Suspended Cell System Research Articles

Organic Pollutant Biodegradation by an Immobilized Mixed Bacterial Culture

Pollution of organic compounds in rivers is generally contributed by wastewater discharge from human activities e.g. untreated black- & greywater, livestock farming, and industry. Without proper control of this waste generation, the river ecosystem will suffer if just rely on its self-purification and dilution by natural runoff to reduce the downstream effect. This research aimed to treat organic wastewater with an immobilized mixed bacterial culture. Immobilization was carried out using calcium alginate in a form of macrocapsule/beads, allowing the degrader microbe to be entrapped in its matrix thus preventing it from washout from the system. 3% (w/v) sodium alginate was mixed with 5 mg (dry) mixed culture and dropwise on 4% (w/v) calcium chloride solution. The results showed that immobilized microbes were still able to grow despite being entangled in a calcium alginate matrix with an initial growth of 6.1 x 105 to 7.4 x 107 colonies/grams and reduced COD content up to 68.3%. Degrader microbe immobilization offers considerable advantages compared to free/ suspended cell system due to the influence of river water flow.

Synergic effect of adsorption and biodegradation enhance cyanide removal by immobilized Alcaligenes sp. strain DN25

A high efficiency and stability polyurethane-foam (PUF)-immobilized cell system was constructed to remove cyanide based on simultaneous adsorption and biodegradation (SAB). The performance of the PUF-immobilized system was evaluated by comparison with the freely suspended cell system. The SAB system exhibited more effective and robust, and could still remain degradation activity even at 40 °C or pH 11.0. The SAB system completely removed 500 mg CN-/L within 8 h at 30 °C, pH 8.0, and 120 rpm, whereas 12 h were required for the free cells system. Moreover, the SAB system showed apparent superiority in removing higher concentration cyanide up to 1200 mg CN-/L. A continuously stirred tank bioreactor (CSTR) was successfully designed and steadily operated with approximately 85% of the total average removal efficiency for 52 days at an influent cyanide concentration of 100-200 mg/L, which demonstrated a favorable reliability. Cyanide removal process could be well described using a pseudo first-order model, and the higher apparent rate constants (k) of the immobilized cells showed the synergic effect of adsorption and biodegradation significantly enhanced cyanide removal. Preliminarily, it was found that the foam characteristic might play a not negligible role on the cyanide-degrading enzyme expression of strain DN25 in the SAB system.

Improved Hydrogen Production from Galactose Via Immobilized Mixed Consortia

In this study, a combined encapsulation and entrapment immobilization strategy was employed to enhance hydrogen production from sewage sludge containing mixed microbial cultures. The results showed that the hydrogen production rate (HPR) and hydrogen yield (HY) of immobilized cells were significantly higher than that of the suspended cells. The peak HPR and HY of 0.76 L/L-d and 1.20 mol/mol galactoseadded attained with the immobilized cell system were comparable to that of the suspended cell system (HPR 0.62 L/L-d and HY 0.88 mol/mol galactoseadded, respectively). The immobilized beads were also found to have efficient hydrogen production upon reuse for more than five cycles, with galactose removal > 85 % in all cases. Soluble metabolic product analysis revealed that fermentation followed a butyrate pathway and the major metabolites produced were acetate and butyrate. The peak total energy production rate and yield were 8.6 kJ/L-d and 308 kJ/mol added, respectively.

Wastewater treatment to enhance the economic viability of microalgae culture

Microalgae culture is still not economically viable and it presents some negative environmental impacts, concerning water, nutrient and energy requirements. In this context, this study aims to review the recent advances on microalgal cultures in wastewaters to enhance their economic viability. We focused on three different culture concepts: (1) suspended cell systems, (2) cell immobilization, and (3) microalgae consortia. Cultures with suspended cells are the most studied. The nutrient removal efficiencies are usually high for wastewaters of different sources. However, biomass harvesting is difficult and a costly process due to the small cell size and lower culture density. On the other hand, the cell immobilization systems showed to be the solution for this problem, having as main limitation the nutrient diffusion from bulk to cells, which results in a reduced nutrient removal efficiency. The consortium between microalgae and bacteria enhances the growth of both microorganisms. This culture concept showed to be a promising technology to improve wastewater treatment, regarding not only nutrient removal but also biomass harvesting by bioflocculation. The aggregation mechanism must be studied in depth to find the process parameters that would lead to an effective and cheap harvesting process.

Kinetic characteristics of biodegradation of methyl orange by Pseudomonas putida mt2 in suspended and immobilized cell systems

Azo dyes, which are considered the most recalcitrant and persistent among all groups of dye, were biodegraded by various types of bacteria. In this study, the decolorization of methyl orange was determined under various conditions by non-immobilized and immobilized Pseudomonas putida mt2. In both systems, the decolorizations were more favorable under anoxic condition. Optimal conditions for decolorization were investigated. The optimal temperature for suspended cell operation is in the range between 33 and 35°C and the decolorization seems not to be suitable in acidic condition. However, the optimal range of pH and temperature in the immobilized cell system were obtained at 7–9 and 35–37°C, respectively. Kinetics parameters of νmax and km were predicted up to 7.5mg/(gh) and 283gL−1, respectively, for non-immobilized system; and 6.3mg/(gh) and 257gL−1, respectively, for immobilized Ca-alginate system. Mass transfer coefficients were calculated as 0.75×10−5 and 6.20×10−5 for each system.

Comparative Evaluation of Pumice Stone as an Alternative Immobilization Material for 1,3-Propanediol Production from Waste Glycerol by Immobilized Klebsiella pneumoniae

In this study, pumice stone (PS), which is a vastly available material in Turkey, was evaluated as an alternative immobilization material in comparison to other commercially available immobilization materials such as glass beads and polyurethane foam. All immobilized bioreactors resulted in much better 1,3-propanediol production from waste glycerol in comparison to the suspended cell culture bioreactor. It was also demonstrated that the locally available PS material is as good as the commercially available immobilization material. The maximum volumetric productivity (8.5g L(-1) h(-1)) was obtained by the PS material, which is 220% higher than the suspended cell system. Furthermore, the immobilized bioreactor system was much more robust against cell washout even at very low hydraulic retention time values.

Use of ceramic-based cell immobilization to produce 1,3-propanediol from biodiesel-derived waste glycerol with Klebsiella pneumoniae

The feasibility of the continuous production of a valuable bioplastic raw material, namely 1,3-propanediol (1,3-PDO) from biodiesel by-product glycerol, using immobilized cells was investigated. In addition, the effect of hydraulic retention time (HRT) was also analysed. Ceramic balls and ceramic rings were used for the immobilization of a locally isolated strain; Klebsiella pneumoniae (GenBank no. 27F HM063413). HRT of 1 h is the best one in terms of volumetric production rate (g 1,3-PDO l(-1) h(-1)). The results indicated that ceramic-based cell immobilization achieved a 2-fold higher production rate (10 g 1,3-PDO l(-1) h(-1)) in comparison with suspended cell system (4·9 g 1,3-PDO l(-1) h(-1)). Continuous cultures with immobilized cells revealed that 1,3-PDO production was more effective and more stable than suspended culture systems. Furthermore, cell immobilization had also obvious benefits especially for resistance of the production for extreme conditions (high organic loading rates, cell washouts). The results were important for understanding the significance of continuous immobilization process among other well-known 1,3-PDO fermentation processes. This work is a promising process for further studies, as the immobilized micro-organism was able to reach high volumetric production rates at short HRT, it has an important role in tolerating and converting glycerol during fermentation. Therefore, HRT is a very significant operational parameter (P value <0·05) directly affecting the bioreactor performance and production rate.

Continuous production of 1,3-propanediol using raw glycerol with immobilized Clostridium beijerinckii NRRL B-593 in comparison to suspended culture

The continuous production of 1,3-propanediol (1,3-PDO) was investigated with Clostridium beijerinckii NRRL B-593 using raw glycerol without purification obtained from a biodiesel production process. Ceramic rings and pumice stones were used for cell immobilization in a packed-bed bioreactor. For comparison purpose, a control bioreactor with suspended culture was also run. The effect of hydraulic retention time (HRT) on the production of 1,3-PDO in both immobilized and suspended bioreactors were also investigated. The study revealed that HRT is an important factor for both immobilized and suspended systems and a HRT of 2h is the best one in terms of volumetric production rate (g 1,3-PDO/L/h). Furthermore, cell immobilization had also obvious benefits especially for the robustness and the reliability of the production. The results indicated that cell immobilization achieved a 2.5-fold higher productivity in comparison to suspended cell system. Based on our results, continuous production of 1,3-PDO with immobilized cells is an efficient method, and raw glycerol can be utilized without any pretreatment.

γ-Alumina Doped Alginate Gel for Cell Immobilization in Fermentation Processes

γ-Alumina (γ-Al 2 O 3 ) doped alginate gel (AEC) was developed as a cell carrier in fermentation processes of Saccharomyces cerevisiae M.30 for ethanol production and Clostridium butyricum DSM 5431 for 1,3-propanediol production. In a single batch system of ethanol fermentation, the final ethanol concentration of suspended cell (SC), immobilized cell on γ-Al 2 O 3 (AC) and AEC cultures were 82.4, 77.1 and 74.6 g/l, respectively. In 4-cycle repeated batch fermentation, the AEC culture demonstrated a good potential of reusability. Its ethanol production and conversion yield of the 1st, 2nd and 3rd repeated batch were comparable to those of the SC and AC cultures with the immobilization yield of 86%. AEC was also found to be effective for the cell immobilization of C. butyricum with the immobilization yield of 83%. However, the strong inhibition effect of cell-γ-Al 2 O 3 immobilization towards 1,3-propanediol production was observed. Moreover, 1, 3-propanediol fermentation stability in the SC, AC and AEC systems tended to be lowered during the repeated batch fermentation. Interfering of positive charge of γ-Al 2 O 3 on the cell membrane was thought to be the cause of the inactivity of C. butyricum DSM 5431 in 1,3-propanediol production.

Identification of bottleneck enzymes with negative dynamic sensitivities: Ethanol fermentation systems as case studies

Dynamic logarithmic gains (normalised time-varying sensitivities) or their products with metabolite concentrations, called an instantaneous bottleneck ranking indicator (instantaneous BR indicator), can express the time courses of bottleneck enzyme ranking. The integrated value of the instantaneous BR indicator, called an overall BR indicator, provides a ranking of bottleneck enzymes over the entire reaction period of time. Owing to the property of the logarithmic gains, these BR indicators are allowed to include both positive and negative values and in turn raise several questions on the identification of a bottleneck enzyme. To eliminate these questions, the present work studies the identification of bottleneck enzymes in four ethanol fermentation systems using suspended and immobilised cells at pH 4.5 and 5.5 as case studies. These systems consist of both intracellular reactions without cell growth (intracellular reaction systems) and those with cell growth (cell growth systems). The analyses of the intracellular reaction systems reveal that both the suspended and immobilised cell systems at pH 5.5 are not robust. In the cell growth systems, therefore, the analyses are conducted only for the suspended and immobilised cell models at pH 4.5. The enzyme glyceraldehyde 3-phosphate dehydrogenase (gapd) initially has the largest logarithmic gains under all conditions, whereas the system becomes insensitive to it over time. Interestingly, the instantaneous BR indicator for gapd is negatively large. Also, other indicators change in positive and/or negative regions. To deal with these values of different signs, all the indicators are integrated to obtain and rank the overall BR indicators in the order of the magnitudes of their absolute values. The result shows that the ranking of enzymes are completely identical to that of the final ethanol concentrations obtained by finitely increasing or decreasing the relevant enzyme activities in accordance with the signs of overall BR indicators. This result indicates that the enzymes must be ranked in the order of the magnitudes of their absolute values to determine a bottleneck enzyme. As a result, the analyses of the intracellular reaction systems reveal that the bottleneck enzyme is switched from a membrane-bound glucose transporter enzyme to phosphofructokinase when the cell-retaining condition is changed from suspension to immobilisation. In the cell growth systems, gapd is identified as the most likely bottleneck enzyme, suggesting that the gapd activity must be decreased to increase the ethanol productivity.

Steroid bioconversion: Towards green processes

There is an increasing trend towards reducing the use of organic solvents in industry due to environmental constraints and the adoption of green chemistry guidelines. To overcome the low volumetric productivity of aqueous bioconversion systems involving sparingly water soluble hydrophobic compounds, processes are being developed and designed to incorporate green solvent such as supercritical fluids, ionic liquids and natural oils, and liquid polymers, among others as an alternative to organic solvents. Moreover, processes are developed and redesigned to use/reuse chemicals and reagents derived from waste or renewable feed stocks in order to diminish E-factors. In this work, the use of green solvents as key components in the bioconversion media for a multi-step microbial bioconversion was assessed in a suspended whole cell system, combined with the use of by-products as raw materials, ultimately used as carbon source for cell growth and as sterol substrate for bioconversion. The model system is the selective cleavage of the side-chain of β-sitosterol performed by free resting cells of Mycobacterium sp. NRRL B-3805, a well-established industrial multi-enzymatic process involving the use of nine catabolic enzymes in a 14-step metabolic pathway. Bioconversion yields in silicone media were higher than the ones obtained in polyethylene glycol (PEG), polypropylene glycol (PPG) and ionic liquids, as well as in dioctyl phthalate (DOP), an organic solvent that has previously been shown to allow high conversion yields. Total conversion of 12 mM substrate in silicone media was consistently obtained at the end of 120-h bioconversion runs. Similar bioconversion profiles were attained during a 50-fold scale-up, maintaining constant the power consumption per unit of volume.

2-Aminobenzothiazole degradation by free and Ca-alginate immobilized cells of Rhodococcus rhodochrous

2-Aminobenzothiazole (ABT) degradation was investigated using free and immobilized systems during photodegradation under solar light in the presence of Fe(III)-nitrilotriacetic acid (FeNTA), biodegradation by Rhodococcus rhodochrous, and during combined conditions. Ca-alginate hydrogel was chosen as a model matrix and some complementary studies were required to characterize this new system. R. rhodochrous metabolism in this type of environment was monitored by NMR spectroscopy. Neither change in intracellular pH values nor in ATP concentrations was observed by in vivo 31P NMR, showing that no metabolic modification occurred between free and immobilized cells. 1H NMR demonstrated that alginate was not used as carbon source by R. rhodochrous. After establishing the pre-treatment protocol by SPE to eliminate solubilised alginate, ABT adsorption on beads and degradation were studied. The same pathways of transformation were observed in suspended and immobilized cell systems. Considering the ABT adsorption phenomenon on alginate beads (8%), the efficiency of the two systems was found to be comparable although the degradation rate was slightly lower with immobilized cells. The most important result was the finding that the positive effect of FeNTA on ABT degradation with immobilized cells was similar to that observed previously with free cells. All these results show that mechanisms observed with free cells can be extrapolated to entrapped cells, i.e. under conditions much closer to those usually encountered in the environment.

Treatment of methylene blue-containing wastewater using microorganisms supported on granular activated carbon under packed column operation

We studied the feasibility of using biological granular activated carbon-packed column in treating methylene blue-containing wastewater. The granular activated carbon with immobilized microbes was packed into a column and fed with 3 liter methylene blue-containing wastewater daily. With initial 1350 mg/l of methylene blue and 1550 mg/l of chemical oxygen demand, it was observed that the colour and chemical oxygen demand removal efficiencies were 99 and 78%, respectively. The high treatment performance of the system could be due to the simultaneous adsorption and biodegradation processes and advantages of immobilized microbes compare to suspended cell system.

Product shifting by controlling medium pH in immobilised Streptomyces coelicolor A3(2) culture

Actinorhodin production by Streptomyces coelicolor A3(2) immobilised naturally in a porous support material was investigated in a 20 l bioreactor. The effect of pH-control on actinorhodin fermentation was conducted by performing different strategies. The combination of pH-control and immobilisation of the cells produced a different result than pH-control with freely suspended cells. The control of pH at 7.2 in an immobilised cell system altered the physiology of the cells shifting the product, actinorhodin, to another pigmented secondary metabolite, undecylprodigiosin. Although the cells were biologically active in such an environment, they were unable to produce actinorhodin. In the freely suspended cell system, however, actinorhodin biosynthesis was not hindered by controlling the medium pH.

Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspensions using flow cytometry.

Flow cytometry was used to measure cell cycle parameters in Solanum aviculare plant cell suspensions. Methods for bromodeoxyuridine (BrdU) labeling of plant nuclei were developed so that cell cycle times and the proportion of cells participating in growth could be determined as a function of culture time and conditions. The percentage of cells active in the cell cycle at 25 degrees C decreased from 52% to 19% within 7.6 d of culture; presence of a relatively large proportion of non-active cells was reflected in the results for culture growth. While the maximum specific growth rate of the suspensions at 25 degrees C was 0.34 d-1 (doubling time: 2.0 d), the specific growth rate of active cells was significantly greater at 0.67 d-1, corresponding to a cell cycle time of 1.0 d. A simple model of culture growth based on exponential and linear growth kinetics and the assumption of constant cell cycle time was found to predict with reasonable accuracy the proportion of active cells in the population as a function of time. Reducing the temperature to 17 degrees C lowered the culture growth rate but prolonged the exponential growth phase compared with 25 degrees C; the percentage of cells participating in the cell cycle was also higher. Exposure of plant cells to different agitation intensities in shake flasks had a pronounced effect on the distribution of cells within the cell cycle. The proportion of cells in S phase was 1.8 times higher at a shaker speed of 160 rpm than at 100 rpm, while the frequency of G0 + G1 cells decreased by up to 27%. Because of the significant levels of intraculture heterogeneity in suspended plant cell systems, flow cytometry is of particular value in characterizing culture properties and behavior.

A spiral bioreactor for removal and recovery of metals from aqueous wastes

Microorganisms from activated sludge were immobilized on a spiral reactor fabricated from a PVC-Silica based biosupport, and used to biosorb lead from synthetic waste streams. Biosorption parameters determined for lead from Freundlich isotherms indicate that 89% of the binding capacity of that observed in suspended cell system is retained in the spiral bioreactor. More than 87% of biosorbed lead was recovered each time after breakthrough by dilute acid washing between the pH of 2 to 2.5. No significant changes in the biosorption pattern was observed over several adsorption and recovery cycles that were conducted. The spiral bioreactor is better than suspended cell systems because it provides comparable surface area as that in the suspended cell system in an immobilized (small volume) form. It is also better than the entrapped microbial cell reactors because it eliminates mass transfer resistance across the external matrix, which may reduce the inherent binding capacity.

Degradation of sodium anthraquinone sulphonate by free and immobilized bacterial cultures

Aerobic biodegradation of a xenobiotic recalcitrant compound sodium anthraquinone-2-sulphonate (SAS), was investigated using as an inoculum a mixed microbial culture, which was activated sludge from industrial and domestic waste-water treatment plants. The difference in SAS degradation was examined using two main systems: (1) suspended cells and (2) immobilized cells, both in batch and in continuous culture. In the suspended cell system, under continuous culture conditions using SAS as a unique source of carbon and energy, it was possible to degrade about 95% of this substrate after 6 days. Maximal SAS removal rates in the suspended-cell system were 593 mg SAS l−1 h−1 and 88.7 mg SAS l−1 h−1 for dilution rates (D) of 0.05 h−1 and 0.075 h−1, respectively. In the immobilized-cell system, almost all SAS was degraded in 6 days and the maximal removal rate reached 88.7 mg SAS l−1 h−1 at D=0.05 h−1. Application of a continuous-flow enrichment procedure resulted in selection of several kinds of micro-organisms and led to a progressive elimination of some species of Aeromonas. A stable microbial community of 11 strains has been established and characterized at D=0.075 h−1. Most of them were Gram-negative and belonged to the genus Pseudomonas.

Inert supports for lactic acid fermentation ?a technological assessment

Production of lactic acid using Lactobacillus delbrueckii NRRL B445 recently renamed L. rhamnosus was studied in continuously recycled packed reactors at pH 6.3 and 42° C. Four inert adsorbent supports were used for immobilization: Raschig rings of sintered glass (Schott, FRG), beads of sintered glass (Schott), beads of porous glass (Poraver; Dennert, FRG) and irregular ceramic particles (Otto Feuerfest, FRG). The best support was found to be the beads of sintered glass, yielding the highest volumetric lactic acid productivity. Zeta potentials of L. rhamnosus showed the cells to be negatively charged at all pHs studied, the charge becoming less negative with increasing ionic strength. The surface charge did not control adhesion. A comparison between the immobilization carried out in batch and continuous tests with the different supports demonstrated that extrapolation from batch adsorption curves to continuous operation can introduce large errors. The effect of dilution rate was also stidied: a saturation concentration of adsorbed cells was achieved at all dilution rates, i.e., the immobilized cell component was almost invariant. Different diameters of Poraver beads were tested; clear evidence for mass transfer limitation was shown. Finally, the effects of pH and substrate concentration under immobilization were evaluated. The results indicate that pHs above or below the optimum for suspended cell systems can be used in the immobilized reactor while maintaining lactic acid productivity. To simplify downstream processing by keeping the glucose concentration close to zero in the effluent, the glucose concentration in the feed has to be chosen in conjunction with the dilution rate.

An experimental study of mass diffusion and reaction rate in an anaerobic biofilm

An experimental reactor consisting of two chambers, separated by a porous ceramic immobilization matrix, was constructed to measure the effective diffusivity of different compounds and the consumption rates of acetate in developing biofilms. In initial experiments, effective diffusivities for acetate, propionate, isopropanol, and lithium salt through the ceramic immobilization matrix in the absence of biofilm were determined to be 40% to 50% less than in water at infinite dilution. The effective diffusivity of the lithium salt was similar to that of acetate. The effective diffusivity of the lithium salt through biofilms of thickness in the range of 200 to 1200 microm was essentially constant with a value of approximately 7% of that in water at infinite dilution. Acetate consumption in the biofilm was linearly proportional to biofilm thickness up to a biofilm depth of 800 microm. Deviation from linearity appeared in biofilm thicknesses greater than 800 microm. Results of these experiments support previous reports that immobilized cell reactors have significantly higher bioconversion rates than suspended cell systems.

Bioreactor considerations for secondary metabolite production from plant cell tissue culture: Indole alkaloids from Catharanthus roseus

Batch shake flask studies with Catharanthus roseus demonstrated that alkaloid production commenced only after growth had slowed or ceased. To obtain high alkaloid productivities for extended periods, a hormone-free production medium was used. To develop a readily scalable process, both immobilized and suspended cell systems were studied. In the immobilized cell systems, growth, glucose utilization, and alkaloid production were suppressed; for the case of membrane entrapped cells this suppression was observed to be reversible. Based on the oxygen requirements of the cells, and the oxygen transfer capabilities of a pneumatically agitated bubble column, conditions were established that allowed the growth and production dynamics observed in shake flasks to be reproduced in the air sparged column reactor. The requirement for the aseptic exchange of growth for production medium was satisfied by using a coarse cotton filter and coupling filtration to aeration. With this filtration system, media can be rapidly and completely exchanged and the filter can be quickly and effectively backwashed. By coupling filtration to aeration, a two-stage batch operation can be employed while requiring only a single bioreactor. Studies with this system demonstrated its capabilities for alkaloid production.