A membrane filter method for the enumeration of aquatic ammonifiers

A standard procedure for the enumeration of heterotrophic bacteria, utilizing heterotrophic plate count medium (m-HPC), has been modified to allow bacterial ammonifier populations also to be counted. Nessler's reagent was included as an outside indicator to ascertain what portion of the population is capable of ammonification. Typically the ammonifier population varied among aquatic habitats, ranging from 0.08 to 37.0% of the heterotrophic population in river water samples, 0.2 to 10.6% for irrigation water samples, and 1.4 to 3.0% for pond and lake water samples.     

Technique for measuring metallic salt effects upon the indigenous heterotrophic microflora of a natural water

The effects of several concentrations of the metallic salts Ag₂SO₄, NaAsO₃, BaCl₂2H₂O, CdCl₂2.5 H₂O, CrCl₂6H₂O, CuCl₂, HgCl₂, NaCl, NiCl₂6H₂O, PbCl₂ and ZnCl₂ upon the indigenous heterotrophic microflora of a natural water were studied. Effects of these salts upon the heterotrophic activity of the microbes were assayed using the heterotrophic technique whereas lethal effects upon the heterotrophic bacteria were determined using nutrient agar plate counts. The heterotrophic activity method is based upon the uptake and mineralization of a radioactively labeled metabolite (in these experiments ¹⁴C-glucose) by the indigenous aquatic microbes and data analysis is by Michaelis—Menten enzyme kinetics equations. Concentrations of metallic salts which resulted in bacterial death also caused erratic uptake and mineralization rates of ¹⁴C-glucose whereas sub-lethal concentrations, as determined by nutrient agar plate counts, caused a non-competitive inhibition of maximum heterotrophic activity and markedly increased the turnover time of the glucose substrate.     

A preliminary note on the sequential decomposition of pectin by aquatic bacteria

In a eutrophic lake, an inverse relationship was observed between total aerobic heterotrophic bacteria and their pectolytic share which consisted mainly of Flavobacterium-Cytophaga species. When grown in a medium containing pectin and yeast extract as organic compounds pure cultures of the most abundant type of this bacterial group showed a diauxic characteristic of growth. Short time before entering the first lag phase of growth, production of pectolytic enzymes increased until the end of the second log phase of growth. This phenomenon may be due to the mechanism of a catabolic repression and derepression.     

Demonstration of mass transfer and pH effects in a nitrifying biofilm

A bench-scale nitrifying trickling filter (surface AREA = 0.5 m²) was developed to permit evaluation of diffusion of oxygen within a biofilm, the pH dependence of ammonium oxidation and external mass transfer. In addition, a biofilm model was developed and verified for homogeneous nitrifying biofilms of varied thickness and for thin nitrifying biofilms covered by heterotrophic biofilms. The model uses literature values for the pH dependence of Monod coefficients for Nitrosomonas and Nitrobacter.

The diffusion coefficient of oxygen in the biofilm was found to be 40–80% of the value in pure water. Due to mass transfer resistance, the biomass ·sees” a lower pH than is measured in the water film passing over it. The surface uptake rate of ammonia is used as an indicator of pH gradients within the biofilm system. With the help of oxygen limitation experiments, the location of nitrifying biomass within mixed biofilms (heterotrophic, autotrophic) can be determined.

The biofilm model predicts ammonium uptake rate of a trickling filter as a function of the bicarbonate concentration in the water film.     

Comparative survival and injury of Candida albicans and bacterial indicator organisms in streams receiving acid mine drainage

Microbiological studies were conducted to assess the survival and injury characteristics of Candida albicans and indicator bacteria in streams impacted by acid mine water (AMW) and organic wastes. Persistence of pure cultures of C. albicans in three AMW-polluted streams was studied in situ using environmental membrane diffusion chambers. Survival of the fungus (at least 3 days in AMW) indicated prolonged tolerance to acid conditions. In comparison, Escherichia coli was killed within several hours of acid stress. Persistence studies also demonstrated that C. albicans was less sensitive to seasonal water temperature fluctuations than E. coli or Streptococcus faecium. In addition to its prolonged survival, C. albicans incurred minimal sublethal injury whereas identical conditions of exposure resulted in significant injury to traditional indicator bacteria. The failure of standard microbiological procedures to detect AMW-damaged bacteria compromises the accuracy of public health safety determinations in these waters. On the other hand, the ability of C. albicans to survive in AMW streams with marked resistance to injury suggests its potential as a favorable alternative sanitary indicator organism for such environments.     

Cometabolic transformation of linear alkylbenzenesulphonates by methanotrophs

Cometabolic transformation of commercial linear alkylbenzenesulphonates (LAS) and pure congeners of 2-[4-(sulphophenyl)]nonan (2C₉LAS) and 2-[4-(sulphophenyl)]decan (2C₁₀LAS) was studied by using a mixed methanotrophic-heterotrophic culture MM1 and a pure culture of type II methanotroph (strain CSC1), isolated from culture MM1. Cultures were grown in shake flasks under conditions promoting expression of the soluble methane monooxygenase (sMMO). Both the mixed culture MM1 and the pure culture CSC1 were capable of transforming LAS with or without methane as the natural substrate. Experiments performed without methane (resting cells) showed faster cometabolic transformation of LAS congeners with longer alkyl side-chains. Both 2C₉LAS and 2C₁₀LAS congeners were transformed significantly faster at 30°C, although the optimum temperature for the growth of mixed culture MM1 was 20°C. Simultaneous methane oxidation and LAS transformation, and their mutual competitive inhibition, indicated the involvement of the MMO enzyme system and possible methanotrophic activity in the initiation of cometabolic LAS transformation. The capability of two heterotrophic species to transform sulphophenyldecanoic acid suggests possible heterotrophic activity in further LAS transformation (β-oxidation). Faster LAS transformation by mixed culture MM1 than by pure culture CSC1 suggested that methanotrophic-heterotrophic interactions are important for transformation of the complex LAS molecule.     

Bacterial glucose mineralization in acid-stressed lake sediments

This paper reports the findings of an investigation in which glucose mineralization by the benthic microbiota of acid-stressed Silver Lake, Ontario, Canada, was quantitated as a function of season. Attempts were also made to measure the mineralization response when the microbiota were subjected to near neutral pH conditions.

Comparisons made on the basis of V_max values revealed that the heterotrophic potential varied according to season, being lowest during the winter and highest during the summer. Also, an unexpected decrease in heterotrophic potential was realized for all samplings at an experimental, circumneutral pH. These results, in combination with observed, higher bacterial cell counts at the experimental pH, indicate that a fraction of the heterotrophic population is incapable of glucose mineralization at the higher pH, and are evidence of the existence of acidophilic bacteria in acid-stressed aquatic environments. Pre-incubation studies showed that the differences in heterotrophic potentials persisted for up to 15 days, the longest pre-incubation period examined.     

Gel immobilization improves survival of Escherichia coli under temperature stress in nutrient-poor natural water

Viable Escherichia coli cells were entrapped in agar gel layers to form artificial biofilms. Sessile-like bacteria and planktonic (suspended) counterparts were exposed for 28 d to natural (spring)water at two temperatures (4°C and 18°C). Culturabilities on non-selective and selective solid media were monitored over the exposure period, together with cell viability that was determined by using a fluorescent viability probe and confocal scanning laser microscopy analysis. The number of planktonic organisms recovered on non-selective medium decreased by 3 logarithmic units during exposure at 4°C and was reduced to an undetectable level at 18°C. Whatever the temperature of the spring water microcosm, however, the immobilized-cell population did not decrease by more than one log unit over the exposure period. Cell counts on coliform-selective, lactose–deoxycholate agar plates confirmed the enhanced resistance of gel-entrapped cells to environment stress as compared to planktonic organisms: at the two tested temperatures, sublethal injury of suspended E. coli reached more than 80% whereas it did not exceed 65% for immobilized cells. Viability studies indicated that planktonic cells rapidly died. These results show that the ability of microorganisms to colonize aquatic ecosystems in the fixed state (i.e. as biofilms) must be considered in studies evaluating cell survival in these environments.     

Effects of substrate concentrations on the growth of heterotrophic bacteria and algae in secondary facultative ponds

This paper presents the effect of substrate concentration on the growth of a mixed culture of algae and heterotrophic bacteria in secondary facultative ponds (SFPs) utilizing settled domestic sewage as a sole source of organic carbon. The growth of the mixed culture was studied at the concentrations ranging between 200 and 800 mg COD/l in a series of batch chemostat reactors. From the laboratory data, the specific growth rate (micro) was determined using the modified Gompertz model. The maximum specific growth rate ( micro(max)) and half saturation coefficients (K(s)) were calculated using the Monod kinetic equation. The maximum observed growth rate ( micro(max)) for heterotrophic bacteria was 3.8 day(-1) with K(s) of 200 mg COD/l. The micro(max) for algal biomass based on suspended volatile solids was 2.7 day(-1) with K(s) of 110 mg COD/l. The micro(max) of algae based on the chlorophyll-a was 3.5 day(-1) at K(s) of 50mg COD/l. The observed specific substrate removal by heterotrophic bacteria varied between the concentrations of substrate used and the average value was 0.82 (mg COD/mg biomass). The specific substrate utilization rate in the bioreactors was direct proportional to the specific growth rate. Hence, the determined Monod kinetic parameters are useful for the definition of the operation of SFPs.     

Cadmium induced adaptive responses of certain biogeochemical cycling bacteria in an aquatic system

The population growth of some biogeochemical cycling bacteria (heterotrophic bacteria (HB), ammonifying bacteria (AB), ammonia oxidizing bacteria (AOB), denitrifying bacteria (DNB) and cellulose decomposing bacteria (CDB)), as well as ammonification, and denitrification rates were determined in simulated pond systems treated with full doses (FDs) and split doses (SDs) of cadmium. Reductions in bacterial density and nitrogen activity rates were more severe for the FD and SD followed by a slight recovery in both. Among the test groups, reductions were maximal in the AOB and DNB and lowest for the CDB populations, suggesting that the latter had greater potential for cadmium resistance. Bicarbonate alkalinity of water was found to exert a profound influence in counteracting cadmium stress in the system, as strong negative correlations between this parameter and bacterial mass were evident in the SD treatment. Though the aquatic microbial populations were greatly altered by cadmium stress, the normal operating range of the system was restored at a later time with the CDB population showing higher degree of adaptive responses.     

Denitrification with natural gas and various new growth media

Biological denitrification was investigated in an attached growth reactor system using several growth media, denitrifying cultures and natural gas (95% methane) as a carbon source. In order to establish a baseline of operation, initial experiments were conducted with a bed of 2–3 mm sand and methanol as a carbon source using a methylotrophic denitrifying culture and then the system was compared with natural gas using various methane utilising cultures. Compared to methanol, performance with methane was considerably lower. In order to improve denitrification with methane a plastic medium (Etapak, surface area 200 m²/m³) was placed above the sand bed (which increased the surface area for bacteria growth in the upper part of the bed), and a new methanotrophic mixed culture (NCIMB-code 11085) was introduced to the system. This combination resulted in a 27% higher denitrification efficiency. Experiments were continued by systematically varying the operating conditions to obtain highest denitrification using methane gas and replacing the Etapak media with different plastic media of higher surface area, but keeping the NCIMB culture unchanged. Other media tested were Pall-rings (surface area 319 m²/m³), IP-spacers (surface area 500 m²/m³) and granular activated carbon (GAC-code: Norit PK 1–3). Best results were obtained with IP-spacers which, surprisingly, are designed for use in the concrete industry rather than as a bacterial support medium. These produced nitrate removal efficiencies of up to 93% at 0.6 m/h or 55% at 1.6 m/h water filtration rates. Run times of 10 days or more to a limiting headloss of about 1.0 m,were usually achieved before “bumping” or back-washing to reduce headloss. Effluent turbidities were generally below 1.0 NTU. Tests for bacteria present with GAC media and COD removal with IP spacers were also carried out. Results are discussed with operational conditions and denitrification efficiencies achieved.     

Microbial pollution indicators in Brazilian tropical and subtropical marine surface waters

The specificity of indicators that depend on elevated incubation temperature as a selective factor for their enumeration was questioned because of the possibility of interference from autochthonous microorganisms adapted to high ambient temperatures. Lactose-fermenting cultures isolated from fecal coliform tests of tropical marine surface waters were identified as consisting of about 70% Escherichia coli, and most of the remaining cultures being Klebsiella, Enterobacter or Citrobacter species. This confirmed the taxonomic specificity of fecal coliform tests for these waters. Fecal and total coliforms, fecal streptococcus, heterotrophic bacteria and yeast counts had correlations of above 99% confidence levels with most other microbial and chemical parameters studied. Waters with fecal coliform counts above 1000 per 100 ml had increased incidence of presumptive pathogenic yeasts, Pseudomonas aeruginosa and Salmonella. Our data support the use of coliforms or fecal streptococci as indicators of recent fecal pollution in tropical marine waters and yeast or heterotrophic bacteria counts as complementary indicator methods for these waters.     

Use of artificial neural networks to evaluate the effectiveness of riverbank filtration

Riverbank filtration (RBF) is a low-cost water treatment technology in which surface water contaminants are removed or degraded as the infiltrating water moves from the river/lake to the pumping wells. The removal or degradation of contaminants is a combination of physicochemical and biological processes. This paper illustrates the development and application of three types of artificial neural networks (ANNs) to estimate the effectiveness of two RBF facilities in the US. The feed-forward back-propagation network (BPN) and radial basis function network (RBFN) model prediction results produced excellent agreement with measured data at a correlation coefficient above 0.99 for filtrate water quality parameters, including temperature as well as turbidity, heterotrophic bacteria, and coliform removal. In comparison, the fuzzy inference system network (FISN) predicted only temperature and bacteria removal with reasonable accuracy. It is shown that the predictive performances of the ANNs depend on the model structure and model inputs.     

Effect of water hardness on the toxicity of cadmium to micro-organisms

A study was made of the effect of water hardness at different concentrations (viz. 0, 80, 120, 160, 240, 320, 400 and 480 mg l⁻¹ as CaCO₃) on the toxicity of cadmium metal (5 mg 1⁻¹) as sulphate to saprophytic and nitrifying bacteria, with respect to the rate constant (K) and ultimate biochemical oxygen demand (L) which were calculated from BOD data (15 days) using the Thomas Graphical Method. Glucose was used as a source of carbon for micro-organisms. It was observed that the toxicity of cadmium to micro-organisms (both saprophytic and nitrifying) decreased with increasing hardness and reached a maximum at 320 mg 1⁻¹ as CaCO₃ for nitrifying and 400 mg l⁻¹ as CaCO₃ for saprophytic bacteria. After these hardness levels, the ultimate BOD (L) and rate constant (K) showed a decrease. Nitrifying bacteria were found to be more sensitive to the metal as well as to its complexation with calcium or with other ions as they retained their normal activity at a lower hardness level as compared to saprophytic bacteria.     

The occurrence and role of Bdellovibrio bacteriovorus in a polluted river

Bdellovibrio bacteriovorus was found to occur in a polluted river in numbers averaging, over an 18 month period, 10–100 parasites/ml. The parasite was not able to grow at river temperatures on host concentrations below about 10⁶–10⁷ bacteria per ml and even at these had a doubling time of only 24–48 h. The results have indicated that the parasite was not important in reducing the bacterial population of the river studied.     

A study of the thermal comfort of primary school children in summer

A study of the thermal comfort and clothing of primary school children aged 7–9 took place during the summers of 1971 and 1972. Of the 641 children who took part, 262 produced records suitable for analysis. The weight of clothing followed the trend of room temperature over several days, but did not respond to diurnal changes of temperature. The subjective warmth followed the variations of room temperature, but was not related to the mean temperatures of the classrooms, which were between 17° and 23°. The children differed significantly among themselves in their subjective response to temperature, but there was no appreciable difference between the responses of the boys and the girls. Thermal comfort distributions are presented and their implications for classroom temperatures are discussed.     

Simultaneous storage and degradation of PHB and glycogen in activated sludge cultures.

Bacteria in activated sludge are subjected to periods of substrate availability and absence of external substrates. The response of bacteria to such dynamic conditions was studied in a 2 L sequencing batch reactor (SBR) by subjecting a mixed microbial population to successive periods of external substrate availability (feast period) and no external substrate availability (famine period). In previous studies, acetate or glucose was used as single substrate leading to the storage of polyhydroxybutyrate or glycogen, respectively. In this study, a mixture of acetate and glucose was used. It appeared that both substrates were consumed simultaneously. The relative contribution of growth and storage processes was in these experiments similar as in the systems fed with a single substrate only. The ratio of substrate uptake over substrate storage was 0.6 Cmol/Cmol for both substrates. The uptake rate of acetate was not influenced by the simultaneous uptake of glucose. The degradation kinetics and rates of the storage compounds were the same as for the systems in which only one compound was stored in the activated sludge. The global performance of the culture grown on mixed substrates could therefore be described as the sum of the conversions observed in cultures fed with the individual substrates.     

Occurrence of nitrifiers and diversity of ammonia-oxidizing bacteria in developing drinking water biofilms

We studied the population dynamics of nitrifying bacteria during the development of biofilms up to 233 or 280 days on polyvinylchloride pipes connected to two full-scale drinking water distribution networks supplying processed and chloraminated surface water. The numbers of nitrifiers in biofilms were enumerated at intervals of 10–64 days by the most probable number (MPN) method at waterworks and at several study sites in distribution network areas. The numbers of nitrifiers increased towards the distal sites. The highest detected MPN counts of ammonia-oxidizing bacteria (AOB) for study areas 1 and 7 were 500 MPN cm⁻² and 1.0×10⁶ MPN cm⁻², and those of nitrite-oxidizing bacteria (NOB) 96 MPN cm⁻² and 2.2×10³ MPN cm⁻², respectively. The diversity of AOB was determined by PCR amplifying, cloning and sequencing the partial ammonia monooxygenase (amoA) gene of selected biofilm samples presenting different biofilm ages. The PCR primers used, A189 and A682, also amplified a fragment of particulate methane monooxygenase (pmoA) gene of methane-oxidizing bacteria. The majority of biofilm clones (24 out of 30 studied) contained Nitrosomonas amoA-like sequences. There were only two pmoA-like sequences of Type I methanotrophs, and four sequences positioned in amoA/pmoA sequence groups of uncultured bacteria. From both study area very similar or even completely identical Nitrosomonas amoA-like sequences were obtained despite of high difference in AOB numbers. The results show that the conditions in newly formed biofilms in drinking water distribution systems favor the growth of Nitrosomonas-type AOB.     

Experimental evaluation of decrease in bacterial activity due to cell death and activity decay in activated sludge

Decrease in bacterial activity (cell decay) in activated sludge can be attributed to cell death (reduction in the amount of active bacteria) and activity decay (reduction in the specific activity of active bacteria). The aim of this study was to experimentally differentiate between cell death and activity decay as a source of decrease in microbial activity. By means of measuring maximal oxygen uptake rates, verifying membrane integrity by live/dead staining and verifying presence of 16S rRNA with fluorescence in-situ hybridization, the decay rates and the death rates of ammonium oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and ordinary heterotrophic organisms (OHOs) were determined respectively in a nitrifying sequencing batch reactor (SBR) and a heterotrophic SBR. The experiments revealed that in the nitrifying system activity decay contributed 47% and 82% to the decreased activities of AOB and NOB and that cell death was responsible for 53% and 18% of decreases in their respective activities. In the heterotrophic system, activity decay took a share of 78% in the decreased activity of OHOs, and cell death was only responsible for 22% of decrease in their activity. The difference between the importance of cell death on the decreased activities of AOB and OHOs might be caused by the mechanisms of substrate storage and/or cryptic growth/death-regeneration of OHOs. The different nutrient sources for AOB and NOB might be the reason for a relatively smaller fraction of cell death in NOB.     

Effect of temperature on the residual compressive strength of laterized concrete

Tests are reported on 100 mm laterized concrete cubes, containing ordinary portland cement, crushed granite, sharp sand and fine laterite in varying proportions. The percentage of sand by weight of total fine aggregate was varied in steps of 25% up to a maximum of 100% corresponding to normal concrete. The mix proportion was . The test specimens were exposed to varying temperatures ranging from 30°C (i.e. room temperature) to 800°C and allowed to cool for 24 hours before crushing. The results showed that unlike normal concrete the residual compressive strength of laterized concrete increaed, by up to 50% of the nominal strength, with increasing temperature up to 200°C before falling to about 20% of the nominal strength at 800°C. The gain in strength depended on the sand content. The results further showed that within the limits of water/cement ratios normally used in concrete works (i.e. 0.55 to 0.65), the residual strength of laterized concrete was independent of the water/cement ratio. Also, the density of laterized concrete was not significantly affected by changes in temperature but that the tangent modulus of elasticity was reduced from 18 kN/mm² at 30°C to 0.12 kN/mm² at 600°C.