Coupled lactic acid fermentation and adsorption

Polyvinylpyridine (PVP) and activated carbon were evaluated for coupled lactic acid fermentation and adsorption, to prevent the product concentration from reaching inhibitory levels. The lactic acid production doubled as a result of periodical circulation of the fermentation broth through a PVP adsorption column. The adsorbent was then regenerated and the adsorbed lactate harvested, by passing 0.1 N NaOH through the column. However, each adsorption-regeneration cycle caused about 14% loss of the adsorption capacity, thus limiting the practical use of this rather expensive adsorbent. Activated carbon was found much more effective than PVP in lactic acid and lactate adsorption. The cells of Lactobacillus delbrueckii subsp. delbrueckii (LDD) also had strong tendency to adsorb on the carbon. A study was therefore conducted using an activated carbon column for simultaneous cell immobilization and lactate adsorption, in a semi-batch process with periodical medium replacement. The process produced lactate steadily at about 1.3 g l(-1)h(-1) when the replacement medium contained at least 2 g l(-1) of yeast extract. The production, however, stopped after switching to a medium without yeast extract. Active lactic acid production by LDD appeared to require yeast extract above a certain critical level (<2 g l(-1)).     

Comparison of different carriers for adsorption of Saccharomyces cerevisiae and Zymomonas mobilis

Spruce sawdust, activated carbon, crushed brick, crushed firebrick and polystyrene have been tested as carriers for adsorption of Saccharomyces cerevisiae and Zymomonas mobilis cells. The isotope technique was used for the determination of the amount of adsorbed cells. The electrochemical nature of the cell surfaces, as well as zeta potentials, specific surface area and average porosity of the carriers were determined. The adsorption of S. cerevisiae and Z. mobilis on the carriers was quite different. The variance in immobilization capacities of the carriers could not be explained by differences in their internal surface areas and porosities. There was a clear tendency to decreasing capacities, with more negative zeta potentials with Z. mobilis. With S. cerevisiae there was no corresponding correlation between immobilization capacity and the carrier's zeta potential. Ethanol fermentation was used as a model to examine the activity, hydrodynamic behaviour and stability of the cell-carrier systems.     

In situ magnetic separation and immobilization of dibenzothiophene-desulfurizing bacteria

In situ cell separation and immobilization of bacterial cells for biodesulfurization were developed by using superparamagnetic Fe₃O₄ nanoparticles (NPs). The Fe₃O₄ NPs were synthesized by coprecipitation followed by modification with ammonium oleate. The surface-modified NPs were monodispersed and the particle size was about 13 nm with 50.8 emu/g saturation magnetization. After adding the magnetic fluids to the culture broth, Rhodococcus erythropolis LSSE8-1 cells were immobilized by adsorption and then separated with an externally magnetic field. The maximum amount of cell mass adsorbed was about 530 g dry cell weight/g particles to LSSE8-1 cells. Analysis showed that the nanoparticles were strongly absorbed to the surface and coated the cells. Compared to free cells, the coated cells not only had the same desulfurizing activity but could also be easily separated from fermentation broth by magnetic force. Based on the adsorption isotherms and Zeta potential analysis, it was believed that oleate-modified Fe₃O₄ NPs adsorbed bacterial cells mainly because of the nano-size effect and hydrophobic interaction.     

Non-porous magnetic supports for cell immobilization

A new method for covering magnetic particles with a stable non-porous layer of a material like zeolite or activated carbon was used for the preparation of support materials with good properties for the immobilization of yeast Saccharomyces cerevisiae cells. The immobilized cells can be used in batch and continuous alcoholic fermentation. A productivity of 35.6 g ethanol/l · h was reached. The adsorption isotherms of the immobilized yeast cells were determined. Yeast cell immobilization on non-porous magnetic supports obeyed the Langmuir isotherm equation. Satisfactory results were obtained also from repeated batch fermentations with fixed cells on supports additionally treated with glutaraldehyde or by simple adsorption.     

Versatility of glutaraldehyde to immobilize lipases: Effect of the immobilization protocol on the properties of lipase B from Candida antarctica

Glutaraldehyde chemistry has been used to immobilize lipase B from Candida antarctica (CALB) under different situations. Using high ionic strength, ionic adsorption is avoided, but CALB is adsorbed on the support via interfacial activation. Using non-ionic detergents (e.g., Triton X-100), the enzyme becomes ionically adsorbed on the activated support. If detergent and salt are simultaneously present during immobilization, a covalent attachment to the support is first produced. In absence of detergent or high ionic strength, a mixture of all of the previous immobilization reasons should coexist. Thus, 5 different CALB biocatalysts were prepared following the previous described protocols, and its stability and activity, pH/activity profile and specificity versus R and S methyl mandelate were analyzed. The existence of covalent attachment of more than 95% of the enzyme molecules was confirmed by washing the biocatalysts in salt and detergent solutions. The glutaraldehyde treatment of the enzyme adsorbed on aminated supports did not produce a significant improvement on the activity of the enzyme versus p-nitrophenylpropinate (pNPB) nor a high stabilization of the enzyme. This differed from the effects of a similar treatment of CAL adsorbed on octyl agarose. However, they were similar to the effects of this treatment on covalently immobilized CALB, suggesting that the immobilization protocol may greatly affect the final effect of a chemical modification on the enzyme properties.Dramatic changes in the enzyme features were observed comparing the different preparations, mainly in the specificity of CALB versus p-NPB and R-methyl mandelate (from 2.5 to 20), or in the enantiospecificity versus R/S methyl mandelate (from 1.8 to 16), confirming that these different immobilization protocols produced biocatalysts with different features. Moreover, changes in experimental conditions produced very different effects on the properties of the different CALB preparations.     

Adhesion of yeast cells to different porous supports, stability of cell-carrier systems and formation of volatile by-products

The aim of our research was to study how the conditions of immobilization influence cell attachment to two different ceramic surfaces: hydroxylapatite and chamotte tablets. Three fermentative yeast strains, namely brewery TT, B4 (ale, lager) and distillery Bc15a strains belonging to Saccharomyces spp., and one strain of Debaryomyces occidentalis Y500/5 of weak fermentative nature, but with high amylolytic activity due to extracellular ??-amylase and glucoamylase, were used in this study. Different media, including cell starvation, were applied for immobilization of yeast strains as well as different phases of cell growth. Immobilization of selected yeasts on a hydroxylapatite carrier was rather weak. However, when incubation of starved yeast cells was conducted in the minimal medium supplemented by calcium carbonate, the scale of immobilization after 24?h was higher, especially for the D. occidentalis strain. Adhesion to hydroxylapatite carriers in wort broth was of reversible character and better results of adhesion were observed in the case of another ceramic carrier-chamotte. The number of immobilized cells was about 10⁶?C10⁷ per tablet and cell adhesion was stable during the whole fermentation process. The comparison of the volatile products that were formed during fermentation did not show any significant qualitative and quantitative differences between the free and the immobilized cells. This is the first time when a cheap, porous chamotte surface has been applied to yeast adhesion and fermentation processes.     

Elucidation of optimum conditions for immobilization of viable cells by using calcium alginate

Different factors which affect the stability of calcium alginate gel beads entrapping viable cells during fermentation were investigated. It was found that among others, the initial population of cells per ml of gel beads, the length of period of incubation in CaCl₂ solution, and the concentration of sodium alginate used for the immobilization were the most important factors affecting the stability of the gel beads during fermentation. By using an initial cell population of about 10⁵ cells per ml of 2.0% sodium alginate, and incubating the beads for at least 22 h in a CaCl₂ solution after immobilization, the percentage of beads which developed cracks during fermentation was highly reduced. Also, without the addition of CaCl₂ into the fermenting broth, the gel beads were stable for nine consecutive batch fermentations.     

Production of butanol from glucose and xylose with immobilized cells of Clostridium acetobutylicum

Pretreated cotton towels were used as carriers to immobilize Clostridium acetobutylicum CGMCC 5234 cells for butanol or ABE production from glucose and xylose. Results showed that cell immobilization was a promising method to increase butanol concentration, yield and productivity regardless of the sugar sources compared with cell suspension. In this study, a high butanol concentration of 10.02 g/L with a yield of 0.20 g/g was obtained from 60 g/L xylose with 9.9 g/L residual xylose using immobilized cells compared with 8.48 g/L butanol and a yield of 0.141 g/g with 20.2 g/L residual xylose from 60 g/L xylose using suspended cells. In mixed-sugar fermentation (30 g/L glucose plus 30 g/L xylose), the immobilized cultures produced 11.1 g/L butanol with a yield of 0.190 g/g, which were 28.3% higher than with suspended cells (8.65 g/L) during which 30 g/L glucose was utilized completely using both immobilized and suspended cells while 3.46 and 13.1 g/L xylose maintained untilized for immobilized and suspended cells, respectively. Based on the results, we speculated that immobilized cells showed enhanced tolerance to butanol toxicity and the cultures preferred glucose to xylose during ABE fermentation. Moreover, the cultures showed obvious difference when grown between high initial concentrations of glucose and those of xylose. Repeated-batch fermentations from glucose with immobilized cells showed better long-term stability than from xylose. At last, the morphologies of free and immobilized cells adsorbed on pretreated cotton towels during the growth cycle were examined by SEM.     

Fibroblast cell behavior on bound and adsorbed fibronectin onto hyaluronan and sulfated hyaluronan substrates

The effect of fibronectin protein (Fn) coating onto polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) on fibroblast cell adhesion was analyzed. The Hyal or HyalS were coated and grafted on the glass substrate by a photolithographic method. The Fn coating was achieved by two different routes: the immobilization of Fn by covalent bond to the polysaccharide layers and the simple adsorption of Fn onto Hyal and HyalS surfaces. AFM, SEM, and ATR-FTIR techniques were used for the chemical and topographical characterization of the surfaces. According to AFM and SEM data, the surface topography was dependent on the method used to cover the polysaccharide layers with the protein. ATR-FTIR analysis supplied information about the rearrangement of Fn after the interaction (adsorption or binding) with the Hyal and the HyalS. The conformational changes of the Fn were minimal when it was simply adsorbed on HyalS surfaces and larger once bound, whereas on the Hyal layer the protein underwent a bigger conformational change once adsorbed and covalently grafted. Then, the biological characterization was carried out by analyzing the human diploid skin fibroblasts adhesion on these surfaces. The morphology of fibroblasts was evaluated by SEM, whereas the dynamics of fibroblasts movement were recorded by a time-lapse system. Cell variations in area, perimeter, and length were analyzed at 2, 4, and 6 h. It was found that the addition of Fn (covalently bound or merely adsorbed) was fundamental in the promotion of fibroblasts adhesion and spreading. The greatest adhesion occurred onto HyalS layers covered by the adsorbed Fn.     

The Interactions Between Monovalent Cations and Calcium During Their Adsorption on Isolated Cell Walls and Absorption by Intact Barley Roots

The adsorption of sodium, potassium, rubidium and calcium ionsat different concentrations was measured on cell walls frombarley roots (Hordeum vulgare L. cv. Union), prepared by detergenttreatment. It was found that sodium and calcium interacted verystrongly during their simultaneous adsorption, whereas potassiumdid not interfere with calcium. This has led us to concludethat calcium and sodium are adsorbed on identical sites in thecell wall, whereas potassium is adsorbed at another site. Rubidiumseems to be less specific for both sites and interferes onlymoderately with calcium. The adsorption on cell walls of thesecations was compared with their adsorption on intact roots at2 °C, where beside the cell wall, sites may be availableat the outer surface of the membrane, and further measurementswere made of absorption at 25 °C. The fact that sodium interactswith calcium and potassium alters the ratio of K to Na in thecell wall compared to their concentrations in the medium. Thepreferential shift towards potassium when calcium is presentcould be very important for the rates of initial uptake in lowsalt barley roots, since the membrane is in contact with a differentproportion of K to Na in the cell wall from the one suppliedin the medium. Hordeum vulgare L., barley, absorption of cations, adsorption, calcium, sodium, potassium rubidium     

Could the improvements in the alcoholic fermentation of high glucose concentrations by yeast immobilization be explained by media supplementation?

Summary The maximal concentration of ethanol produced during the fermentation of 320 g/l glucose bySaccharomyces bayanus was higher when the yeast cells were immobilized either by adsorption on celite or by entrapment in k-carrageenan beads (from 10.5% with free cells up to 14.5% and 13.1% (v/v) respectively). This increase was due to medium supplementation with the compounds present in the immobilization supports.     

Cell immobilization: Application to alcohol production

The immobilization of whole cells is a technique that can be used in several production processes, among them alcoholic fermentation. By concentrating an active cell biomass in a bioreactor, the efficiency of bioconversion increases, as does the reactor productivity which, in turn, results in the reduction of the reactor size for a given production rate. Immobilization can be carried out in different ways; adsorption and entrapment in gelatinous matrices are the methods most commonly used. These techniques can be applied to practically all of the viable and non-viable whole cells systems of interest. Several reactor configurations are used sucessfully; many more have been proposed. Fermentor design imposes limitations on immobilization processes, requiring study of such aspects as mass-transfer, growth of cells in the reactor, aeration and backmixing.     

产糖化酶黑曲霉的固定化研究

采用多孔聚酯材料作为固定化载体,考察并比较了载体吸附固定化黑曲霉菌丝细胞的条件,当菌丝体细胞与载体预培养的条件为pH值5.0、孢子浓度为105个/ml、固液比为1/75时,有利于菌丝体的生长、吸附固定及发酵产酶.在产糖化酶的发酵过程中,与游离菌丝体细胞相比,发酵过程持续产酶时间有一定程度的延长,产糖化酶活力始终高于游离菌丝体.     

Characterization of production of free gluconic acid by Gluconobacter suboxydans adsorbed on ceramic honeycomb monolith

Gluconobacter suboxydans IFO 3290 was immobilized by adsorption on ceramic honeycomb monolith and continuous production of free gluconic acid from glucose was performed in an aerated reactor. The effects of reactor residence time, aeration rate, and glucose concentration were investigated on the gluconic acid yield. Observation of SEM photographs revealed that the cells were adsorbed with a high density not only on the outer surface of the support but also on the inner surface of large pores. From measurement of the number of the adsorbed cells, it was elucidated that the biofilm comprised a monolayer or bilayer of the cells. Maximum specific rate of growth was estimated for the free and adsorbed cells, and the adsorbed cells were found to grow at a fast rate compared with the free cells. In the continuous fermentation performed for one month at the glucose concentration of 100 kg/m(3), reactor residence time of 3.5 h and aeration rate of 900 cm(3)/min, the activity of the adsorbed cells was appreciably stable. The high productivity of 26.3 kg/(m(3)-reactor . h) was attained with the gluconic acid yield of 84.6% and glucose conversion of 94%.     

Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation

At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu²⁺ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu²⁺ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu²⁺ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress.     

Immobilization of Cells for Application in the Food Industry

Abstract

Immobilization of cells offers advantages to the food process industries, including enhanced fermentation productivity and cell stability and reduced downstream processing costs due to facilitated cell recovery and recycle. This article summarizes the varied immobilization methodologies, including adsorption, entrapment, covalent binding, and microencapsulation. Examples of interest to the food industry are provided, together with a review of the physiological effects of immobilization. Topics in process engineering include immobilized cell bioreactor configurations and the scale-up potential of the various immobilization techniques.     

Lactate production in an integrated process configuration: reducing cell adsorption by shielding of adsorbent

The problem of binding of microbial cells to an adsorbent matrix during in situ recovery of bioproducts from a fermentation broth has been addressed by shielding the adsorbent with a thin layer of a non-ionic polymer. Extractive bioconversion of lactic acid by integrating ion-exchange adsorption with the fermentation stage was studied. The effect of coating of the ion-exchanger with agarose on product recovery and cell adsorption was evaluated. Extractive fermentation with both uncoated and coated resin resulted in an increase in reactor productivity as compared to the normal fermentation. The free cell density in the system with agarose-coated beads was similar to that in control fermentation, but was significantly lower in the system with the uncoated ion-exchanger. Electron microscopic scanning of the bead surface after passage of the fermentation broth showed cells attached to the native adsorbent but not to the coated one.     

Receptor elements for biosensors in two ways of methylotrophic yeast immobilization

Receptor elements for biosensors based on Hansenula polymorpha NCYC 495 ln yeast cells for ethanol assay were developed using two ways of cell immobilization, i.e., physical adsorption on a glass fiber membrane and covalent binding on a modified nitrocellulose membrane. The linear diapason of ethanol assays for a biosensor based on yeast cells adsorbed on glass fiber was 0.05–1.18; for a biosensor based on yeasts immobilized on a nitrocellulose membrane, 0.2–1.53 mM. Receptor elements based on sorbed cells possessed 2.5 times higher long-term stability. The time response was 1.5 times less for cells immobilized using DEAE-dextran and benzoquinone. The results of ethyl alcohol assays using biosensors based on cells immobilized via adsorption and covalent binding, as well as using the standard areometric method, had high correlation coefficients (0.998 and 0.997, respectively, for the two ways of immobilization). The results indicate the possibility to consider the described models of receptor elements for biosensors as prototypes for experimental samples for practical use.     

新的分离纯化青霉素酰化酶方法的研究

按0．6％(w／v)的比例将皂土加到青霉索酰化酶发酵上清液中,可将酶100％吸附,而吸附的蛋白质仅占发酵上清液中的10％左右。吸附时的pH和无机盐对酶的吸附影响不大。使用不同pH和种类的缓冲液洗涤皂土－酶复合物,不能将酶洗脱,但可洗脱15％左右吸附的杂蛋白。使用含10％以上的PEG和NaCl的磷酸缓冲液可将酶全部洗脱．酶纯化25倍,浓缩6倍左右。此法特点是简便,酶活力收率高,可在常温下操作,也可直接从未除菌体的发酵液中提取酶,具有工业应用价值。     

Growth Kinetics of Thiobacillus thiooxidans on the Surface of Elemental Sulfur

The growth kinetics of Thiobacillus thiooxidans on elemental sulfur in batch cultures at 30(deg)C and pH 1.5 was studied by measuring the time courses of the concentration of adsorbed cells on sulfur, the concentration of free cells suspended in liquid medium, and the amount of sulfur oxidized. As the elemental sulfur was oxidized to sulfate ions, the surface concentration of adsorbed cells per unit mass of sulfur approached a maximum value (maximum adsorption capacity of sulfur particles) whereas the concentration of free cells continued to increase with time. There was a close relationship between the concentrations of free and adsorbed cells during the microbial sulfur oxidation, and the two cell concentrations were well correlated by the Langmuir isotherm with adsorption equilibrium constant K(infA) and maximum adsorption capacity X(infAm) of 2.10 x 10(sup-9) ml per cell and 4.57 x 10(sup10) cells per g, respectively. The total concentration of free and adsorbed cells increased in parallel with the amount of sulfate formed. The total growth on elemental sulfur gave a characteristic growth curve in which a linear-growth phase followed the period of an initial exponential phase. The batch rate data collected under a wide variety of inoculum levels (about 10(sup5) to 10(sup8) cells per ml) were consistent with a kinetic model assuming that the growth rate of adsorbed bacteria is proportional to the product of the concentration, X(infA), of adsorbed cells and the fraction, (theta)(infV), of adsorption sites unoccupied by cells. The kinetic and stoichiometric parameters appearing in the model were estimated from the experimental data, and the specific growth rate, (mu)(infA), and growth yield, Y(infA), were 2.58 day(sup-1) and 2.05 x 10(sup11) cells per g, respectively. The proposed model and the parameter values allowed us to predict quantitatively the surface attachment of T. thiooxidans cells on elemental sulfur and the bacterial growth in both initial exponential and subsequent linear phases. The transition from exponential to linear growth was a result of two competing factors: an increase in the adsorbed-cell concentration, X(infA), permitted a decrease in the unoccupied-site fraction, (theta)(infV).