of semisolid olive mill residues called “alperujo” (AL), by four strains of Phanerochaete chrysosporium, Trametes versicolor, Pycnoporus cinnabarinus and Aspergillus niger in solid state frementation (SSF). Fungal strains were selected by their ability to grow on olive mill waste water. The treatment of AL with P. chrysosporium led to higher removal of organic matter and decolorization than P. cinnabarinus and T. versicolor. The strain A. niger leads to a relative improvement of the biodegradability of OMW. Using P. chrysosporium, the treatment of AL substrate in SSF showed an efficient decolorization and an appreciable COD and phenolic content removal only in the presence of a support. Sugarcane bagasse used as support improved the oxygen transfer in the culture. The extracellular fluid of P. chrysosporium (composed mainly of LiP) exhibited high ability to decolorize AL showing the efficiency of the enzyme produced. A percentage of sugarcane bagasse equal to 30% was an optimal condition to improve the growth and the decolorization of AL. The results showed good prospects of using the three basidiomycetes, in particular P. chrysosporium, for the Decolorization of AL.
Keywords: Alperujo; Decolorization; Phanerochaete chrysosporium; Respirometry; Solid state fermentation;

Kinetics of soluble and immobilized horseradish peroxidase-mediated oxidation of phenolic compounds by V. Vojinović; R.H. Carvalho; F. Lemos; J.M.S. Cabral; L.P. Fonseca; B.S. Ferreira (126-135).
Horseradish peroxidase (HRP) is an intensively used enzyme in biochemical and medical analyses. It is also proposed for wastewater processing. A bioprocess monitoring method based on co-oxidation of phenol-4-sulfonic acid (PSA) and 4-aminoantipyrine (4-AAP) by immobilized HRP was recently described. In this work a mathematical model that describes a set of reactions that occur in phenol/4-AAP/HRP system upon addition of hydrogen peroxide has been used for the evaluation of the PSA/4-AAP/HRP system by estimating and comparing the apparent kinetic parameters for different reaction systems. The parameters thus obtained were compared for three phenolic compounds (phenol, p-chlorophenol and PSA) and for immobilized HRP in the PSA/4-AAP system. The model was able to capture distinctive features of the system, such as increasing initial reaction rates for increasing concentrations of H2O2; a plateau upon reactant exhaustion; and decreasing initial reaction rates at high H2O2 concentrations. The performance of the PSA/4-AAP/HRP system expressed as the initial reaction rate was found to be less than 12% that of the conventional phenol/4-AAP/HRP system. The estimated parameters for the kinetic model provided the basis for the mechanistic explanation for the lower performance observed in the system containing PSA.
Keywords: Horseradish peroxidase; Kinetic parameters; Modelling; 4-Aminoantipyrine; Immobilization; Biosensors;

Optimization of xylanase production by Chaetomium thermophilum in wheat straw using response surface methodology by Petros Katapodis; Vassiliki Christakopoulou; Dimitris Kekos; Paul Christakopoulos (136-141).
A 32 central composite experimental design was performed with the aim of optimizing xylanase production by Chaetomium thermophilum IMI 291753 grown on wheat straw in submerged cultures. Various carbon and nitrogen sources were consecutively optimized, and wheat straw and sodium nitrate were the selected substrates to test the effect of two variables on xylanase production. A second-order quadratic model and a response surface method showed that the optimum conditions for xylanase production were 3.9% (w/v) wheat straw and 0.7% (w/v) sodium nitrate.
Keywords: Wheat straw; Response surface method; Chaetomium thermophilum; Xylanase;

Evaluation of composts as biofilter packing material for treatment of gaseous p-xylene by Jae Woong Hwang; Seok Jin Jang; Eun Yeol Lee; Cha Yong Choi; Sunghoon Park (142-149).
Composts are often employed as a packing material of a biofilter for treating gaseous streams contaminated with volatile organic compounds or malodorous chemicals. In order to select a good packing material for a biofilter, two commercially available composts in Korea, pig manure compost (PMC) and food waste compost (FWC), were studied during the treatment of gaseous p-xylene. The composts were used as purchased or after washed with water to remove water-soluble nutrients and microorganisms on the compost surface. Washed composts exhibited a shorter start-up period for a biofilter to reach a high and steady degradation level of p-xylene. Nitrogen limitation was experienced when a biofilter was operated without adding a supplemental nitrogen source. The soluble nitrogen content was initially higher in PMC than in FWC, but the latter had a higher content of insoluble nitrogen and a lower activity of the nitrification which converts NH3-N to NO3-N. The biofilter packed with FWC yielded a lower loss of nitrogen as leachate and exhibited a more stable performance for a longer period of 150 days with the maximal elimination capacity of 21 g  p-xylene m−3  h−1.
Keywords: Biofiltration; Compost; Nitrogen limitation; Nitrification activity; p-Xylene;

The thermodynamic deactivation kinetics, ΔH *, ΔS *, E and ΔG * were used to understand the stability behaviour of the novel chitinases (I–III) of the marine isolate Pantoea dispersa in a range of salts and varying pH conditions (pH 4–9). The deactivation rate decreased with increasing half-life of chitinase I–III in the presence of salts at 30–60 °C with the following effect on the order of stability evident NaCl > KH2PO4  > KBr > NH4NO3  > KNO3  > MgSO4·7H2O > CaCl2. With increasing pH the deactivation rate decreased whilst the half-life of chitinase I–III increased. This lead to increased stability at high pH's with the following order of stability evident 9 ≥ 8 > 7 > 6 > 5 > 4 at 30–60 °C. With increasing pH's and addition of each salt ΔH * of chitinase I–III increased whilst deactivation energy decreased. Furthermore, ΔS * was found to be comparatively lower than untreated chitinase I–III. The free energy of chitinase I–III decreased with increasing pH and different salts in the same order as described above. The greater stability of chitinase I (t 1/2: 270, 225, 185 and 135 min), chitinase II (t 1/2: 225, 210, 195 and 135 min) and chitinase III (t 1/2: 210, 150, 120 and 105 min) at 30–60 °C respectively were observed at pH 8. With the addition of NaCl, the following stabilities of chitinase I (t 1/2: 270, 225, 185 and 135), chitinase II (t 1/2: 225, 210, 195 and 135) and chitinase III (t 1/2: 210, 150, 120 and 105) at 30–60 °C respectively were observed which is greater than the chitinase of Trichoderma harzianum a recognised commercial biocontrol agent.
Keywords: Chitinase; Pantoea dispersa; pH; Salts; Stability; Thermodynamic; Temperature;

Polypeptide linkers suitable for the efficient production of dimeric scFv in Escherichia coli by Yoichi Kumada; Tomomi Kawasaki; Yasufumi Kikuchi; Shigeo Katoh (158-165).
Single chain variable fragment antibodies (scFvs) were selected from a phage library displaying scFvs consisting of the VH and VL domains of an anti-bisphenol A monoclonal antibody and 20 randomized amino acids linkers by biopanning using an antigen bisphenol A. After four rounds of biopanning selections, seven scFvs with different polypeptide linkers were isolated. The scFvs expressed by the Escherichia coli transformants predominantly formed dimeric structures, and were found in soluble fractions at higher concentrations than those of scFvs containing flexible linkers (G4S)1–3, because the transformants expressing these scFvs could grow to higher cell concentration after the induction of scFv production with IPTG. Consequently, five times higher productivities of soluble scFvs were attained compared with scFvs having the flexible linkers. The antigen binding activities of these scFvs were confirmed by affinity chromatography using an antigen-coupled column and ELISA. The selected polypeptide linkers will be useful for the production of soluble scFv dimers in E. coli.
Keywords: ScFv; Polypeptide linker; Phage display; Dimeric scFv;

The combination of non-mechanical (chemical) and mechanical methods such as high pressure homogenisation (HPH) can increase the release of intracellular components and decrease the exposure to mechanical disruption required for the breakage to attain maximum intracellular release. Through these, the energy requirement of microbial cell disruption can be decreased along with the reduction in the micronisation of cell debris. Pretreatments to permeabilise or weaken the cell envelope were selected and the optimum conditions determined through a screening process. The permeabilisation of Escherichia coli with EDTA was successful in achieving maximum intracellular protein release at a lower pressure of 13.8 MPa on high pressure homogenisation, compared with 34.5 MPa in the absence of EDTA. Significant reduction in energy input required was observed with the use of this combination method. Pretreatment with guanidium hydrochloride (G-HCl) and Triton X-100 also resulted in increased intracellular release and decreased energy usage. Chemical pretreatment can be useful in enhancing mechanical disruption, however, careful selection of pretreatment conditions is required to avoid protein deactivation and chemical interference in the protein assay.
Keywords: Cell disruption; Downstream processing; Protein recovery; Chemical pretreatment; EDTA; High pressure homogenisation;

Aerobic granules with a mean size of 1 mm were used as biosorbent to remove soluble nickel ion from aqueous solution. Investigation of the mechanisms of Ni2+ biosorption by aerobic granules showed that Ni2+ biosorption was associated with a significant release of Ca2+ ion, e.g., 1 meq of Ni2+ adsorbed would result in a release of 0.68 meq of Ca2+. Compared to fresh aerobic granules, analysis by powder X-ray diffraction (XRD) further revealed that no new crystal was formed in the aerobic granules after Ni2+ biosorption. These seem to imply that a chemical precipitation mechanism would not be largely involved in the Ni2+ biosorption by aerobic granules. The contribution of extracellular polymeric substances (EPS) to the Ni2+ biosorption by aerobic granules was also examined. It was found that the EPS-associated Ni2+ biosorption by aerobic granules only accounted for about 14.2% of the total Ni2+ removed. The biosorption isotherm equation previously derived from a thermodynamic principle was applied to this study and it can provide a satisfactory description for Ni2+ biosorption by aerobic granules at various temperatures. It was found that the equilibrium constants of the Ni2+ biosorption increased with increase of temperature from 25 to 55 °C, i.e., the biosorption of Ni2+ by aerobic granules is favored at high temperature. The thermodynamic parameters (ΔG°, ΔH° and ΔS°) were also determined, and it was found that the Ni2+ biosorption by aerobic granules was an endothermic process. Meanwhile, the energy dispersive X-ray (EDX) analysis indicated that Ni2+ ion could penetrate into the core of the aerobic granule, and the distribution of the adsorbed Ni2+ in the aerobic granule seemed uniform.
Keywords: Biosorption; Aerobic granule; Equilibrium isotherm; Nickel; Thermodynamics; Ion exchange; EPS; Precipitation;

A new and simple voltammetric assay method for DNA hybridization events using a disposable DNA sensor was developed. The DNA sensor was prepared by self-assembling horseradish peroxidase (HRP)-linked single-stranded DNA (HRP-ssDNA) onto gold nanoparticles-modified composite membrane at a carbon paste electrode (CPE). With a non-competitive format, the injected sample containing the complementary DNA sequence (cDNA) was produced transparent hybridization reaction with the immobilized HRP-ssDNA. The formed double strand DNA (dsDNA) inhibited partly the active center of HRP, and decreased the immobilized HRP to H2O2 reduction. The fabrication procedure of the DNA sensor was characterized by using scanning electron microscopy (SEM) and cyclic voltammetry (CV). The performance and factors influencing the performance of the DNA sensor were investigated. The hybridization reaction between the probe and its complementary sequence as the target was measured by differential pulse voltammetry (DPV). Under optimal conditions, the current change obtained from the binding HRP relative to H2O2 system was proportional to the cDNA concentration in the range of 1.5 × 10−10 to 9.5 × 10−9  M with a detection limit of 5.0 × 10−11  M (at 3δ). Tests relating to the detection of cDNA demonstrated that the developed DNA sensor exhibited short assay time, high sensitivity, acceptable reproducibility and long-term stability.
Keywords: DNA biosensor; Enzyme; Gold nanoparticles; Voltammetric detection;

Dibenzothiophene biodesulfurization in resting cell conditions by aerobic bacteria by Ainhoa Caro; Karina Boltes; Pedro Letón; Eloy García-Calvo (191-197).
Rhodococcus erythropolis IGTS8 and the genetically modified strain Pseudomonas putida CECT 5279 are effectively capable to carry out the oxidative non-destructive desulfurization of dibenzothiophene (DBT), called 4S pathway, so that sulfur is released after four enzymatic steps, without carbon skeleton breakdown. Biodesulfurization (BDS) process yields were studied both in aqueous and biphasic media, with hexadecane as model oil. All batch assays were carried out at Erlenmeyer flasks scale, under different experimental conditions such as oil fraction phase percentages, substrate concentrations and cellular densities, using resting cell as operation mode. Providing that cell densities were not too high, both biocatalysts achieved better DBT conversion with higher biomass concentrations and lower oil fractions and DBT concentrations. Comparing aqueous and biphasic media it has been also proved that P. putida CECT 5279 strain was more sensitive for DBT mass transfer limitation, determining the preferable use in biphasic systems of IGTS8 to practical BDS process as well. Furthermore, the experimental results suggested there could be inhibitions effects by product accumulation in aqueous media, but these effects were not so clear in biphasic condition, because 2-hydroxybiphenyl (HBP) oil–water partition coefficient is very high.
Keywords: Biodesulfurization; Dibenzothiophene; Resting cell; Mass transfer; Pseudomonas sp. CECT 5279; Rhodococcus sp. IGTS8;

Organic solvents effect on the secondary structure of araujiain hI, in different media by Evelina Quiroga; Gerardo Camí; José Marchese; Sonia Barberis (198-202).
The aim of this work was to carry out a comparative study of the secondary structure of araujiain hI (cystein plant protease) in aqueous–organic media (N,N-dimethylformamide (50%) and hexane (50%)) and in Tris–HCl buffer (0.1 M, pH 8), using FT-IR spectroscopy. The changes in the enzyme structure were correlated with the changes in the preferences and enzyme activities in such media. The enzyme activity observed in the organic media was higher than those obtained in buffer. Although, the substrate preferences of araujiain hI exhibit different patterns in each medium, the enzyme always showed a high preference for glutamine derivative. According to the study of the secondary structure, it was possible to conclude that a secondary structure with a high α-helical character was responsible for the highest activity of araujiain hI in the studied organic media. Although, the correlation between enzyme structure and catalytic activity requires direct measurement of active-site structure and of the effect of the reaction medium on the transition state of the reaction, it is clear that the non-covalent forces which maintain the native secondary structure of enzyme were modified when araujiain hI was suspended in such aqueous–organic media.
Keywords: Cystein plant proteases; Araujiain hI; Secondary structure; Aqueous–organic media; Enzyme activity; FT-IR spectroscopy;

The ability of cell-bound enzyme of Aspergillus flavus Link 44-1 for production of kojic acid was studied in resuspended cell system. Cell material was produced in batch fermentation using 2 L stirred tank fermenter. The cell mycelia were then resuspended into 250 mL shake flask containing various carbon sources solution. Among the carbon sources tested, glucose gave the highest kojic acid yield based on carbon consumed (0.365 g/g) followed by sucrose (0.279 g/g), starch hydrolysate (0.212 g/g) and fructose (0.195 g/g). The rate of biotransformation was increased with increasing mycelial cell. Kojic acid production was also varied with different glucose and sucrose concentrations. The highest production was obtained at 100 g/L glucose and 100 g/L sucrose with a final kojic acid concentration of 45.3 and 33.4 g/L, respectively. The rate of biotransformation of glucose and sucrose to kojic acid followed the Michaelis–Menten equation, suggesting that the biotransformation rate vary with substrate concentration similar to the behaviour of many enzymes reaction.
Keywords: Kojic acid; Biotransformation; Cell-bound enzyme; Resuspended cell system; Aspergillus flavus;

In this work, batch and fed-batch cultivations of a recombinant Escherichia coli (E. coli), which expressed Interleukin-11 (IL-11) as a fusion protein, were performed in a 12 L bioreactor. Time profiles of total RNA, total protein, rhIL-11 protein, and cell and glucose concentrations were obtained and the results were simulated with a three-compartment model designed previously, a new empirical model, and a new four-compartment model. Numerical simultaneous integration of the model equations was done using a fourth order Runga-Kutta method. Although the three-compartment model was able to fit most of the batch fermentation results reasonable well, it failed to depict cell growth delay caused by induction when induction effects were serious. With only 7 model parameters, the newly developed empirical model was able to fit both pre-induction processes and post-induction ones with reasonable accuracy. It was therefore used to model both batch and fed-batch fermentation results where growth inhibition caused by induction was pronounced. The four-compartment model was constructed to characterize induction effects in a profound way. Based on the simulation results, this model exhibited similar performance to that of the empirical model in fitting the fermentation data. In addition it was successfully used for providing guidance to the determination of optimal induction time.
Keywords: Structured modeling; Compartment model; Fermentation; Fed-batch; Interleukin-11;

A kinetic study of reuterin production by Lactobacillus reuteri PRO 137 in resting cells by M. Tobajas; A.F. Mohedano; J.A. Casas; J.J. Rodríguez (218-225).
This work deals with the production of reuterin by Lactobacillus reuteri PRO 137 in a two-step batch fermentation process. First, L. reuteri cells were grown anaerobically in a glucose-containing media, and in a second stage reuterin was produced by the starved resting cells in glycerol-containing media. In order to maximize biomass production, the influence of temperature (31–43 °C) and pH (5.0–6.5) was evaluated. A simple model has been proposed to describe the microbial growth and substrate consumption. The values of the kinetic constant for growth rate, yield of biomass on substrate and maximum biomass concentration indicate that the optimum operating conditions were 40 °C and pH 5.5. Biomass produced in those growth conditions was harvested, washed and resuspended in a medium containing glycerol as the only carbon source for reuterin production by resting cells. Reuterin production was evaluated for several biomass (2 and 5 CDW g/L) and glycerol (150–300 mM) concentrations. A model describing the kinetics of the production process has been proposed. The model allows to describe simultaneously the concentrations of viable cells, glycerol, reuterin and 1,3-propanediol. A multi-response nonlinear parameter estimation procedure using the Marquardt algorithm and numerical integration of the differential equations by Runge-Kutta method was used to obtain the values of the fitting parameters.
Keywords: Batch processing; Lactobacillus reuteri; Microbial growth; Modelling; Production kinetics; Reuterin;

Optimization of pre-treated palm oil mill effluent digestion in an up-flow anaerobic sludge fixed film bioreactor: A comparative study by A.A.L. Zinatizadeh; A.R. Mohamed; M.D. Mashitah; A.Z. Abdullah; M. Hasnain Isa (226-237).
An up-flow anaerobic sludge fixed film (UASFF) bioreactor was used to treat physically and chemically pre-treated palm oil mill effluent (POME) under different operating conditions. In physical pre-treatment, POME was pre-settled for 2 h and the supernatant was fed to the reactor. In chemical pre-treatment, optimum dosages of a cationic and an anionic polymer were used. Experiments of pre-treated POME digestion were conducted based on a central composite face-centered design (CCFD) with two independent operating variables, feed flow rate (Q F) and up-flow velocity (V up). The operating variables were varied to cover a wide range of organic loading rates (OLR) from 3.8 to 29 g COD/(l d). Six dependent parameters were either directly measured or calculated as response. These parameters were total COD (TCOD) removal, effluent pH, effluent total volatile fatty acid (TVFA), effluent bicarbonate alkalinity (BA), methane yield (Y M), and solids retention time (SRT). The performance of the reactor was compared for the pre-settled and chemically pre-treated POME. The chemical pre-treatment approach was shown to be more predictable, reliable and practical as the sludge produced was very compressible and was easy to separate. At a comparable range of Q F and V up, the pre-settled POME yielded slightly better reactor performance in terms of COD removal (%), bicarbonate alkalinity and methane yield. At an OLR of about 16.5 g COD/(l d), higher COD removal efficiency (90–94%) was achieved compared to that of the chemically pre-treated POME (82–88%) despite about 33% of organics in the pre-settled POME was contributed by suspended solids. The optimum conditions for digestion of the pre-settled and chemically pre-treated POME were determined as Q F of 1.65 l/d, V up of 0.6 and Q F of 2.45 l/d, V up of 0.75, respectively. The experimental findings were in close agreement with the model prediction.
Keywords: POME; Pre-treatment; UASFF bioreactor; Central composite face-centered design (CCFD);

Cerein 8A production in soybean protein using response surface methodology by A.P.M. Dominguez; D. Bizani; F. Cladera-Olivera; A. Brandelli (238-243).
In this work a 23 factorial design was performed with the aim of optimizing the bacteriocin (cerein 8A) production by Bacillus cereus 8A, using response surface methodology (RSM). The bacteriocin production was first tested on different substrates, like soybean meal, soybean protein, fish meal, industrial fibrous soybean residue and cheese whey. Soybean protein was the selected medium to test the effect of three variables (temperature, initial pH and soybean protein concentration) by RSM. Statistical analysis of results showed that, in the range studied, the three variables have a significant effect on cerein 8A production. Response-surface data showed maximum bacteriocin production at initial pH between 6.5 and 9.0, temperature between 22 and 34 °C and soybean protein concentration higher than 20 g l−1. It was not possible to determine a relation between bacteriocin production and optimal conditions for bacterial growth in terms of pH and temperature, although increased bacteriocin yields and bacterial growth were obtained with higher soybean protein concentration.
Keywords: Aerobic processes; Bacteriocin; Experimental design; Modeling; Soybean protein; Submerged culture;

SMHP (strontium magnesium hydrogen phosphate) crystals were grown in silica gel medium in three growth environments using different gel densities, various concentrations of phosphoric acid and supernatant solutions. The gel pH plays an important role in the formation of different HPO4 species in the phosphoric system. The pH ranges in which HPO4 2− ion dominate were considered, which in turn is necessary for the growth of SMHP crystals. The SMHP crystals are grown in three different growth environments by applying various growth parameters and found the optimum growth environment. The characterization of grown crystals were studied by FTIR, TGA/DTA, SEM, XRD and etching.
Keywords: SMHP; Laser light; Calculi; Surface morphology; Growth parameters; Trace elements and isotopes;