Degradation of phenol by polymer entrapped microorganisms

Summary A Pseudomonas sp. which was isolated from phenol-containing soil was immobilized in alginate and polyacrylamide-hydrazide (PAAH) and cultivated in a special airlift fermenter.The immobilized Pseudomonas sp. was able to degrade phenol at initial concentrations up to 2 g/l in less than 2 days, although the free cells did not grow at this concentration.The immobilization materials act as a protective cover against phenol, PAAH being more effective than alginate. The degradation activity as well as the outgrowth of bacteria can be manipulated by the concentration of the immobilization material, the temperature and the nitrogen content in the medium.The cells grew predominantly in microcolonies in the outer area of the beads when nitrogen was available as 1.0g NH₄NO₃/l and 0.5g (NH₄)₂SO₄/l.Prof. Dr. A. Fiechter dedicated to his 60th birthday     

Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel beads.     

Biological control of phytophagous ladybird beetles Epilachna vigintioctopunctata (Col., Coccinellidae) by chitinolytic phylloplane bacteria Alcaligenes paradoxus entrapped in alginate beads

The chitinase secreting strain KPM‐012A of Alcaligenes paradoxus was isolated from tomato leaves and vitally entrapped in sodium alginate gel beads to provide a new method for biocontrol of phytophagous ladybird beetles Epilachna vigintioctopunctata. First, the peritrophic membrane was dissected from the adult ladybird beetles that ingested the suspension of KPM‐012A after starvation to observe degradation of the midgut surface by the bacteria under electron microscopy. The peritrophic membrane around the bacteria was degraded, suggesting the release of chitinase from the ingested bacteria. Large amounts of chitinase were successfully released from KPM‐012A‐entrapped calcium alginate beads. This chitinase release from the microbial beads was sustained for 1 week and was sufficient to digest the peritrophic membrane. Daily supply of tomato leaves treated with the microbial beads caused considerable suppression of leaf feeding and oviposition by the adult ladybird beetles, indicating that this method is effective for decreasing population of insect pests in the subsequent generation. Thus, the present study provided an experimental basis for the biocontrol measures of herbivorous insect pests by the chitinolytic bacteria entrapped in alginate beads.     

Alginate beads provide a beneficial physical barrier against native microorganisms in wastewater treated with immobilized bacteria and microalgae

When the freshwater microalga Chlorella sorokiniana and the plant growth-promoting bacterium Azospirillum brasilense were deployed as free suspensions in unsterile, municipal wastewater for tertiary wastewater treatment, their population was significantly lower compared with their populations in sterile wastewater. At the same time, the numbers of natural microfauna and wastewater bacteria increased. Immobilization of C. sorokiniana and A. brasilense in small (2–4 mm in diameter), polymer Ca-alginate beads significantly enhanced their populations when these beads were suspended in normal wastewater. All microbial populations within and on the surface of the beads were evaluated by quantitative fluorescence in situ hybridization combined with scanning electron microscopy and direct measurements. Submerging immobilizing beads in wastewater created the following sequence of events: (a) a biofilm composed of wastewater bacteria and A. brasilense was created on the surface of the beads, (b) the bead inhibited penetration of outside organisms into the beads, (c) the bead inhibited liberation of the immobilized microorganisms into the wastewater, and (d) permitted an uninterrupted reduction of ammonium and phosphorus from the wastewater. This study demonstrated that wastewater microbial populations are responsible for decreasing populations of biological agents used for wastewater treatment and immobilization in alginate beads provided a protective environment for these agents to carry out uninterrupted tertiary wastewater treatment.     

Stabilization of Aspergillus parasiticus cytosine deaminase by immobilization on calcium alginate beads improved enzyme operational stability

Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life of 1.10?h at 37°C, was stabilized by immobilization on calcium alginate beads. The immobilized CD had pH and temperature optimum of 5 and 50°C respectively. The immobilized enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the apparent K_M values of 0.60?mM and 0.65?mM respectively, displaying activation energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads gave the highest yield of apparent enzymatic activity of 51.60% of the original activity and the enzymatic activity was lost exponentially at 37°C over 12?h with a half-life of 5.80?h. Hence, the operational stability of native CD can be improved by immobilization on calcium alginate beads.     

Improvement of biodesulfurization activity of alginate immobilized cells in biphasic systems

The immobilization of Pseudomonas delafieldii R-8 in calcium alginate beads has been studied in order to improve biodesulfurization activity in oil/water (O/W) biphasic systems. A gas jet extrusion technique was performed to produce immobilized beads. The specific desulfurization rate of 1.5 mm diameter beads was 1.4-fold higher than that of 4.0 mm. Some nonionic surfactants can significantly increase the activity of immobilized cells. The desulfurization rate with the addition of 0.5% Span 80 increased 1.8-fold compared with that of the untreated beads. The rate of biodesulfurization was markedly enhanced by decreasing the size of alginate beads and adding the surfactant Span 80, most likely resulting from the increasing mass transfer of substrate to gel matrix.     

Use of immobilized cells of the strainCorynebacterium sp. for 4-nitrophenol degradation

4-Nitrophenol degrading bacterial strainCorynebacterium sp. 8/3 was isolated from chemically polluted soil. The product of cometabolic transformation of 4-nitrophenol was identified as 4-nitrocatechol., Effect of immobilization (encapsulation in calcium alginate) ofCorynebacterium sp. cells on the process of 4-nitrophenol transformation was investigated. 4-Nitrophenol was converted by encapsulated cells and encapsulation had a protective effect, on 4-nitrophenol degrading bacteria in repeated cycles of incubation. Transformation of 4-nitrophenol to 4-nitrocatechol by encapsulated cells was influenced by pH of medium but was not influenced by concentration of alginate and CaCl₂. The count of viable cells in alginate beads declined approximately by one order of magnitude after 10 d of incubation. Presented at the 4th Mini-Symposium on Biosorption and Microbial Degradation, Prague, Czech Republic, November 26–29, 1996.     

Immobilization of whole cells ofStreptomyces kanamyceticus containing glucose isomerase by a combination of heat treatment in the presence of minerals and entrapment in calcium alginate gels

A method of immobilization of whole cells ofStreptomyces kanamyceticus containing glucose isomerase was devised, based on techniques of heat fixation in the presence of minerals and, entrapment in calcium alginate gels. The optimum activity of the enzyme was obtained when the cells were heat-fixed at 60°C for 10 min in the presence of 50 mmol/L MgSO₄·7H₂O and 5 mmol/L CoCl₂·6H₂O and then cast into calcium alginate beads using 2% sodium alginate.     

Immobilization of laccase from Streptomyces psammoticus and its application in phenol removal using packed bed reactor

Laccase was produced from Streptomyces psammoticus under solid-state fermentation. The enzyme was partially purified by ammonium sulphate precipitation and was immobilized in alginate beads by entrapment method. Calcium alginate beads retained 42.5% laccase activity, while copper alginate beads proved a better support for laccase immobilization by retaining 61% of the activity. Phenol and colour removal from a phenol model solution was carried out using immobilized laccase. Batch experiments were performed using packed bed bioreactor, containing immobilized beads. Reusability of the immobilized matrix was studied for up to 8 successive runs, each run with duration of 6 h. The system removed 72% of the colour and 69.9% of total phenolics from the phenol model solution after the initial run. The immobilized system maintained 50% of its efficiency after eight successive runs. The degradation of phenolic compounds by immobilized laccase was evaluated and confirmed by Thin layer chromatography and nuclear magnetic resonance spectroscopy.     

包埋法固定化对硫氧化微生物菌群结构和功能的影响

【目的】为探讨包埋法固定化过程对硫氧化菌群硫化物去除能力及菌群微生物群落结构的影响,【方法】以聚乙烯醇-海藻酸钠-活性炭为载体,对硫氧化菌群进行了固定化,并采用富含硫化物的无机盐培养基,对比固定化与非固定化硫氧化菌群对硫化物的氧化去除能力。同时,利用PCR-DGGE技术,探讨硫氧化菌群在固定化前后以及在硫化物氧化去除过程中微生物群落结构变化。【结果】在对硫氧化菌群进行固定化之后,12 h之内对硫化物的最大去除能力从1000 mg/L下降为600 mg/L。硫氧化菌群的微生物群落结构发生了明显变化,但菌群中的硫氧化菌Catenococcus thiocycli未受影响,硫氧化菌Thioclava pacifica在菌群中的地位反而得到了强化。【结论】受制于底物在载体材料中的扩散迁移效率,硫氧化菌群对硫化物的氧化去除能力在固定化之后有所下降。由于不同微生物对固定化形成的微环境的适应能力以及对载体附着能力的不同,固定化对硫氧化菌群的微生物群落结构产生较大影响。     

Increasing the stability of immobilizedLactococcus lactis cultures stored at 4 °C

Summary Immobilized cell technology was used to prepare concentrated cultures ofLactococcus lactis that lost only 22% of viability over a 30-day storage period at 4°C. Concentrated cultures ofL lactis CRA-1 were immobilized in calcium alginate beads and added to glycerol, NaCl or sucrose-NaCl solutions in order to obtain a_w readings ranging from 0.91 to 0.97. The suspensions were subsequently placed at 4°C and viability (CFU g^–1 of bead) was followed during storage. Viability losses were high at a_w readings of 0.95 and 0.97 and pH dropped significantly (up to one unit) in the unbuffered solutions. Addition of 1% soytone or glycerophosphate helphed stabilize pH, and a beneficial effect on viability during storage was observed in the glycerol-soytone mix when the beads were added to the conservation solutions immediately following immobilization. When beads were added to the conservation solution immediately following immobilization, a 70% drop in cell counts occurred during the first 5 days of incubation. Dipping theL lactis-carrying beads in milk for 2h before mixing with the glycerolsoytone 0.93 a_w solution reduced this initial 5-day viability loss. Cultures grown in the alginate beads also had good stability in the 0.93 a_w glycerol-soytone solution, where 78% of the population was viable after 30 days at 4°C. The process could be used to store immobilized cells at a processing plant, or by suppliers of lactic starters who wish to ship cultures without freezing or drying.     

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 l (+) lactic acid by Lactobacillus delbrueckii immobilized in functionalized alginate matrices

The role of functionalized alginate gels as immobilized matrices in production of l (+) lactic acid by Lactobacillus delbrueckii was studied. L. delbrueckii cells immobilized in functionalized alginate beads showed enhanced bead stability and selectivity towards production of optically pure l (+) lactic acid in higher yields (1.74Yp/s) compared to natural alginate. Palmitoylated alginate beads revealed 99% enantiomeric selectivity (ee) in production of l (+) lactic acid. Metabolite analysis during fermentation indicated low by-product (acetic acid, propionic acid and ethanol) formation on repeated batch fermentation with functionalized immobilized microbial cells. The scanning electron microscopic studies showed dense entrapped microbial cell biomass in modified immobilized beads compared to native alginate. Thus the methodology has great importance in large-scale production of optically pure lactic acid.     

Optimization of α‐Amylase Immobilization in Calcium Alginate Beads

Abstract

α‐Amylase enzyme was produced by Aspergillus sclerotiorum under SSF conditions, and immobilized in calcium alginate beads. Effects of immobilization conditions, such as alginate concentration, CaCl₂ concentration, amount of loading enzyme, bead size, and amount of beads, on enzymatic activity were investigated. Optimum alginate and CaCl₂ concentration were found to be 3% (w/v). Using a loading enzyme concentration of 140 U mL^?1, and bead (diameter 3 mm) amount of 0.5 g, maximum enzyme activity was observed. Beads prepared at optimum immobilization conditions were suitable for up to 7 repeated uses, losing only 35% of their initial activity. Among the various starches tested, the highest enzyme activity (96.2%) was determined in soluble potato starch hydrolysis for 120 min at 40°C.     

Carbon nanotubes promote Cr(VI) reduction by alginate-immobilized Shewanella oneidensis MR-1

Bioreduction of hexavalent chromium (Cr(VI)) into trivalent one (Cr(III)) based on microbial immobilization techniques has been recognized as a promising way to remove Cr contaminants from wastewater. However, such a bioreduction process is inefficient due to limited electron transfer through the immobilization matrix. In this study, a modified immobilization process was proposed by impregnating carbon nanotubes (CNTs) into Ca-alginate beads, which were then used to immobilize Shewanella oneidensis MR-1 for enhanced Cr(VI) reduction. Compared with the free cells and the beads without CNTs, the AL/CNT/cell beads showed up to 4 times higher reduction rates, mainly attributed to an enhanced electron transfer by the CNTs. In addition, the dose of CNTs greatly improved the stability of beads, suggesting a high feasibility of the AL/CNT/cell beads for repeated use. The optimized CNT concentration, temperature and pH for Cr(VI) reduction by the AL/CNT/cell beads were 0.5%, 30 °C and 6.0–7.0, respectively.     

Cell immobilization using PVA crosslinked with boric acid

A new cell immobilization technique is described in which polyvinyl alcohol is crosslinked with boric acid, with the addition of a small amount of calcium alginate. The presence of the calcium alginate improves the surface properties of the beads, preventing agglomeration. A pure culture of phenol-degrading Pseudomonas was immobilized in the PVA-alginate beads. Phenol was successfully degraded in a fluidized bed of the beads, indicating that cell viability was maintained following the immobilization procedure. The PVA-alginate beads proved to be very strong and durable, with no noticeable degradation of the beads after 2 weeks of continuous operation of the fluidized bed.     

A new method for immobilization of acetylcholinesterase

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V _max) and Michaelis–Menten constant (K _m) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca–alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 °C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.     

Microbial transformations of N-(4-chlorphenyl)-benzoisothiazolone

Summary The microbial degradation of N-(4-chlorphenyl)-benzoisothiazolone (1) was studied by Streptomyces species in analogy to the metabolism of this drug in animals. As main metabolite 2-thiomethyl-N-(4-chlorphenyl)-benzamide (3) was found. The corresponding sulfoxide (4) and the sulfone (5) were obtained as further transformation products. The unmethylated 2-sulfhydryl compound (2) could be isolated in only small amounts. The degradation pathway follows by these results via a reductive cleavage of the S-N-bond of the isothiazolone ring with subsequent methylation of the sulfhydryl group and further oxidation of the resulting thiomethyl substituent.     

Resolution of N-(2-ethyl-6-methylphenyl) alanine by using microgel beads containing Pseudomonas cepacia lipase

Abstract

Pseudomonas cepacia lipase (PCL) was immobilized in alginate microgel beads by electrostatic dispersion. The high electrical potential applied in the immobilization process could significantly decrease the droplet size. The optimum conditions for lipase immobilization were 2% (w/v) alginate, 100 mM CaCl₂, 8 mg/mL enzyme, 4 kV electrical potential and 200 μm mean bead size. Under these conditions, 78.2 U/g of immobilized PCL activity was obtained with 39.1% retained activity and 57.2% immobilization efficiency. The immobilized PCL (PCL-CA) was subsequently used in the enantioselective hydrolysis of (R, S)-N-(2-ethyl-6-methylphenyl) alanine methyl ester. Although PCL-CA exhibited slightly lower activity than free PCL, it preserved the high enantioselectivity (E-value >?200), which afforded enantiomerically pure (R)-acid (99% e.e._p). Furthermore, PCL-CA exhibited higher thermal stability, storage and medium stability than that of free PCL. Batch-wise operational stability studies demonstrated that PCL-CA retained its initial activity for at least 10 cycles of hydrolysis.     

Degradation of 2,4,6-Trichlorophenol by Phanerochaete chrysosporium: Involvement of Reductive Dechlorination

Under secondary metabolic conditions, the lignin-degrading basidiomycete Phanerochaete chrysosporium mineralizes 2,4,6-trichlorophenol. The pathway for the degradation of 2,4,6-trichlorophenol has been elucidated by the characterization of fungal metabolites and oxidation products generated by purified lignin peroxidase (LiP) and manganese peroxidase (MnP). The multistep pathway is initiated by a LiP- or MnP-catalyzed oxidative dechlorination reaction to produce 2,6-dichloro-1,4-benzoquinone. The quinone is reduced to 2,6-dichloro-1,4-dihydroxybenzene, which is reductively dechlorinated to yield 2-chloro-1,4-dihydroxybenzene. The latter is degraded further by one of two parallel pathways: it either undergoes further reductive dechlorination to yield 1,4-hydroquinone, which is ortho-hydroxylated to produce 1,2,4-trihydroxybenzene, or is hydroxylated to yield 5-chloro-1,2,4-trihydroxybenzene, which is reductively dechlorinated to produce the common key metabolite 1,2,4-trihydroxybenzene. Presumably, the latter is ring cleaved with subsequent degradation to CO₂. In this pathway, the chlorine at C-4 is oxidatively dechlorinated, whereas the other chlorines are removed by a reductive process in which chlorine is replaced by hydrogen. Apparently, all three chlorine atoms are removed prior to ring cleavage. To our knowledge, this is the first reported example of aromatic reductive dechlorination by a eukaryote.