Destruction of coliforms in water and sewage water by dye-sensitized photooxidation.

A new approach to disinfection of water and sewage water, by oxidative destruction of microorganisms by photosensitization, is described. Samples of water and sewage water, to which an inoculum of fecal Escherichia coli had been added, were exposed to solar radiation in the presence of a dye sensitizer, under continuous aeration. The effects of the sensitizer methylene blue at concentrations of 0 to 10 mg/liter, different radiation times from 0 to 2 h, and sunlight intensities of 0, 68, and 2,030 muE/m2 per s were investigated. In laboratory-scale experiments, 1.3 X 10(9) coliforms in 100 ml of oxidation pond municipal sewage water containing 0.5 mg of methylene blue were destroyed in about 30 min. Similar results were obtained with nonchlorinated potable water. These results demonstrate the possibilities available for the disinfection of water and sewage water by this method.     

Injured coliforms in drinking water.

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.     

Quantitative determination of Escherichia coli from coliforms and faecal coliforms in sea water.

Escherichia coli concentration in sea water was determined by the MUG test after primary growth on membrane filters used to determine total coliforms or faecal coliforms. A good correlation (r = 0.86) was found between E. coli obtained from coliforms versus those from faecal coliforms. Verification procedures showed that all the MUG-positive colonies obtained on both media were E. coli. Evaluation of this data and the literature indicated that this technique for estimation of E. coli in sea water is a useful addition to laboratory procedures without generally increasing the time and the expense of the analysis of recreational water.     

Fecal coliforms in sewage. II. Transfer of markers and the presence of plasmids

The transference of the genetic markers and the presence of DNA plasmidial in 240 cultures of Escherichia coli was investigated. The strains were originated from Waste Treatment Plant (inffluent and effluents) located in Ilha do Governador, Rio de Janeiro. By conjugation analysis, E. coli K 12 allowed the isolation of the transconjugants resistant to antibiotics Su, Sm, Tc, Cm, Ap; heavy metals (Cu, Hg and Zn) and colicinogenic factors (Ia, Ib and V) mainly in coliforms isolated Cm and Ap from the terminals of the treatment plant. The percentual distribution of the plasmids was prevalent in the cultures of E. coli originated from material collected in the effluents and reached a rate higher than 65%.     

Validity of fecal coliforms, total coliforms, and fecal streptococci as indicators of viruses in chlorinated primary sewage effluents.

Quantities of combined chlorine that usually destroyed more than 99.999% of the indigenous fecal coliforms, total coliforms, and fecal streptococci in primary sewage effluents destroyed only 85 to 99% of the indigenous viruses present. Viruses were recovered from five of eight chlorinated primary effluents from which fecal coliforms were not recovered by standard most-probable-number procedures. The limited volumes of such chlorinated effluents that can be tested for indicator bacteria with currently available multiple-tube and membrane filter techniques restrict the value of fecal coliforms, fecal streptococci, and even total coliforms as indicators of viruses in these effluents. Although fecal coliforms and fecal streptococci are useful indicators of viruses in effluents from which these bacteria are recovered, the absence of these bacteria and even total coliforms from disinfected effluents (in standard tests) does not assure that viruses are also absent.     

Membrane filter method for recovery of fecal coliforms in chlorinated sewage effluents.

The standard one-step M-FC broth-membrane-filter procedure for recovery of fecal coliforms from chlorinated sewage effluents is much less effective than the multiple-tube (most-probable-number) technique. A two-step membrane-filter method, using a pre-enrichment technique with phenol red lactose broth and incubation at 35 degrees C for 4 h, followether 18+/-2 h, enhanced fecal coliform recovery from chlorinated effluents. The results of 126 comparisons using chlorinated effluents from five wastewater plants showed that fecal coliform recovery by using the two-step membrane-filter method is comparable to that using the multiple-tube procedure.     

Validity of fecal coliforms, total coliforms, and fecal streptococci as indicators of viruses in chlorinated primary sewage effluents.

Quantities of combined chlorine that usually destroyed more than 99.999% of the indigenous fecal coliforms, total coliforms, and fecal streptococci in primary sewage effluents destroyed only 85 to 99% of the indigenous viruses present. Viruses were recovered from five of eight chlorinated primary effluents from which fecal coliforms were not recovered by standard most-probable-number procedures. The limited volumes of such chlorinated effluents that can be tested for indicator bacteria with currently available multiple-tube and membrane filter techniques restrict the value of fecal coliforms, fecal streptococci, and even total coliforms as indicators of viruses in these effluents. Although fecal coliforms and fecal streptococci are useful indicators of viruses in effluents from which these bacteria are recovered, the absence of these bacteria and even total coliforms from disinfected effluents (in standard tests) does not assure that viruses are also absent.     

Frequency distribution of coliforms in water distribution systems.

Nine small water distribution systems were sampled intensively to determine the patterns of dispersion of coliforms. The frequency distributions of confirmed coliform counts were compatible with either the negative-binomial or the lognormal distribution. They were not compatible with either the Poisson or Poisson-plus-added zeroes distribution. The implications of the use of the lognormal distributional model were further evaluated because of its previous use in water quality studies. The geometric means from 14 data sets ranged from 10(-6) to 0.2 coliforms per 100 ml, and the geometric standard deviations were between 10 and 100, with one exception. If the lognormal model is representative of the coliform distribution; the arithmetic mean sample count is a poor estimator of the true mean coliform density, and the probability of water in a distribution system containing small patches with large coliform densities without detection by routine monitoring is finite. These conclusions have direct bearing on the interpretation of microbiological quality standards for drinking water.     

Injured coliforms in drinking water

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.     

Inactivation of enterococci and fecal coliforms from sewage and meatworks effluents in seawater chambers.

Inactivation in sunlight of fecal coliforms (FC) and enterococci (Ent) from sewage and meatworks effluents was measured in 300-liter effluent-seawater mixtures (2% vol/vol) held in open-topped chambers. Dark inactivation rates (kDs) were measured (from log-linear survival curves) in enclosed chambers and 6-liter pots. The kD for FC was 2 to 4 times that for Ent, and inactivation was generally slower at lower temperatures. Sunlight inactivation was described in terms of shoulder size (n) and the slope (k) of the log-linear portion of the survival curve as a function of global solar insolation and UV-B fluence. The n values tended to be larger for Ent than for FC, and the k values for FC were around twice those for Ent in both effluent-seawater mixtures. The combined sunlight data showed a general inactivation rate (k) ranking in effluent-seawater mixtures of meatworks FC > sewage FC > meatworks Ent > sewage Ent. Describing 90% inactivation in terms of insolation (S90) gave far less seasonal variation than T90 (time-dependent) values. However, there were significant differences in inactivation rates between experiments, indicating the contribution to inactivation of factors other than insolation. Inactivation rates under different long-pass optical filters decreased with the increase in the spectral cutoff wavelength (lambda 50) of the filters and indicated little contribution by UV-B to total inactivation. Most inactivation appeared to be caused by two main regions of the solar spectrum--between 318 and 340 nm in the UV region and > 400 nm in the visible region.     

Fecal coliforms in sewage waters. I. Resistance to antibiotics, heavy metals and colicinogeny

Qualitative bacteriological analysis was carried out in two sewage treatment plants in the city of Rio de Janeiro during the period 1984-1985. Specific points of the plants were selected for the collection of affluent and effluent samples. The study involved the isolation and the identification of 540 cultures of Escherichia coli that were analyzed for their resistance to eight antibiotics (sulfadiazine, streptomycin, tetracycline, chloramphenicol, kanamycin, ampicillin, nalidixic acid and gentamycin), and three heavy metals (copper sulphate, mercuric chloride and zinc sulphate) as well as colicinogeny. About 95% of the isolated cultures from the effluents had genetic markers while the samples originated from the affluents showed 70%.     

Detection of fecal coliforms in water by using [14C]mannitol.

Interest in rapid bacterial detection methods for sanitary indicator bacteria in water prompted a study of the use of [U-14C]mannitol to detect fecal coliforms (FC). A simple method which used m-FC broth, membrane filtration, and two-temperature incubation (35 degrees C for 2 h followed by 44.5 degrees C for 2.5 h) was developed. [U-14C]mannitol was added to the medium, and the temperature was raised to 44.5 degrees C after 2 h at 35 degrees C. 14CO2 was collected as Ba14CO3 and assayed by liquid scintillation spectroscopy. Correlations were examined between FC cell numbers at the start of incubation (standard 24-h FC test) and Ba14CO3 counts per minute after 4.5 h. Results indicated that FC numbers ranging from 1 x 10(1) to 2.1 x 10(5) cells could be detected in 4.5 h. Within-sample reproducibility at all cell concentrations was good, but sample-to-sample reproducibility was variable. Comparisons between m-FC broth and m-FC broth modified by substituting D-mannitol for lactose indicated that the standard m-FC broth was the better test medium. Results from experiments in which dimethyl sulfoxide was used to increase permeability of FC to [U-14C]mannitol indicated no increase in 14CO2 production due to dimethyl sulfoxide. Detection of FC by this method may be useful for rapid estimation of FC levels in freshwater recreational areas, for estimating the quality of potable source water, and potentially for emergency testing of potable water, suspected of contamination due to distribution line breaks or cross-connections.     

Evaluation of coliforms as indicators of water quality in India.

The total and thermotolerant coliform counts in rural drinking water derived from ground water, piped supplies and surface water are reported for a number of areas in India. To evaluate these counts as indicators of recent faecal contamination the total coliforms and thermotolerant coliforms isolated have been identified. Thermotolerant Escherichia coli formed 11.7% of the total coliforms and 75.1% of the thermotolerant coliforms. Citrobacter sp. (20.4%) and Klebsiella sp. (50.9%) were the other common total coliforms isolated and, among the thermotolerant coliforms, Klebsiella sp. (16.4%) was the only other organism frequently encountered. The total coliform counts were significantly correlated with water temperature. The applicability in tropical areas of standards developed for temperate climates is discussed.     

Detection of singlet oxygen and its role in dye-sensitized photooxidation in aqueous and micellar solutions

Indirect methods for the detection of singlet oxygen in dye-sensitized photooxidation based on its interception by some singlet oxygen acceptors in aqueous and micellar solutions are discussed. Mechanistic aspects and some applications of a very sensitive method using p-nitrosodimethylaniline in the presence of imidazole (RNO + imidazole method) are also treated. The technique of competition kinetics with a singlet oxygen quencher N-3 which can serve for the determination of the role of singlet oxygen is discussed as well. Such competition with tryptophan and guanosine shows that these substrates react exclusively or predominantly via the singlet oxygen mechanism in the presence of hematoporphyrin as sensitizing dye.     

Rapid detection of total and fecal coliforms in water by enzymatic hydrolysis of 4-methylumbelliferone-beta-D-galactoside.

Three fluorogenic methylumbelliferone (MU) substrates were evaluated for rapid detection of total and fecal coliform bacteria (TC and FC) in drinking water. 4-MU-beta-D-galactoside, MU-heptanoate, and MU-glucuronide were used to determine enzyme activity as a surrogate measure of coliform concentration. Coliforms occurring in river water and in potable water artificially contaminated with raw sewage were tested. The initial rate of hydrolysis (delta F) of MU-beta-D-galactoside showed promise as an indicator of TC and FC within 15 min. delta F of MU-glucuronide was insufficient in the 15-min assay, and combinations of the MU substrates did not enhance delta F. A direct membrane filter method incorporating MU-beta-D-galactoside into an agar medium allowed the detection of as few as 1 FC per 100 ml within 6 h.     

Failure of the most-probable-number technique to detect coliforms in drinking water and raw water supplies.

A procedure was developed to detect false-negative reactions (interference) in the standard most-probable-number (S-MPN) technique for coliform enumeration of untreated surface water and potable water supplies. This modified MPN (M-MPN) procedure allowed a quantitative assessment of the interference with coliform detection in untreated surface water and potable water supplies. Coliform interference was found to occur in the presumptive, confirmed, and completed tests of the S-MPN technique. When coliforms were present, interference with their detection occurred in over 80% of the samples. The inferior nature of the S-MPN was revealed by the 100% increase in the incidence of completed coliform-positive drinking water samples obtained with the M-MPN technique. The M-MPN procedure was also superior to the standard membrane filter technique. Eight different species of coliforms were recovered from false-negative tests, including Citrobacter, Enterobacter, Klebsiella, and Escherichia coli (in decreasing order of occurrence). The use of standard MPN techniques for monitoring potable water supplies may lead to a false security that the drinking water supply is potable, i.e., free from indicator bacteria.     

Selective detection and enumeration of fecal coliforms in water by potentiometric measurement of lipoic acid reduction.

Water samples of various origins were inoculated into a specific coliform-selective lactose broth provided with lipoic (thioctic) acid, and the time evolution of the redox potential of the cultures was monitored during incubation at 41 degrees C by use of gold versus reference electrodes. Positive potential-time responses, i.e., 100-mV potential shifts recorded within 20 h of inoculation, were related to the initial number of fecal coliforms in the broth determined by control enumeration techniques, and the organisms responsible were isolated and identified by conventional procedures. A total of 30 samples of wastewater, 38 of surface water, 553 of groundwater, and 110 of drinking water were tested successively. A total of 240 natural water samples, including 172 groundwater samples, and 1 drinking water sample were found to be positive in the potentiometric test. The majority (i.e., 92.5%) of the relevant potentiometric detection times were shorter than 15 h, and 96% of these could be attributed to Escherichia coli. Fifteen hours corresponded to the limit for detecting 1 E. coli cell per 100 ml of water. About 78% of the potentiometric responses occurring after 15 h were induced by fecal coliforms other than E. coli (Enterobacter cloacae, Klebsiella pneumoniae, and Citrobacter freundii). Calibration curves relating detection times shorter than 15 h to fecal coliform (i.e., E. coli) concentrations were constructed for the natural water samples tested. There were minor variations in the average growth rate of the organisms in the relation to the contamination level of the water tested. The number of false-positive samples in the potentiometric test was equivalent to that of false-negative samples (groundwater or drinking water).