Degradation of benzene by a Rhodococcus sp. using immobilized cell systems

The continuous degradation of benzene by a Rhodococcus sp. using free and immobilized cell systems was compared. Cell entrapment in calcium and strontium alginate beads and adhesion on support materials such as glass beads were found to be unsatisfactory. Degradation of benzene by cells immobilized in either ceramic or cellulose carriers proved to be more efficient than its non-immobilized counterpart. A retention time of 36 h was required to effect a 97% degradation of benzene using suspended free cells while cells immobilized on cellulose or ceramic carriers effected 97% degradation at 24 and 18 h, respectively. Recycling of the ceramic carriers was also possible and resulted in an even shorter retention time of 12h to effect a 97% degradation of benzene. Cell adhesion on the support materials was confirmed by scanning electron microscopy.     

New application of single and mixed immobilized cells for furfural biodegradation

In this study, a new application of immobilized microbial cells for biodegradation of furfural in aqueous solution was investigated using spouted bed bioreactor. Pseudomonas sp., as a single type specie as well as activated sludge as mixed cultures were individually immobilized in 3 different bio-carrier matrices which were prepared by reinforcement of natural polysaccharides including sodium alginate, guar-gum and agar-agar with polyvinyl alcohol. The results demonstrated a complete removal (100%) of furfural from aqueous solutions using immobilized cells (IC) of Pseudomonas sp., and mixed cultures as well. Recycling of used IC for furfural removal in successive treatment cycles provided significant removal rates up to 96%. In general, results revealed that IC exhibited better performance compared to free cells in regard with the removal rate of furfural, duration of biodegradation process, as well as the ability for recycling and sustaining the high concentrations of furfural.     

Microbial transformation of validamycin A to valienamine by immobilized cells

Immobilized Pseudomonas sp. HZ519 cells have been used for transformation of validamycin A to valienamine and the degradation pathway of validamycin A by Pseudomonas sp. HZ519 has also been studied. Substrate inhibition in immobilized cell system was avoided. An average of 8.6 g L^?1 valienamine concentration was obtained when concentration of validamycin A was increased up to 120 g L^?1. Through a treatment of the immobilized cells with 0.3 mol L^?1 substrate, the activity of the immobilized cells was increased distinctly. Compared with free cells, the productivity of valienamine by CA-immobilized cells was improved about three times. The reusability of the immobilized cells was evaluated with repeated–batch degradation experiments. The Tiele modulus was obtained from the experimental effectiveness factor. The result showed that the degradation process in the immobilized system was governed by intraparticle diffusion and chemical reaction.     

Microbial transformation of validamycin A to valienamine by immobilized cells

Immobilized Pseudomonas sp. HZ519 cells have been used for transformation of validamycin A to valienamine and the degradation pathway of validamycin A by Pseudomonas sp. HZ519 has also been studied. Substrate inhibition in immobilized cell system was avoided. An average of 8.6 g L^-1 valienamine concentration was obtained when concentration of validamycin A was increased up to 120 g L^-1. Through a treatment of the immobilized cells with 0.3 mol L^-1 substrate, the activity of the immobilized cells was increased distinctly. Compared with free cells, the productivity of valienamine by CA-immobilized cells was improved about three times. The reusability of the immobilized cells was evaluated with repeated-batch degradation experiments. The Tiele modulus was obtained from the experimental effectiveness factor. The result showed that the degradation process in the immobilized system was governed by intraparticle diffusion and chemical reaction.     

Biosensor for acrylamide based on an ion-selective electrode using whole cells of Pseudomonas aeruginosa containing amidase activity

The aim of this work was to develop a biosensor for toxic amides using whole cells of Pseudomonas. aeruginosa containing amidase activity, which catalyses the hydrolysis of amides such as acrylamide producing ammonia and the corresponding organic acid. Whole cells immobilized in several types of membrane in the presence of glutaraldehyde and an ammonium ion-selective electrode, were used for biosensor development. This biosensor exhibited a linear response in the range of 0.1–4.0×10^?3 M of acrylamide, a detection limit of 4.48×10^?5 M acrylamide, a response time of 55 s, a sensitivity of 58.99 mV mM^?1 of acrylamide and a maximum t_1/2 of 54 days. The selectivity of this biosensor towards other amides was investigated, which revealed that it cross-reacted with acetamide and formamide, but no activity was detected with phenylacetamide, p-nitrophenylacetamide and acetanilide. It was successfully used for quantification of acrylamide in real industrial effluents and recovery experiments were carried out which revealed an average substrate recovery of 93.3%. The biosensor is cheap since whole cells of P. aeruginosa can be used as source of amidase activity.     

Influence of phenol on the biodegradation of pyridine by freely suspended and immobilized Pseudomonas putida MK1

AIMS: To study the effect of co-contaminants (phenol) on the biodegradation of pyridine by freely suspended and calcium alginate immobilized bacteria. METHODS AND RESULTS: Varying concentrations of phenol were added to free and calcium alginate immobilized Pseudomonas putida MK1 (KCTC 12283) to examine the effect of this pollutant on pyridine degradation. When the concentration of phenol reached 0.38 g l(-1), pyridine degradation by freely suspended bacteria was inhibited. The increased inhibition with the higher phenol levels was apparent in increased lag times. Pyridine degradation was essentially completely inhibited at 0.5 g l(-1) phenol. However, immobilized cells showed tolerance against 0.5 g l(-1) phenol and pyridine degradation by immobilized cell could be achieved. CONCLUSIONS: This works shows that calcium alginate immobilization of microbial cells can effectively increase the tolerance of P. putida MK1 to phenol and results in increased degradation of pyridine. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of wastewater stream can be negatively affected by the presence of co-pollutants. This work demonstrates the potential of calcium alginate immobilization of microbes to protect cells against compound toxicity resulting in an increase in pollutant degradation.     

Novel strategy for biodegradation of 4-nitrophenol by the immobilized cells of Pseudomonas sp. YPS3 with Acacia gum

The mass organic compound 4-nitrophenol with low molecular is involved in many chemicals processes and most common organic pollutants. 4-Nitrophenol (4-NP) existing in soils and water bodies, thereby causing severe environmental impact and health risk. Even low concentrations are harmful to health and potential mutagenic and carcinogenic. Though the existing methods of biodegradation though effective, their popularity is hindered due to high cost. Hence, in the present study a less expensive method involving the use of Pseudomonas sp. with gum arabic (PAA) was tested. The biodegradation of 4-NP was thoroughly investigated by progressive characterization methods. The promising Pseudomonas sp. YPS 3 was identified with biochemical and molecular identification process. The average particle sizes of stable crystalline PAA was 8–20 nm. The experiments were conducted with optimized parameters viz., pH (7.0), concentration (30 ppm), temperature (37 °C) and time (6 h). The study was tested as adsorbent particle size on 4-NP concurrent adsorption-biodegradation. In addition, these Pseudomonas sp. YPS3 and its PAA are used as an eco-friendly for removal of toxic organic 4-NP pollutant from the ecosystems.     

Degradation kinetics of phenol by immobilized cells of Candida tropicalis in a fluidized bed reactor

Degradation kinetics of phenol by free and agar-entrapped cells of Candida tropicalis was studied in batch cultures. The initial phenol degradation rate achieved with free cells was higher than that obtained with immobilized cells, when phenol concentrations up to 1000 mg l^–1 were used. However, at higher phenol concentrations, the behaviour was quite different. The initial degradation rate of the immobilized yeast cells was about 10 times higher than that of the free cells, at a phenol concentration of 3500 mg l^–1. The semicontinuous and continuous degradation of phenol by immobilized yeast cells was also investigated in a multi-stage fluidized bed reactor. The highest phenol removal efficiencies and degradation rates as well as the lowest values of residual phenol and chemical oxygen demand were obtained in the semicontinuous culture when phenol concentrations up to 1560 mg l^–1 were used.     

19F NMR study on the biodegradation of fluorophenols by various Rhodococcus species

Of all NMR observable isotopes 19F is the one perhaps most convenient for studies on biodegradation of environmental pollutants. The reasons underlying this potential of 19F NMR are discussed and illustrated on the basis of a study on the biodegradation of fluorophenols by four Rhodococcus strains. The results indicate marked differences between the biodegradation pathways of fluorophenols among the various Rhodococcus species. This holds not only for the level and nature of the fluorinated biodegradation pathway intermediates that accumulate, but also for the regioselectivity of the initial hydroxylation step. Several of the Rhodococcus species contain a phenol hydroxylase that catalyses the oxidative defluorination of ortho-fluorinated di- and trifluorophenols. Furthermore, it is illustrated how the 19F NMR technique can be used as a tool in the process of identification of an accumulated unknown metabolite, in this case most likely 5-fluoromaleylacetate. Altogether, the 19F NMR technique proved valid to obtain detailed information on the microbial biodegradation pathways of fluorinated organics, but also to provide information on the specificity of enzymes generally considered unstable and, for this reason, not much studied so far.     

Effect of elicitation on the accumulation of solasodine by immobilized cells of Solanum eleagnifolium Cav.

The effect of a fungal elicitor obtained from Alternaria sp. on growth and solasodine production by free and alginate-entrapped cells of Solanum eleagnifolium Cav. was studied. Fourteen-day-old cultures were elicited with 1% FW/V autoclaved homogenates. The solasodine production increased from 0.9 to 1.5 mg g^-1 DW (65%) in suspension cultures and from 0.75 to 1.4 mg g^-1 DW (about 95%) in entrapped cells. The maximum accumulation was obtained after 72 h of elicitation. In order to induce alkaloid release from cells (suspension and entrapped cells), permeabilization with 10% dimethylsulfoxide (DMSO) for 30 min was used. In both cases (free and entrapped cells), about 50–60% of intracellular solasodine was released into the medium. The reuse of elicited and permeabilized entrapped cells was also carried out for three production cycles.     

Influence of phenol on biodegradation of p-nitrophenol by freely suspended and immobilized Nocardioides sp. NSP41

The effect of the presence of an alternate toxiccompound (phenol) on the p-nitrophenol(PNP)-degrading activity of freely suspended andcalcium alginate immobilized Nocardioides sp.NSP41 was investigated. In the single substrateexperiments, when the concentration of phenol and PNPwas increased to 1400 mg l^-1 and 400 mg l^-1,respectively, the initial cell concentrations in thefreely suspended cell culture should be higher than1.5 g dry cell weight l^-1 for completedegradation. In the simultaneous degradationexperiment, when the initial concentration of phenolwas increased from 100 to 400 mg l^-1, thespecific PNP degradation rate at the concentration of200 mg l^-1 was decreased from 0.028 to 0.021h^-1. A freely suspended cell culture with a highinitial cell concentration resulted in a highvolumetric degradation rate, suggesting the potentialuse of immobilized cells for simultaneous degradation.In the immobilized cell cultures, althoughsimultaneous degradation of PNP and phenol wasmaintained, the specific PNP and phenol degradationrate decreased. However, a high volumetric PNP andphenol degradation rate could be achieved byimmobilization because of the high cell concentration.Furthermore, when the immobilized cells were reused inthe simultaneous degradation of PNP and phenol, theydid not lose their PNP- and phenol-degrading activityfor 12 times in semi-continuous cultures. Takentogether, the use of immobilized Nocardioidessp. NSP41 for the simultaneous degradation of PNP andphenol at high concentrations is quite feasiblebecause of the high volumetric PNP and phenoldegradation rate and the reusability of immobilizedcells.     

Production of acrylamide using immobilized cells of<Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> M33

The cellsof Rhodococcus rhodochrous M33, which produce a nitrile hydratase enzyme, were immobilized in acrylamide-based polymer gels. The optimum pH and temperature for the activity of nitrile hydratase in both the free and immobilized cells were 7.4 and 45°C, respectively, yet the optinum temperature for acrylamide production by the immobilized cells was 20°C. The nitrile hydratase of the immobilized cells was more stable with acrylamide than that of the free cells. Under optimal conditions, the final acrylamide concentration reached about 400 g/L with a conversion yield of almost 100% after 8 h of reaction when using 150 g/L of immobilized cells corresponding to a 1.91 g-dry cell weight/L. The enzyme activity of the immobilized cells rapidly decreased with repeated use. However, the quality of the acrylamide produced by the immobilized cells was much better than that produced by the free cells in terms of color, salt content, turbidity, and foam formation. The quality of the aqueous acrylamide solution obtained was found to be of commercial use without further purification.     

Kinetics of biodegradation of p-xylene and naphthalene and oxygen transfer in a novel airlift immobilized bioreactor

The scope of this study included the biodegradation performance and the rate of oxygen transfer in a pilot-scale immobilized soil bioreactor system (ISBR) of 10-L working volume. The ISBR was inoculated with an acclimatized population of contaminant degrading microorganisms. Immobilization of microorganisms on a non-woven polyester textile developed the active biofilm, thereby obtaining biodegradation rates of 81 mg/L x h and 40 mg/L x h for p-xylene and naphthalene, respectively. Monod kinetic model was found to be suitable to correlate the experimental data obtained during the course of batch and continuous operations. Oxygen uptake and transfer rates were determined during the batch biodegradation process. The dynamic gassing-out method was used to determine the oxygen uptake rate (OUR) and volumetric oxygen mass transfer, K(L) a. The maximum volumetric OUR of 255 mg O(2)/L x h occurred approximately at 720-722 h after inoculation, when the dry weight of biomass concentration was 0.67 g/L.     

Ethanol production from whey permeate by immobilized yeast cells

The ability of two yeast strains to utilize the lactose in whey permeate has been studied. Kluyveromyces marxianus NCYC 179 completely utilized the lactose (9.8%), whereas Saccharomyces cerevisiae NCYC 240 displayed an inability to metabolize whey lactose for ethanol production. Of the two gel matrices tested for immobilizing K. marxianus NCYC 179 cells, sodium alginate at 2% (w/v) concentration proved to be the optimum gel for entrapping the yeast cells effectively. The data on optimization of physiological conditions of fermentation (temperature, pH, ethanol concentration and substrate concentration) showed similar effects on immobilized and free cell suspensions of K. marxianus NCYC 179, in batch fermentation. A maximum yield of 42.6 g ethanol l^?1 (82% of theoretical) was obtained from 98 g lactose l^?1 when fermentation was carried at pH 5.5 and 30°C using 120 g dry weight l^?1 cell load of yeast cells. These results suggest that whey lactose can be metabolized effectively for ethanol production using immobilized K. marxianus NCYC 179 cells.     

PVA-卡拉胶混合载体固定化大肠杆菌-酵母菌混合体系生产谷胱甘肽

提出了ＰＶＡ－卡拉胶混合载体固定化微生物细胞的技术,确定了较好的制备工艺,并用该载体对大肠杆菌－酵母菌混合体系进行了固定化研究。结果表明：在ＰＶＡ浓度１０％,卡拉胶浓度０．５％,成型剂的ｐＨ值６．４,菌体量０．５ｇ／ｇ固定化细胞,固化时间３６ｈ的条件下,固定化细胞具有较好的机械强度和较高的酶活力。     

酰胺生物降解机制的分子模拟研究

为探索酰胺生物降解酶的微观降解机制,用分子对接的方法模拟了酰胺与酰胺酶的相互作用,得到其复合物结构的理论模型,根据打分函数最低原则筛选出的RhAmidase与L-Methioninamide之间最佳构象打分函数为-86.741 9,二次打分函数为-76.022 4。同时,应用LPC/CSU Server研究了最佳构象的相互作用情况,结果表明,酰胺与酰胺酶之间以疏水作用数量最多,酰胺酶的ARG256 A、LEU353 A、TYR346 A、ARG225 A、THR218 A和PRO222 A在催化过程中起到了重要作用。     

Biotransformation of codeinone to codeine by immobilized cells of papaver somniferum

Papaver somniferum (opium poppy) cells were immobilized in calcium alginate, where they continued to live with their biological activity for 6 months. The immobilized living cells performed the biotransformation of (?)-codeinone to (?)-codeine in both a shake flask and a column bioreactor. The biotransformation ratio in the shake flask (70.4%) was higher than that in the cell suspension (60.8%). Furthermore, 88% of the codeine converted was excreted in the medium. The column bioreactor was functional for 30 days under optimal conditions (20°, 3.75 vvm in aeration), whereas the ratio was 41.9%.     

Degradation of benzotrifluoride via the dioxygenase pathway in Rhodococcus sp. 065240

We previously isolated Rhodococcus sp. 065240, which catalyzes the defluorination of benzotrifluoride (BTF). In order to investigate the mechanism of this degradation of BTF, we performed proteomic analysis of cells grown with or without BTF. Three proteins, which resemble dioxygenase pathway enzymes responsible for isopropylbenzene degradation from Rhodococcus erythropolis BD2, were induced by BTF. Genomic PCR and DNA sequence analysis revealed that the Rhodococcus sp. 065240 carries the gene cluster, btf, which is highly homologous to the ipb gene cluster from R. erythropolis BD2. A mutant strain, which could not catalyze BTF defluorination, was isolated from 065240 strain by UV mutagenesis. The mutant strain had one mutation in the btfT gene, which encodes a response regulator of the two component system. The defluorinating ability of the mutant strain was recovered by complementation of btfT. These results suggest that the btf gene cluster is responsible for degradation of BTF.