Verification of enhanced phosphate removal capability in pure cultures of<Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> under anaerobic/aerobic conditions in an SBR

Laboratory experiments were conducted using pure cultures ofAcinetobacter under anaerobic/aerobic cyclic conditions to explain the release and uptake of soluble phosphate in an activated sludge process showing enhanced biological phosphate removal (EBPR). Under anaerobic/aerobic cyclic conditions in a Sequencing Batch Reactor (SBR), COD uptake concurrent with soluble phosphate release byAcinetobacter was not significant during the anaerobic periods, indicating that EBPR would not be established in pure cultures. However,Acinetobacter cells accumulated higher phosphate content (5.2%) in SBR than that obtained (4.3%) from batch experiments. These results suggest thatAcinetobacter sp. may not follow the proposed pattern of behavior of poly-P bacteria in EBPR activated sludge plants.     

Phosphorus removal by pure and mixed cultures of microorganisms

The ability to remove inorganic phosphate from synthetic wastewater was tested with about 40 microbial strains, and Pseudomonas aeruginosa IAM 1007 was found to give good performance under aerobic conditions. However, the phosphate removal under batch anaerobic/aerobic (A/O) treatment was not satisfactory in pure cultures of several strains including P. aerginosa, and Aceinetobacter calcoaceticus, but the activated sludge from a plant with an A/O process almost depleted the phosphate. Mixed cultures of P. aeruginosa in the presence of the facultativelu anaerobic strains of A-1 or A-8 isolated from the activated sludge showed enhanced phosphate removal. This suggests a symbiotic effect among microbial species on biological removal of inorganic phosphate in the A/O process.     

Role of anaerobiosis in virulence of Salmonella typhimuirium

Intestinal pathogens are exposed to various stress conditions during their infectious cycle. Anaerobiosis, one of such hostile condition, is offered by the host within gut and intestinal lumen, where survival, multiplication and entry into intestinal epithelial cells is priority for the invading pathogen. In the present study, a virulent strain of S. typhimurium (1402/84) was grown under anaerobic conditions and its virulence characteristics such as host cell binding, penetration and intracellular survival were compared with aerobic S. typhimurium. Anaerobically grown S. typhimurium showed significantly higher binding to immobilized mice enterocytes and intestinal mucus as compared to bacteria grown aerobically. Anaerobic bacteria also showed an early penetration of mucus and subsequent binding to underlying immobilized enterocytes, in vitro. Anaerobic S. typhimurium exhibited increased intracellular survival within spleen macrophages of mice and caused significantly higher fluid accumulation in ligated rabbit ileal loops as compared to aerobic bacteria. LD₅₀ of anaerobic S. typhimurium was also observed to be 2 fold lower when compared to aerobic bacteria. Cell surface hydrophobicity of anaerobic S. typhimurium was also found to be significantly higher than aerobic bacteria. Thus, it appears that exposure of S. typhimurium to anaerobiosis results in its enhanced virulence, adhesion and penetration of host cells.     

Simultaneous anaerobic and aerobic degradation of the sulfonated azo dye Mordant Yellow 3 by immobilized cells from a naphthalenesulfonate-degrading mixed culture

The naphthalenesulfonate-oxidizing bacterium Sphingomonas sp. BN6 was immobilized in calcium alginate. These beads were incubated under aerobic conditions in a medium with the sulfonated azo dye, Mordant Yellow 3 (MY3), and glucose. The immobilized cells converted MY3, but only a marginal turnover of the dye was found under these conditions with freely suspended cells of Sphingomonas sp. BN6. Under anaerobic conditions, suspended cells of Sphingomonas sp. BN6 reductively cleaved the azo bond of MY3 to 6-aminonaphthalene-2-sulfonate (6A2NS) and 5-aminosalicylate. The turnover of MY3 by the immobilized cells under aerobic conditions resulted in the formation of more than equimolar amounts of 5-aminosalicylate, but almost no (6A2NS) was detected. Cells of Sphingomonas sp. BN6 aerobically oxidize 6A2NS to 5-aminosalicylate. It was therefore concluded that the cells in the anaerobic center of the alginate beads reduced MY3 to 6A2NS and 5-aminosalicylate and that 6A2NS was oxidized to 5-aminosalicylate by those cells that were immobilized in the outer aerobic zones of the alginate beads. The presence of oxygen gradients within the alginate beads was verified by using oxygen micro-electrodes. A coimmobilisate of Sphingomonas sp. BN6 with a 5-aminosalicylate degrading bacterium completely degraded MY3. The immobilized cells also converted the sulfonated azo dyes Amaranth and Acid Red␣1. Received: 6 May 1996 / Received revision: 6 August 1996 / Accepted: 12 August 1996     

Production of alkaline protease with immobilized cells of bacillus subtilis PE-11 in various matrices by entrapment technique

The purpose of this investigation was to study the effect ofBacillus subtilis PE-11 cells immobilized in various matrices, such as calcium alginate, k-Carrageenan, ployacrylamide, agar-agar, and gelatin, for the production of alkaline protease. Calcium alginate was found to be an effective and suitable matrix for higher alkaline protease productivity compared to the other matrices studied. All the matrices were selected for repeated batch fermentation. The average specific volumetric productivity with calcium alginate was 15.11 U/mL/hour, which was 79.03% higher production over the conventional free-cell fermentation. Similarly, the specific volumetric productivity by repeated batch fermentation was 13.68 U/mL/hour with k-Carrageenan, 12.44 U/mL/hour with agar-agar, 11.71 U/mL/hour with polyacrylamide, and 10.32 U/mL/hour with gelatin. In the repeated batch fermentations of the shake flasks, an optimum level of enzyme was maintained for 9 days using calcium alginate immobilized cells. From the results, it is concluded that the immobilized cells ofB subtilis PE-11 in calcium alginate are more efficient for the production of alkaline protease with repeated batch fermentation. The alginate immobilized cells ofB subtilis PE-11 can be proposed as an effective biocatalyst for repeated usage for maximum production of alkaline protease. Published: October 21, 2005     

Effects of immobilization on growth,substrate consumption, β-galactosidase induction,and byproduct formation inEscherichia coli

Summary Some metabolic properties of both suspended and immobilized aerobically and anaerobically growingEscherichia coli cells were investigated. Metabolic activity was found to be substantially different whenE. coli cells were immobilized in alginate. Cells grown immobilized in alginate, and then released from the gel, synthesized 1.6 (aerobic growth) and 4.9 (anaerobic growth) times as much -galactosidase per cell in response to induction as did suspended cells. Under both aerobic and anaerobic conditions, the cell yield from glycerol for immobilized cells was half that for suspended cells. At specific growth rates that were not significantly different from those of suspended cells, immobilized cells consumed glycerol at twice the rate of suspended cells. Immobilized cells produced elevated quantities of acetate, pyruvate, and lactate. Interpretation of these findings is discussed in terms of the kinetics of energy metabolism and the regulation of inducible protein synthesis inE. coli.     

Starter culture of <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> strain B for aerobic granulation

Aggregation of bacterial cells is used in formation of microbial granules. Aerobically grown microbial granules can be used as the bio-agents in the treatment of wastewater. However, there are problems with start up of microbial granulation and biosafety of this process. Aim of this research was selection and testing of safe microbial strain with high cell aggregation ability to shorten period of microbial granules formation. Five bacterial strains with cell aggregation index higher than 50% have been isolated from the granules. Strain of Pseudomonas veronii species was considered as most probably safe starter culture for granulation because other strains belonged to the species known as human pathogens. The microbial granules were formed after 3 days of cultivation in case when P. veronii strain B was applied to start-up aerobic granulation process using model wastewater. The granules were produced from activated sludge after 9 days of cultivation. Microbial aggregates produced from starter culture of P. veronii strain B were more compact (sludge volume index was 70 ml/g) than those produced from activated sludge (sludge volume index was 106 ml/g). It is a first proof that application of selected safe starter pure culture with high cell aggregation ability can accelerate and enhance formation of microbial granules.     

Utilization in alginate beads for Cu(II) and Ni(II) adsorption of an exopolysaccharide produced by <Emphasis Type="Italic">Chryseomonas luteola</Emphasis> TEM05

Copper and nickel adsorption onto calcium alginate, sodium alginate with an extracellular polysaccharide (EPS) produced by the activated sludge bacterium Chryseomonas luteola TEM05 and the immobilized C. luteola TEM05 from aqueous solutions were studied. After that, the multi metal ions containing these ions together were prepared and partial competitive adsorptions of these mixtures were also investigated. The metal adsorption of gel beads were carried out at pH 6.0, 25 °C. The maximum adsorption capacities in Langmuir isotherm for calcium alginate, calcium alginate + EPS, calcium alginate + C. luteola TEM05 and calcium alginate + EPS + C. luteola TEM05 were 1.505, 1.989, 1.976, 1.937 mmol/g dry weight for Cu(II) and 0.996, 1.224, 1.078, 1.219 mol/g dry weight for Ni(II), respectively.The competitive biosorption capacities of the carrier for all metal ions were lower than single conditions.     

Phosphate Release and Uptake by Pure Cultures of Acinetobacter sp.: Effect of the Volatile Fatty Acids Concentration

Phosphorus release and uptake by pure cultures of Acinetobacter strains were investigated under anaerobic and aerobic conditions respectively. Tests were performed to study the relationship between phosphorus release-storage reaction and behavior of extracellular organic substrates: acetic, propionic, and butyric acids have been used at four concentrations (50, 100, 500, and 1000 mg · L⁻¹) in the anaerobic step of biological phosphorus removal. The results obtained depend on the strain and the volatile fatty acid (VFA) used. Phosphorus released under anaerobic condition was not always related to the amount of VFA or phosphorus consumed. Phosphorus uptake (P-uptake) in the aerobic step was found to be independent of phosphorus release rates. The best phosphorus uptake rates were obtained by Acinetobacter lwoffi ATCC21130 and Acinetobacter calcoaceticus Genoespecie SUCT-5 with butyric acid as carbon source. Received: 20 May 1996 / Accepted: 8 July 1996     

Copper ions and the regulation ofSaccharomyces cerevisiae metallothionein genes under aerobic and anaerobic conditions

We have previously reported that theSaccharomyces cerevisiae CRS5 metallothionein gene is negatively regulated by oxygen. The mechanism of this repression was the focus of the current study. We observed that the aerobic repression ofCRS5 is rapid and occurs within minutes of exposing anaerobic cultures to air. Furthermore, theCUP1 metallothionein gene ofS. cerevisiae was found to be subject to a similar down-regulation of gene expression. We provide evidence that the aerobic repression of yeast metallothioneins involves copper ions and Ace1, the coppertrans-activator ofCUP1 andCRS5 gene expression. A functional Ace1 binding site was found to be necessary for the aerobic repression ofCRS5. Moreover, the aerobic down-regulation of the metallothioneins was abolished when cells were treated with elevated levels of copper. Our studies show that anaerobic cultures accumulate higher levels of copper than do aerobic cells and that this copper is rapidly lost when cells are exposed to air. In fact, the kinetics of this copper loss closely parallels the kinetics ofCUP1 andCRS5 gene repression. The yeast metallothionein genes, therefore, serve as excellent markers for variations in copper accumulation and homeostasis that occur in response to changes in the oxidative status of the cell.     

Ethanol production from sulphuric acid wood hydrolysate ofPinus radiata using free and immobilized cells ofPichia stipitis

Summary Ethanol was produced by a strain ofPichia stipitis adapted to an inhibitory acid wood hydrolysate ofPinus radiata. The best ethanol productivity for batch cultures was 0.21 g/l h at 0.7% ethanol. Varying culture conditions increased ethanol concentration to 0.76%, however the productivity decreased to 0.18 g/l h. A decrease in ethanol concentration in the culture fluid was noted late in the batch which suggested ethanol catabolism. Values of kinetic parameters (K _m,K _s, _max, andV _max) were evaluated for this system. The use of calcium alginate immobilized cells in a continuous-flow stirred tank reactor lead to enhanced fermentative performance, namely a maximum productivity of 0.27 g/l h and 1.13% ethanol yield. The immobilized cells in continuous flow reactors represent an attractive option for fermenting sugars released by sulphuric acid hydrolysis ofP. radiata wood.     

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

Summary 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.     

In situ measurement of algal growth potential in aquatic ecosystems by immobilized algae

Cells of the green algaSelenastrum capricornutum were immobilized in alginate beads. The alga was able to grow inside these beads without being grazed by zooplankton. For P-limited immobilized cells, however, a lower µ _m and initial slope of the Monod growth curve µ _m /K _s were found than for free cells.To study the feasibility of immobilized algae to estimate algal growth potentialin situ in aquatic ecosystems, a series of experiments were conducted in indoor model ecosystems (microcosms) and in a small stream. The use of immobilized algae allowed a continuous registration of algal growth potential integrated over periods with natural fluctuations in the environment. The method of encapsulation of the algae can, however, still be improved. The alginate matrix is exposed to marked degradation by microorganisms when incubated in polluted streams for a period longer than two weeks. The applicability of other types of matrices should be tested.Author for correspondence     

Intracellular Accumulation of the Monomeric Precursors of Polyphosphates and Polyhydroxyalkanoates in Acinetobacter calcoaceticusand Escherichia coliCells

The formation of polyhydroxyalkanoates granules in anaerobically grown Escherichia coliM-17 cells was found to be preceded by the intracellular accumulation of carbonic acids (predominantly, acetic acid), amounting to 9% of the cytosol. The intracellular concentration of acidic metabolites increased after the lyophilization of the bacterial biomass and decreased after its long-term storage (3.5–13.5 years). The decrease in the concentration of acidic metabolites is likely due to the dehydration of dimeric carbonic acids in the viscoelastic cytosol of resting bacterial cells. The hydrophobic obligately aerobic cells of Acinetobacter calcoaceticusIEGM 549 are able to utilize a wide range of growth substrates (from acetate and citrate to hydrophobic hydrocarbons), which is considerably wider than the range of the growth substrates of E. coli(predominantly, carbohydrates). The minimal essential and optimal concentrations of orthophosphates in the growth medium of A. calcoaceticuswere found to be tens of times lower than in the case of E. coli.The intracellular content of orthophosphates in A. calcoaceticuscells reached 35–77% of the total phosphorus content (P_total), providing for the intense synthesis of polyphosphates. The P_totalof the A. calcoaceticuscells grown in media with different proportions between the concentrations of acetate and phosphorus varied from 0.7 to 3.3%, averaging 2%. This value of P_totalis about two times higher than that observed for fermenting E. colicells. Lowering the cultivation temperature of A. calcoaceticusfrom 37–32 to 4°C augmented the accumulation of orthophosphates in the cytoplasm, presumably owing to a decreased requirement of growth processes for orthophosphate. In this case, if the concentration of phosphates in the cultivation medium was low, they were completely depleted.     

Semicontinuous production of red pigment by immobilized cells of<Emphasis Type="Italic">Bacillus</Emphasis> sp. BH-99 using column bioreactor

The semicontinuous production of red pigment by immobilized cells ofBacillus sp. BH-99 was investigated in comparison with free cells. The red pigment produced highest productivity under the conditions of aeration of 0.2 mL/min and 2 mm diameter of gel beads by using 3.0% sodium alginate. Semicontinuous production by immobilized cells showed the highest productivity with replacement of fresh production medium in every 72 h for fourth fermentation cycle following the conditions of red pigment productivity.     

Investigations on neomycin production with immobilized cells of<Emphasis Type="Italic">Streptomyces marinensis</Emphasis> Nuv-5 in calcium alginate matrix

The purpose of this investigation was to study the effect ofStreptomyces marinensis NUV-5 cells immobilized in calcium alginate for the production of neomycin. The effect of various parameters, such as the effect of alginate concentration (1%, 2%, 3%, 4%, and 5% wt/vol), the effect of cation (caCl₂, BaCl₂, and SrCl₂), the concentration of cation (0.01M, 0.125M, 0.25M, 0.375M, and 0.5M), the curing times (1, 6, 11, 16, and 21 hours), and the diameter of the bead (1.48, 2.16, 3.24, 4.46, and 5.44 mm), on neomycin production and bead stability were studied. The effect of maltose (4%, 3%, 2%, and 1% wt/vol) and sodium glutamate (0.6%, 0.3%, 0.15%, and 0.075%) wt/vol) concentration on neomycin production was also studied. Better neomycin production was achieved with optimized parameters, such as alginate at 2% wt/vol, 0.25M CaCl₂, 1-hour curing time, and 3.24 mm bead diameter. Effective neomycin production was achieved with 3% wt/vol maltose and 0.6% wt/vol sodium glutamate concentration. The repeated batch fermentations were conducted (every 96 hours) using the optimized alginate beads, employing the production medium with 3% wt/vol maltose and 0.6% wt/vol sodium glutamate along with minerals salts solution. The increase in antibiotic production was observed up to the 5th cycle, and later gradual decrease in antibiotic production was observed. Comparison of the total antibiotic production with free cells and immobilized cells was also done. An enhanced antibiotic productivity of 32% was achieved with immobilized cells over the conventional free-cell fermentation, while 108% more productivity was achieved over the washed free-cell fermentation. From these results it is concluded that the immobilized cells ofS marinensis NUV-5 in calcium alginate are more efficient for the production of neomycin with repeated batch fermentation.     

Screening of encapsulated microbial cells for the degradation of inorganic cyanides

Summary Different encapsulation matrices were screened to encapsulate cells ofPseudomonas putida for degradation of inorganic cyanides. Degradation of NaCN by free cells and cells immobilized in agar, alginate or carrageenan matrices was studied. The rate of NaCN degradation was monitored for 120 h by measuring pH, bacterial growth, dissolved and gaseous NH₃ and gaseous CO₂. Alginate-immobilized cells degraded NaCN more efficiently than free cells or agar- or carrageenan-immobilized cells.     

Degradation of Mono-Fluorophenols by an Acclimated Activated Sludge

Acclimated activated sludge was examined for its ability to degrade mono-fluorophenols as the sole carbon source in aerobic batch cultures. The acclimated activated sludge degraded fluorophenol efficiently. It degraded 100 mg/l 3-fluoropheno and 4-fluorophenol in 16 h with, respectively, 99.85% and 99.91% fluoride anion release and it degraded 50 mg/l 2-fluorophenol in 15 h with 99.26% fluoride anion release. The aerobic biodegradability of the mono-fluorophenols decreased in the order: 4-fluorophenol > 3-fluorophenol > 2-fluorophenol, resulting mainly from a different octanol/water partition coefficient and different steric parameter of the fluorophenols. The mechanism study revealed that the initial step in the aerobic biodegradation of mono-fluorophenols by the activated sludge was their transformation to fluorocatechol. Following transformation of the fluorophenol to fluorocatechol, ring cleavage by catechol 1, 2-dioxygenases proceeded via an ortho-cleavage pathway, then defluorination occurred.     

Phenol Degradation by Immobilized Cells of Arthrobacter citreus

An aerobic microorganism with an ability to utilize phenol as carbon and energy source was isolated from a hydrocarbon contamination site by employing selective enrichment culture technique. The isolate was identified as Arthrobacter citreus based on morphological, physiological and biochemical tests. This mesophilic organism showed optimal growth at 25°C and at pH of 7.0. The phenol utilization studies with Arthrobacter citreus showed that the complete assimilation occurred in 24 hours. The organism metabolized phenol up to 22 mM concentrations whereas higher levels were inhibitory. Thin layer chromatography, UV spectral and enzyme analysis were suggestive of catechol, as a key intermediate of phenol metabolism. The enzyme activities of phenol hydroxylase and catechol 2,3-dioxygenase in cell free extracts of Arthrobacter citreus were indicative of operation of a meta-cleavage pathway for phenol degradation. The organism had additional ability to degrade catechol, cresols and naphthol. The degradation rates of phenol by alginate and agar immobilized cells in batch fermentations showed continuous phenol metabolism for a period of eight days.     

Continuous production of N-carbamyl-D-alanine byPeptococcus anaerobius adsorbed on activated charcoal

Summary The strict anaerobic bacteriumPeptococcus anderobius produced a D-hydantoinase which was used to catalyse the formation of N-carbamyl-D-alanine from DL-5-methylhydantoin. Whole cells ofP. anaerobius adsorbed on granular activated charcoal were stable for up to seven days when used in two bioreactor systems (percolation and fluidization) operated continuously under aerobic conditions at 50°C.