A rapid screening method for bacteria degrading polycyclic aromatic hydrocarbons

Aims:  A rapid procedure was developed to screen for bacteria that are able to grow on polycyclic aromatic hydrocarbons (PAHs).
Methods and Results:  A drop of ethyl ether-dissolved PAH is spread on a sterilized cellulose acetate/nitrate filter lying on the top of a mineral salts agarose plate. After the evaporation of ethyl ether, a serially diluted sample is spread over the filter and incubated. Subsequently, the PAH degrading bacteria can be counted and isolated.
Conclusions:  This procedure is a simple method for screening bacterial isolates for the ability to grow with PAHs.
Significance and Impact of the Study:  This technique is rapid to screen and/or count PAH-degrading bacteria and is also used to streak cultivation without disrupting the PAH layer on plate.     

A method for screening bacteria: Aerobically degrading chlorinated short-chain hydrocarbons

A method for screening of short-chain chlorinated-hydrocarbon-degrading bacteria is described. It uses as a criterium for selection the liberation of protons and change in color of a pH indicator rather than growth on the compound as sole carbon source. The usefulness of indicator plates is demonstrated with several bacteria, known to degrade certain chlorinated hydrocarbons. Bacteria-degrading volatile compounds can be isolated with a medium containing a second carbon source. A correlation between proton liberation and liberation of chloride or disappearance of 2-chloroethanol from the medium is demonstrated as an example. The method should be useful in isolating bacteria for the decontamination of respective commodities.     

苯酚降解菌的分离及培养特性研究

对南充市郊炼油厂活性污泥进行富集,驯化筛选得到2株能以苯酚作为唯一碳源和能源生长的菌株,编号为S1,S2,两菌株可耐10,000mg/L左右的苯酚浓度,实验得出其最佳生长条件为pH7-8,温度25℃-30℃,在适宜条件下,对苯酚有较好的降解能力,而且苯对两菌株的生长表现为抑制作用。     

Isolation and characterization of thermophilic bacteria capable of degrading dehydroabietic acid.

Using a semi-continuous enrichment method, we isolated two thermophilic bacterial strains, which could completely degrade abietane resin acids, including dehydroabietic acid (DhA). Strain DhA-73, isolated from a laboratory-scale bioreactor treating bleached kraft mill effluent at 55 degrees C, grew on DhA as sole carbon source; while DhA-71, isolated from municipal compost, required dilute tryptic soy broth for growth on DhA. DhA-71 grew on DhA from 30 degrees C to 60 degrees C with maximum growth at 50 degrees C; while, DhA-73 grew on DhA from 37 degrees C to 60 degrees C with maximum growth at 55 degrees C. At 55 degrees C, the doubling times for DhA-71 and DhA-73 were 3.3 and 3.7 h, respectively. DhA-71 and DhA-73 had growth yields of 0.26 and 0.19 g of protein per g of DhA, respectively. During growth on DhA, both strains converted DhA to CO2, biomass, and dissolved organic carbon. Analyses of the 16S-rDNA sequences of these two strains suggest that they belong to two new genera in the Rubrivivax subgroup of the beta subclass of the Proteobacteria. Strains DhA-71 and DhA-73 are the first two bacteria isolated and characterized that are capable of biodegradation of resin acids at high temperatures. This study provided direct evidence for biodegradation of resin acids and feasibility for biotreatment of pulp mill effluent at elevated temperatures.     

Teratogenic effects of aliphatic nitriles

Intraperitoneal injection of acrylonitrile at 1.51-2.26 mmole/kg (80-120 mg/kg) or propionitrile at 0.54-1.51 mmole/kg (30-83 mg/kg) on the morning of Day 8 of gestation in the hamster induced exencephaly, encephalocoeles, and rib fusions and bifurcations in the offspring. These doses of the aliphatic nitriles also resulted in obvious toxicity to the dams. Multiple intraperitoneal injections of sodium thiosulfate at 4.03 mmole/kg (1 gm/kg) protected both dams and embryos against toxicity. When the larger doses of either acrylonitrile or propionitrile were given in the presence of sodium thiosulfate, teratogenic effects were observed in the absence of overt signs of maternal poisoning. A survey of the literature describes many studies which demonstrate that acrylonitrile and propionitrile are converted in vivo to toxicologically significant concentrations of cyanide and that sodium thiosulfate, an established cyanide antagonist, can provide protective actions against poisoning by either acrylonitrile or propionitrile. The observations suggest that the teratogenic effects of both acrylonitrile and propionitrile are related to the metabolic release of cyanide.     

A rapid screening method for micro-organisms degrading polycyclic aromatic hydrocarbons in microplates

A method for enumerating micro-organisms degrading polycyclic aromatic hydrocarbons (PAHs) was developed. The micro-organisms present in water samples are incubated in 96-well microplates, in which the desired PAHs are available as sole carbon source in a liquid mineral-salts medium (MSM). Walls and bottoms of the wells in the microplates are covered with PAHs by dissolving them in a non-polar solvent and pipetting this solution into the wells. After solvent elimination under vacuum, the PAHs remain on the surface of the wells. The formation of coloured products during microbial degradation of PAHs causes colouring of the MSM, thus allowing evaluation of the cell titre by determining the most probable number. Usage of an electronic multichannel pipette makes the work faster and more effective. This allows the inoculation of several microplates pre-treated with different PAHs out of one serial dilution. On the one hand, this method is very effective in screening the usability spectrum of different PAHs microorganisms; on the other hand it allows the additional employment of other sources of hydrocarbons. Correspondence to: M. Stieber     

Selection and characterization of aerobic bacteria capable of degrading commercial mixtures of low-ethoxylated nonylphenols

Aims:  Isolation and characterization of new bacterial strains capable of degrading nonylphenol ethoxylates (NP n EO) with a low ethoxylation degree, which are particularly recalcitrant to biodegradation.
Methods and Results:  Seven aerobic bacterial strains were isolated from activated sludges derived from an Italian plant receiving NP n EO-contaminated wastewaters after enrichment with a low-ethoxylated NP n EO mixture. On the basis of 16S rDNA sequence, the strains were positioned into five genera: Ochrobactrum , Castellaniella , Variovorax , Pseudomonas and Psychrobacter . Their degradation capabilities have been evaluated on two commercial mixtures, i.e. Igepal CO-210 and Igepal CO-520, the former rich in low ethoxylated congeners and the latter containing a broader spectrum of NP n EO, and on 4- n -nonylphenol (NP). The strains degraded Igepal CO-210, Igepal CO-520 and 4- n -NP all applied at the initial concentration of 100 mg l⁻¹, by 35–75%, 35–90% and 15–25%, respectively, after 25 days of incubation.
Conclusions:  Some of the isolated strains, in particular the Pseudomonas strains BCb12/1 and BCb12/3, showed interesting degradation capabilities towards low ethoxylated NP n EO congeners maintaining high cell vitality.
Significance and Impact of the Study:  Increased knowledge of bacteria involved in NP n EO degradation and the possibility of using the isolated strains in tailored process for a tertiary biological treatment of effluents of wastewater treatment plants.     

Glucose and plant exudate enhanced enumeration of bacteria capable of degrading polycyclic aromatic hydrocarbons

Enumerating environmental microbial isolates capable of polycyclic aromatic hydrocarbon (PAH) degradation can provide insight into the microbe-plant interactions that facilitate PAH removal. We examined a known PAH degrader ( Pseudomonas putida G7), a nondegrader ( Agrobacterium tumefaciens LBA4404), and several microorganisms isolated from the environment by using a PAH cocktail in an enumeration medium?with or without 0.025% (m/v) glucose and (or) root exudates. Compared with the standard most probable number (MPN), the addition of glucose and root exudates in a modified MPN method resulted in a 3- to 11-fold enhancement of PAH degraders being enumerated among microorganisms found in PAH-contaminated soils. High-performance liquid chromatography analysis verified that PAH levels were reduced using this modified enumeration method. Low levels of glucose, perhaps in concert with other materials in exudates, may promote microbial metabolism, thereby enhancing PAH degradation.     

A novel screening method of cellulase-producing bacteria

Cellulase is the key to utilize the renewable and abundant cellulose resource, cellulase-producing microorganism is an important source of cellulase. The traditional screening method of cellulase-producing microorganism is low efficacy and not macroscopic. The screening method in this study was based on the interactive culture character between cellulase-producing bacteria and Phytophthora parasitica var. nicotianae on plates, the results indicated that the inhibition zone and cellulase activity of bacterial strains are conformity on the whole, so the screening method is very quickly and apparent.     

A new microtitre plate screening method for evaluating the viability of aerobic respiring bacteria in high surface biofilms

Aims: It is difficult to determine the effects of bactericidal compounds against bacteria in a biofilm because classical procedures for determining cell viability require several working days, multiple complicated steps and are frequently only applicable to cells in suspension. We attempt to develop a compact, inexpensive and versatile system to measure directly the extent of biofilm formation from water systems and to determine the viability of respiring bacteria in high surface biofilms. Methods and Results: It has been reported that the reduction of tetrazolium sodium salts, such as XTT (sodium 3,3′‐[1‐[(phenylamino)carbonyl]‐3,4‐tetrazolium]Bis(4‐methoxy)‐6‐nitro)benzene sulfonic acid hydrate), during active bacterial metabolism can be incorporated into a colorimetric method for quantifying cell viability. XTT is reduced to a soluble formazan compound during bacterial aerobic metabolism such that the amount of formazan generated is proportional to the bacterial biomass. Conclusions: We show here, for the first time, that this colorimetric approach can be used to determine the metabolic activity of adherent aerobic bacteria in a biofilm as a measure of cell viability. This technique has been used to estimate viability and proliferation of bacteria in suspension, but this is the first application to microbial communities in a real undisturbed biofilm. Significance and Impact of the Study: This simple new system can be used to evaluate the complex biofilm community without separating the bacteria from their support. Thus, the results obtained by this practice may be more representative of the circumstances in a natural system, opening the possibility to multiple potential applications.     

A simple and rapid method for screening amylolytic bacteria

A laboratory class was designed for the study of the ecology of amylolytic bacteria in soil, although other sources may be equally suitable for this purpose. Groups of three students carried out the following: (a) preparation and sterilization of medium and plates, (b) collection and preparation of soil samples, spreading the samples on the plates, (c) incubation of the plates at 37 degrees C overnight, a further 1 h incubation at 60 degrees C to observe amylolytic activity due to thermophilic bacteria, and (d) interpretation and discussion of the results. These tasks are accomplished in two periods of 4h on consecutive days. No sophisticated instruments are required for these experiments, which can be carried out in three classes of 4h each. On the first day the students prepare culture media, buffers and reagents, as well as collect and grow soil samples. The second day is spent for both taxonomic identification of colonies and the HAI determination.     

Selection and isolation of bacteria capable of degrading dinoseb (2-sec-buty-4,6-dinitrophenol)

Dinoseb (2-sec-butyl-4,6-dinitrophenol) has been a widely used herbicide that persists in some contaminated soils, and has been found in groundwaters, causing health and environmental hazards. Persistence in some soils may stem from a lack of dinoseb-degrading organisms. We established a chemostat environment that was strongly selective for aerobic (liquid phase) and anaerobic (sediment phase) bacteria able to degrade dinoseb. The chemostat yielded five taxonomically diverse aerobic isolates that could transform dinoseb to reduced products under microaerophilic or denitrifying conditions, but these organisms were unable to degrade the entire dinoseb molecule, and the transformed products formed multimeric material. The chemostat also yielded an anaerobic consortium of bacteria that could completely degrade dinoseb to acetate and CO₂ when the Eh of the medium was less than-200 mV. The consortium contained at least three morphologically different bacterial species. HPLC analysis indicated that dinoseb was degraded sequentially via several as yet unidentified products. Degradation of these intermediates was inhibited by addition of bromoethane sulfonic acid. GC-MS analysis of metabolites in culture medium suggested that regiospecific attacks occurred non-sequentially on both the nitro groups and the side-chain of dinoseb. The consortium was also able to degrade 4,6-dinitro-o-cresol, 3,5-dinitrobenzoic acid, 2,4-dinitrotoluene, and 2,6-dinitrotoluene via a similar series of intermediate products. The consortium was not able to degrade 2,4-dinitrophenol. To our knowledge, this is the first report of strictly anaerobic biodegradation of an aromatic compound containing a multicarbon, saturated hydrocarbon side chain.Abbreviations BESA bromoethane sulfonic acid - RAMM reduced anaerobic mineral medium     

A fast screening method for surface layers on Gram-positive bacteria

A two-step screening method is described to identify regularly arranged surface layers (S layers) on Gram-positive bacterial strains. A non-destructive release of S-layer sheets is achieved by enzymatic hydrolysis of the underlying peptidoglycan using lysozyme. The existence of regular S layers is then directly confirmed by scanning force microscopy or transmission electron microscopy. This method requires a minimal amount of bacterial cells and may be used as a `quick test' for demonstrating the presence of S layers.     

A rapid plate culture method for screening of amylase producing micro-organisms

A simple and rapid method is described for the detection of amylase-producing micro-organisms on solid media using Remazol Brilliant Blue-starch as substrate. The blue starch is incorporated into nutrient agar and amylase production is detected by the disappearance of the colour around the colonies. The method which is non-destructive, allows for direct visualization and isolation of amylolytic micro-organisms from the environment without prior replication of colonies.     

A rapid colony screening method for the detection of arsenate-reducing bacteria

A rapid and simple method has been developed for the detection of arsenate reducing bacteria based on the presence of arsenite [As (III)], the end product of anaerobic arsenate [As (V)] respiration. Confirmation of As (III) product is made by the reduction of starch-iodine complex. The method can be used over a large pH range (5.5–9.0) and can easily be determined at arsenite concentration as low as 0.025 mM. Major advantages of this technique are that a large number of samples can be analyzed easily at a time.     

A rapid bactometer method for screening of biocides against sulfate-reducing bacteria

A rapid and efficient bactometer method was developed for screening biocides against sulfate-reducing bacteria. The method is based on impedance microbiology principles and uses double-layer API (American Petroleum Institute) agar medium supplemented with 0.1% sodium thioglycolate as a reducing agent. Compared to the conventional API procedure, which requires 28 days, the present technique takes only 1 day to obtain test results. Excellent linear correlation (r=–0.98) was found between the impedance detection time and log initial cell concentration. The results of the bactometer test were comparable to that of the API bottle test.     

Insulin-degrading enzyme is capable of degrading receptor-bound insulin

In the investigation of the intracellular sites of insulin degradation, it might be important whether receptor-bound insulin could be a substrate for insulin-degrading enzyme (IDE). Insulin receptor and IDE were purified from rat liver using a wheat germ agglutinin column and monoclonal anti-IDE antibody affinity column, respectively. [125I]insulin-receptor complex was incubated with various amounts of IDE at 0 degree C in the presence of disuccinimidyl suberate and analyzed by reduced 7.5% SDS-PAGE and autoradiography. With increasing amounts of IDE, the radioactivity of 135 kd band (insulin receptor alpha-subunit) decreased, whereas that of 110 kd band (IDE) appeared then gradually increased, suggesting that IDE could bind to receptor-bound insulin. During incubation of insulin-receptor complex with IDE at 37 degrees C, about half of the [125I]insulin was dissociated from the complex. However, the time course of [125I]insulin degradation in this incubation was essentially identical to that of free [125I]insulin degradation. Cross-linked, non-dissociable receptor-bound [125I]insulin was also degraded by IDE. Rebinding studies to IM-9 cells showed that the receptor binding activity of dissociated [125I]insulin from insulin-receptor complex incubated with IDE was significantly (p less than 0.001) decreased as compared with that without the enzyme. These results, therefore, show that IDE could recognize and degrade receptor-bound insulin, and suggest that IDE may be involved in insulin metabolism during receptor-mediated endocytosis through the degradation of receptor-bound insulin in early neutral vesicles before their internal pH is acidified.