Alginate-immobilized Cells Research Articles

Deep Eutectic Solvent Pretreatment of Water Hyacinth for Improved Holocellulosic Saccharification and Fermentative Co-Production of Xylitol and Lipids Using Rhodosporidium toruloides NCIM 3547

In this study, delignification of water hyacinth (WH) using a mild ionic liquid-like chemical deep eutectic solvent (DES) synthesized using choline chloride and urea was conducted and the process parameters were optimized by Box–Behnken design (BBD)-based response surface methodology (RSM). From the results, a delignification of 64.32 ± 4.08% (w/w) was obtained under 1:12.5 (biomass:DES ratio), 4.63 h (time) and 87 °C (temperature). Further, a dilute sulphuric acid (2%, v/v) hydrolysis was carried out to destabilize the hemicellulose that resulted in 23.7 ± 0.50 g/L of xylose. Fermentation of the obtained xylose was carried out using a red oleaginous yeast, Rhodosporidium toruloides NCIM 3547, with free and Ca2+-alginate-immobilized cells for xylitol production under microaerophilic conditions and obtained yields of 4.73 ± 0.40 g/L (168 h) and 9.18 ± 0.10 g/L (packed bed reactor with a retention time of 18 h), respectively. Further, when the same fermentation was performed under aerobic conditions about 40.93 ± 0.73% lipid accumulation was observed with free cells. For saccharification, Aspergillus-niger-derived cellulase was used and this resulted in a yield of 27.45 ± 0.04 g/L of glucose. The glucose-enriched hydrolysate was supplemented for fermentation under nitrogen starved conditions from which 46.81 ± 2.60% (w/w) lipid content was obtained.

Immobilization of engineered E. coli cells for asymmetric reduction of methyl acetoacetate to methyl-(R)-3-hydroxybutyrate

The efficient asymmetric bio-synthesis of chiral β-hydroxy esters is of great importance for industrial production. In this work, a simple and productive engineered E.coli cell-immobilized strategy was applied for the asymmetric reduction of MAA to (R)-HBME with high enantioselectivity. Compared with the corresponding inactivated free cells, the alginate-immobilized cells remained 45% of initial activity at 50 ℃ and 65% after reuse of 10 times. After 60 days of storage at 4 ℃, the immobilized cells maintained more than 80% relative activity. Immobilization contributed significantly to the improvement of thermal stability, pH tolerance, storage stability and operation stability without affecting the yield of product. The immobilized recombinant E. coli cell had absolute enantioselectivity for the asymmetric reduction of MAA to (R)-HBME with e.e. > 99.9%. Therefore, microbial cell immobilization is a perspective approach in asymmetric synthesis of chiral β-hydroxy esters for industrial applications.Graphical

Response of Pseudokirchneriella subcapitata in Free and Alginate Immobilized Cells to Heavy Metals Toxicity.

Effects of 12 heavy metals on growth of free and alginate-immobilized cells of the alga Pseudokirchneriella subcapitata were investigated. The tested metals ions include Al, As, Cd, Co, Cr, Cu, Hg, Se, Ni, Pb, Sr, and Zn. Toxicity values (EC50) were calculated by graphical interpolation from dose-response curves. The highest to the lowest toxic metals are in the order Cd > Co > Hg > Cu > Ni > Zn > Cr > Al > Se > As > Pb > Sr. The lowest metal concentration (mg L−1) inhibiting 50% (EC50) of algal growth of free and immobilized (values in parentheses) algal cells were, 0.018 (0.09) for Cd, 0.03 (0.06) for Co, 0.039 (0.06) for Hg, 0.048 (0.050) for Cu, 0.055 (0.3) for Ni, 0.08 (0.1) for Zn, 0.2 (0.3) for Cr, 0.75 (1.8) for Al, 1.2 (1.4) for Se, 3.0 (4.0) for As, 3.3 (5.0) for Pb, and 160 (180) for Sr. Free and immobilized cultures showed similar responses to Cu and Se. The free cells were more sensitive than the immobilized ones. Accordingly, the toxicity (EC50) of heavy metals derived only form immobilized algal cells might by questionable. The study suggests that batteries of alginate-immobilized algae can efficiently replace free algae for the bio-removal of heavy metals.

Quantitative estimation of complex phosphate - solubilization by immobilized or free Azospirillum Lipoferum (H3) as vital vaccine in Pikovskaya broth (PVK) medium

Azospirillum species were collected from the rhizosphere and free soil of different plants in Al-Jabal El- Akhdar region. The isolates were identified according to biochemical activities. Isolate (H3) which showed the higher solubilization efficiency (SE) on Pikovskaya medium (PVK) containing insoluble phosphate (inorganic phosphate) Ca3(po4)2 Isolate (H3) was identified as Azospirillum lipoferum (H3), which used as an inoculum as free cell suspension or as alginate formulation. Phosphate solubilization was measured by A.lipoferum (H3) as free or alginate immobilized cells in (PVK) liquid medium and recording the pH of the medium at the same time. The results showed in the phosphorus content by immobilized bacteria in liquid (PVK) broth medium from 2nd day of incubation (2.48 µg/ ml) to 10th day (3.70 µg/ ml) and the free bacteria from 2nd day (8.81 µg/ ml) to 10th day (4.49 µg/ ml). pH of the liquid (PVK) broth medium was recorded from 2nd day to 10th day after incubation which decreased by immobilized A.lipoferum (H3) from 7.00 pH to 4.47 on the 10th day and by the free A.lipoferum (H3) to 6.00, which improved the production of organic acids from sugars which was response of decreasing the pH of the medium. This present study contribute to make agriculture more productive with less harm to the environment and for developing countries where the use of fertilizers is costly, and to encourage of using the biofertilizers ‘plant growth promoting rhizobacteria’ (PGPR) instead of the chemical fertilizers to enhance the plant growth which is the main goal to increase the food production in a healthy way.

Chlorine toxicity to Navicula pelliculosa and Achnanthes spp. in a flow-through system: The use of immobilised microalgae and variable chlorophyll fluorescence

Chlorination is a widely used antifouling method for freshwater and marine applications. Chlorine added to seawater reacts to form oxidants that are toxic to biofouling organisms. Further, the oxidants that result are short-lived, but may nevertheless affect non-target species in waterbodies receiving the antifouling effluent. This study evaluated the toxicity of chlorinated seawater (e.g. following sodium hypochlorite addition) on two different species of marine benthic diatoms (Achnanthes spp., and Navicula pelliculosa), which are representative of microphytobenthos communities – an important component in coastal habitats that may be exposed to chlorinated seawater. To evaluate the growth inhibition over a 72 h period, algae were immobilised in alginate beads and exposed to different levels of chlorination in a flow through system. Growth rates and physiological condition of the microalgae were evaluated using a Fast Repetition Rate fluorometer (FRRf). To determine whether alginate influenced the sensitivity of algal response, studies were also conducted in a static test system (without renewal of test solutions) using both free cells and immobilised cells with initial chlorine added to achieve a similar range of concentrations as those used in the flow-through study. Within the first hour of the exposure period there was an indication that, for both species, the free algal cells in the static system were more sensitive to exposure to chlorinated seawater than were alginate-immobilised cells in the flow through system. Immobilised cells in a static system with a single addition of chlorine were also less sensitive to chlorination than free algal cells. However, for periods of 24 h or more due to decay of TRO in the static system the exposure of immobilised algae in the flow through system had a greater impact and hence lower effect concentrations. For the flow-through studies Achnanthes spp. was the most sensitive after 72 h exposure with a potential no effect concentration EC10 value of 0.02 mg l−1 as Cl2 equivalents expressed as total residual oxidants (TRO) compared 0.04 mg l−1 TRO for N. pelliculosa. Immobilisation of algal cells in alginate was found to be an effective means of determining the impact of chlorination and is likely to be effective for other non-persistent substances. Based on the data produced, the extent and significance of ecological effects of chlorination upon algal species typical of microphytobenthos are likely to be limited providing discharges comply with a maximum allowable concentration of 0.01 mg l−1 TRO at the edge of an agreed mixing zone.

Effect of Nitrogen Source on Dye Decolouration by Alginate-immobilized Cells of Pseudomonas aeruginosa and Bacillus subtilis

Effect of Nitrogen Source on Dye Decolouration by Alginate-immobilized Cells of Pseudomonas aeruginosa and Bacillus subtilis

Immobilization of Methylosinus trichosporium OB3b for methanol production.

Due to the natural gas boom in North America, there is renewed interest in the production of other chemical products from methane. We investigated the feasibility of immobilizing the obligate methanotrophic bacterium Methylosinus trichosporium OB3b in alginate beads, and selectively inactivating methanol dehydrogenase (MDH) with cyclopropane to produce methanol. In batch cultures and in semi-continuous flow columns, the exposure of alginate-immobilized cells to cyclopropane or cyclopropanol resulted in the loss of the majority of MDH activity (>80%), allowing methanol to accumulate to significant concentrations while retaining all of M. trichosporium OB3b's methane monooxygenase capacity. Thereafter, the efficiency of methanol production fell due to recovery of most of the MDH activity; however, subsequent inhibition periods resulted in renewed methanol production efficiency, and immobilized cells retained methane-oxidizing activity for at least 14 days.

Bioenergetics of lactate vs. acetate outside TCA enhanced the hydrogen evolution levels in two newly isolated strains of the photosynthetic bacterium Rhodopseudomonas.

Two local hydrogen-evolving strains of purple nonsulfur bacteria have been isolated, characterized, and identified as Rhodopseudomonas sp. TUT (strains Rh1 and Rh2). Lactate followed by succinate and malate supported the highest amounts of H2 production, growth (O.D.660nm, proteins and bacteriochlorphyll contents), nitrogenase activity, and uptake hydrogenase; the least of which was acetate. Alginate-immobilized cells evolved higher hydrogen amounts than free cell counterparts. Rh1 was more productive than Rh2 at all circumstances. Lactate-dependent hydrogen evolution was more than twice that of acetate, due to ATP productivity (2/-1, respectively), which is limiting to the nitrogenase activity. The preference of lactate over other acids indicates the feasibility of using these two strains in hydrogen production from dairy wastewater.

Production of precursors for anti-Alzheimer drugs: Asymmetric bioreduction in a packed-bed bioreactor using immobilized D. carota cells

ABSTRACT(S)-1-Phenylethanol derivatives, which are the precursors of many pharmacological products, have also been used as anti-Alzheimer drugs. Bioreduction experiments were performed in a batch and packed-bed bioreactor. Then, the kinetics constants were determined by examining the reaction kinetics in the batch system with free and immobilized carrot cells. Also, the effective diffusion coefficient (De) of acetophenone in calcium alginate-immobilized carrot cells was investigated. Kinetics constants for free cells, which are intrinsic values, are reaction rate Vmax = 0.052 mmol L−1 min−1, and constants of the Michaelis–Menten KM = 2.31 mmol L−1. Kinetics constants for immobilized cells, which are considered apparent values, are Vmax, app = 0.0407 mmol L−1 min−1, KM, app = 3.0472 mmol L−1 for 2 mm bead diameter, and Vmax, app = 0.0453 mmol L−1 min−1, KM, app = 4.9383 mmol L−1 for 3 mm bead diameter. Average value of effective diffusion coefficient of acetophenone in immobilized beads was determined as 1.97 × 10−6 cm2 s−1. Using immobilized carrot cells in an up-flow packed-bed reactor, continuous production of (S)-1-phenylethanol through asymmetric bioreduction of acetophenone was performed. The effects of the residence time and concentrations of substrate were investigated at pH 7.6 and 33°C. Enantiomerically pure (S)-1-phenylethanol (ee > 99%) was produced with 75% conversion at 4-hr residence time.

Parameters and kinetics of olive mill wastewater dephenolization by immobilized Rhodotorula glutinis cells

Olive mill wastewater (OMW) with total phenol (TP) concentration range of 300–1200 mg/L was treated with alginate-immobilized Rhodotorula glutinis cells in batch system. The effects of pellet properties (diameter, alginate concentration and cell loading (CL)) and operational parameters (initial TP concentration, agitation rate and reusability of pellets) on dephenolization of OMW were studied. Up to 87% dephenolization was obtained after 120 h biodegradations. The utilization number of pellets increased with the addition of calcium ions into the biodegradation medium. The overall effectiveness factors calculated for different conditions showed that diffusional limitations arising from pellet size and pellet composition could be neglected. Mass transfer limitations appeared to be more effective at high substrate concentrations and low agitation rates. The parameters of logistic model for growth kinetics of R. glutinis in OMW were estimated at different initial phenol concentrations of OMW by curve-fitting of experimental data with the model.

Efficacy and reusability of alginate-immobilized live and heat-inactivated Trichoderma asperellum cells for Cu (II) removal from aqueous solution

Cu(II) removal efficacies of alginate-immobilized Trichoderma asperellum using viable and non-viable forms were investigated with respect to time, pH, and initial Cu(II) concentrations. The reusability potential of the biomass was determined based on sorption/desorption tests. Cu(II) biosorption by immobilized heat-inactivated T. asperellum cells was the most efficient, with 134.22mg Cu(II) removed g−1 adsorbent, compared to immobilized viable cells and plain alginate beads (control) with 105.96 and 94.04mg Cu(II) adsorbed g−1 adsorbent, respectively. Immobilized non-viable cells achieved equilibrium more rapidly within 4h. For all biosorbents, optimum pH for Cu(II) removal was between pH 4 and 5. Reusability of all biosorbents were similar, with more than 90% Cu(II) desorbed with HCl. These alginate-immobilized cells can be applied to reduce clogging and post-separation process incurred from use of suspended biomass.

The removal of ρ-chlorophenol in aqueous cultures with free and alginate-immobilized cells of the microalga Tetraselmis suecica

The present study aimed at evaluating the ability of some isolated cyanobacterial and microalgal strains for the removal of ρ-chlorophenol (ρ-CP), an environmentally harmful contaminant. To identify the most efficient species, a screening program was carried out using 15 microalgal and cyanobacterial strains. Among them, Tetraselmis suecica was able to remove 67 % of the ρ-chlorophenol at an initial concentration of 20 mg L−1 from the medium within a 10-day period. The efficacy of the process was dependent on the ρ-chlorophenol concentration. At concentrations above 60 mg L−1 of the pollutant, no removal was observed due to the inhibitory effect of ρ-chlorophenol on the T. suecica cells. The effect of cell immobilization in alginate beads on T. suecica removal capacity was also examined. Using this technique, the removal efficacy for the initial ρ-CP concentration of 20 mg L−1 increased up to 94 %.

Removal and biodegradation of nonylphenol by immobilized Chlorella vulgaris

The removal and biodegradation of nonylphenol (NP) by alginate-immobilized cells of Chlorella vulgaris were compared with their respective free cultures. The effects of four cell densities of 104 per algal bead were investigated, as were the four algal bead concentrations, with regard to the removal and biodegradation of NP. Although immobilization significantly decreased the growth rate and NP’s biodegradation efficiency of C. vulgaris, NP removal over a short period was enhanced. The NP removal mechanism by immobilized cells was similar to that by free cells, including adsorption onto alginate matrix and algal cells, absorption within cells and cellular biodegradation. The optimal cell density and bead concentration for the removal and biodegradation of NP was 50–100×104cells algal bead−1 and 2–4beadsml−1 of wastewater, respectively. These results demonstrated that immobilized C. vulgaris cells under optimal biomass and photoautotrophic conditions are effective in removing NP from contaminated water.

Application of the yeast pheromone system for controlled cell-cell communication and signal amplification

The aim of the work is to exploit the yeast pheromone system for controlled cell-cell communication and as an amplification circuit in technical applications, e.g. biosensors or sensor-actor systems. As a proof of principle, we developed recombinant Saccharomyces cerevisiae cells that express enhanced green fluorescent protein (EGFP) in response to different concentrations of the alpha(α)-factor mating pheromone. A respective reporter construct allowing the pheromone-driven expression of EGFP was transformed into the S. cerevisiae strains BY4741 and BY4741 bar1Δ. Upon addition of synthetic α-factor, the fluorescence strongly increases after 4 h. Furthermore, cells with constitutive α-factor expression were able to induce the expression of EGFP in co-cultivation with sensor cells only if both cell types were deleted for the gene BAR1, encoding α-factor protease. For technical applications, the immobilization of functionalized cells may be beneficial. We show that pheromone-induced expression of EGFP is effective in alginate-immobilized cells. Based on S. cerevisiae α-factor, we developed a controlled cell-cell communication system and amplification circuit for pheromone-driven expression of a target protein. The system is effective both in suspension and after cell immobilization. The developed set of recombinant yeast strains is the basis to apply the yeast pheromone system for signal production and amplification in biosensors or sensor-actor systems.

Phenol degradation by Aureobasidium pullulans FE13 isolated from industrial effluents

The degradation of phenol (2–30 mM) by free cells and by alginate-immobilized cells of Aureobasidium pullulans FE13 isolated from stainless steel effluents was studied in batch cultures with saline solution not supplemented with nutrients or yeast extract. The rate at which the immobilized cells degrade phenol was similar to the rate at which the suspended cells could degrade phenol, for a concentration of up to 16 mM of phenol. The maximum phenol volumetric degradation rate for 16 mM phenol was found to be 18.35 mg l −1 h −1 in the assays with free cells and 20.45 mg l −1 h −1 in the assays with alginate-immobilized cells, 18 mM phenol and cellular concentration of 0.176 g/l. At concentrations higher than this, an inhibitory effect was observed, resulting in the lowering of the phenol degradation rates. The immobilization was detrimental to the catechol 1,2-dioxygenase activity. However, the immobilized cells remained viable for a longer period, increasing the efficiency of phenol degradation. The yeast showed catechol 1,2-dioxygenase activity only after growth in the phenol, which was induced at phenol concentrations as low as 0.05 mM and up to 25 mM at 45 h of incubation at 30 °C. Phenol concentrations higher than 6 mM were inhibitory to the enzyme. Addition of glucose, lactate, succinate, and benzoate reduced the rate at which phenol is consumed by cells. Our results suggest that inoculants based on immobilized cells of A. pullulans FE13 has potential application in the biodegradation of phenol and possibly in the degradation of other related aromatic compounds.

Repeated-batch production of galactooligosaccharides from lactose at high concentration by using alginate-immobilized cells of Sporobolomyces singularis YIT 10047

We investigated the production of galactooligosaccharides (GOS) from lactose by alginate-immobilized cells of Sporobolomyces singularis YIT 10047 (IM-SS). beta-Galactosidase activity was stable at 30 to 50 degrees C but decreased dramatically between 50 and 60 degrees C and disappeared at 70 degrees C in acetate buffer. The enzyme activity remaining was no more than 20% of that of unheated samples after incubation in distilled water at 55 degrees C, whereas its value was about 60% at the same temperature under buffered condition. However, activity was maintained more than 80% with 10% to 50% (w/w) lactose after incubation at 55 degrees C without buffer. In a single-batch reaction, GOS yield was 41.0% with free cells and 40.4% with IM-SS. We attempted a repeated-batch reaction using IM-SS with 600 g L(-1)lactose. IM-SS produced GOS stably for 20 batches (22 h/batch, 440 h in total) at 55 degrees C and pH 5.0 or 6.0. IM-SS produced GOS at 242 g L(-1), at a rate of 8.72 g L(-1) h(-1). Both GOS yield and production rate were higher than those in published experiments on GOS production using immobilized biocatalysts. The repeated-batch reaction with IM-SS would be an ideal system for GOS production because of its stability and high productivity.

Investigation of continuous cyclodextrin glucanotransferase production by the alginate-immobilized cells of alkalophilic Bacillus sp. in an airlift reactor

Cyclodextrin glucanotransferase (CGTase) production by free and calcium alginate-immobilized cells was investigated in an airlift reactor. The average volumetric productivity with continuous fermentation (13.65 U ml −1 h −1) was found to be greater than the free cells (6.89 U ml −1 h −1). The total CGTase enzyme produced with continuous fermentation was found to be about 98% greater than with that of free cells. The immobilized Bacillus particles showed a half-life of 18 days during continuous fermentation under airlift conditions.

Degradation of 1-butanol by solvent-tolerant Enterobacter sp. VKGH12

Abstract Biodegradation of solution-phase 1-butanol by solvent-tolerant Enterobacter sp. VKGH12 (NCIM 5221), which utilizes 1-butanol as its sole source of carbon and energy, was investigated. At an initial loading of 0.4% and 0.8% (v/v) 1-butanol, freely suspended cells degraded 76% and 26%, whereas calcium alginate-immobilized cells degraded 96% and 92% 1-butanol, respectively, in batch cultures. At these loadings, alginate-immobilized cells could be reused for 10 and 7 cycles, respectively, without significant loss in degradation efficiency. A packed-bed reactor containing immobilized cells efficiently degraded 1-butanol at a flow rate of 25–75 ml h −1 at an initial loading of 0.4% (v/v), and could be continuously operated for 10 days at an influent concentration of 0.4% (v/v) 1-butanol without a loss in degradation efficiency. The results suggest the application of immobilized solvent-tolerant bacterium in treatment of solvent waste streams.

Efficiency of a fixed-bed and a gas-lift three-column reactor for continuous production of ethanol by pectate-and alginate-immobilized Saccharomyces cerevisiae cells

AbstractEthanol-tolerant and thermo-tolerant yeast strain Saccharomyces cerevisiae C11-3 cells immobilized in calcium pectate and calcium alginate gels were used for ethanol fermentation in a three-reactor system with a gradient temperature control. The fermentation process has been tested in a fixed-bed and a gas-lift arrangement. The gas-lift system was more efficient due to a better mass transport between the phases. Abrasion was more evident in calcium alginate particles, while calcium pectate beads were not significantly damaged. Two different concentrations of alginate were tested and calcium pectate gel was demonstrated to be more suitable as an immobilization material in comparison with calcium alginate due to its mechanical resistance and favourable diffusion parameters, providing an ethanol production of more than 7.5 g dm−3 h−1 over a period of 630 h.

Parametric Studies on Batch Degradation of a Plasticizer Di-n-Butylphthalate by Immobilized Bacillus sp

Di-n-butylphthalate (DBP) is one of the phthalate esters (PAEs) used in the manufacture of plasticizers, insect repellents and synthetic fibres and contributes to environmental pollution. We report a novel bacterium belonging to the genus, Bacillus (NCIM 5220), which has the ability to utilize DBP as the sole source of carbon and energy. This bacterium was immobilized in alginate. The degradation of DBP by immobilized cells was compared with free cells. The effects on the degradation of DBP of different factors like gel (alginate) concentration, gel bead size, temperature, and pH were investigated. Oxygen uptake in the presence of DBP by free and immobilized cells was also studied. The results showed that the degradation of DBP by immobilized cells was more efficient than by free cells. Further, the effect of various factors tested on the degradation of DBP by alginate-immobilized cells showed that the degradation of DBP was remarkably affected by alginate concentration between 2 and 5% and drastically decreased between bead size 2 and 5 mm. A change of 10 °C of reaction temperature from 30 to 40 °C did not alter the degradation of DBP, and maximum degradation was appeared to be favoured over a broad pH range of 6.5–7.5 for immobilized cells as compared to free cells, which showed an optimum temperature of about 35 °C and pH of 7.0. The immobilized cells showed higher oxidation of DBP than free cells. Thus more efficient degradation of DBP could be achieved by immobilizing Bacillus sp. in alginate beads.