The Role of Protozoa in the Activated-Sludge Process

Ciliates are the dominant protozoa in activated sludge, andthose which either attach themselves to or crawl over the surfaceof the sludge are the most common. Experimental work has demonstratedthat ciliated protozoa are essential for the production of agood quality effluent. Without ciliates, highly turbid, lowquality effluents are obtained. Experimental evidence furthersuggests that the predatory activities of the ciliates uponthe dispersed growths of bacteria are responsible for clarificationand for the reduction of coliform numbers during the activated-sludgeprocess. Mathematical models have been devised which take intoaccount both the bacterial and the protozoan populations. Thepopulations have been simulated on a computer and the resultsobtained in this way have helped to explain many of the observationsthat have been made upon the microbial populations found infull-scale activated-sludge plants.     

Bacteriophages of l-Glutamic Acid-Producing Bacteria

Several new phages were obtained from the abnormally fermented broths and from the air in l-glutamic acid fermentation factory using Br. lactofermentum. These twenty-one phages were classified into five serological groups on the basis of cross-neutralization tests with homologous and heterologous antisera. Group I contained ten phages, i.e., P61, P114, P401, P465, P468I, P508, P650, P204, Ap615 and L2. Group II contained five phages, i.e., P468II, Ap85II, Ap62, Ap72 and SI. Group III contained P468III, Ap85III, Ap93 and Ap518, and groups IV and V one phage each, P4 and L1, respectively.

In view of serological similarities and of differences in the host specificity, the phages of group I are considered as host range mutants derived from a plaque type mutant, P114, of original phage P61.     

Bacteriophages of l-Glutamic Acid-Producing Bacteria

Antiphage properties of many kinds of chemicals such as antibiotics, surface-active agents and chelating agents were examined on Brevibacterium lactofermentum No. 2256—phage P465 system using double-layer agar method, as a part of the basic study, for preventing phage infection in the industrial fermentation.

A great majority of inhibitors which were selected were usually nonspecific and inhibited also bacterial growth. Among about 200 chemicals tested, 5 antibiotics such as chloramphenicol and tetracycline, 6 chelating agents such as phytic acid and 19 surface- active agents such as PEG monoester and POE alkyl ether showed the selective inhibitions for phage infection at the concentrations which did not affect bacterial growth, or at the subbactericidal concentrations that suppressed bacterial growth slightly.

Of the above chemicals which showed selective inhibitions for phage infection, a possible mechanism of chelating agents chiefly of phytic acid was investigated. When 0.1 to 0.2% of phytic acid was present in the medium, the effect of inhibition was most remarkable; this could be applied to the actual phage-infected l-glutamic acid fermentation. Phytic acid had no direct phagocidal action, nor did it inhibit the late step of the phage multiplication; but it prevented the adsorption of phages, which required inorganic cofactors such as Mg²⁺ or Ca²⁺ in this step, to the host bacteria. Moreover, a part of the infected bacteria was made incapable of forming plaques in the presence of phytic acid. These results suggested that the chelation between Mg²⁺ or Ca²⁺ and phytic acid would remove the metal ions essential for phage adsorption and prevent the phage adsorption and infection of phage DNA, consequently, the phage infection.

The effect of the non-ionic surface-active agents (SAA) on the infection of phage P465 of Br. lactofermentum was examined by adsorption and one-step growth experiments as a part of the basic study on the prevention of phage-infection in the industrial fermentation. Among various SAA tested, polyoxyethylene stearyl ether (POE-SE), polyethylene glycol monooleate (PEG-MO) and polyoxyethylene sorbitan monostearate (Tween 60) had remarkably demonstrated the selective inhibition of phage infection.

The effect of the above three SAA was apparently restricted to the initial adsorption step of phage infection, for the phage already adsorbed would not be affected by exposure to SAA. However, the results of one-step growth experiment indicated that Tween 60 inhibited not only the phage-adsorption, but also the maturation of phage already adsorbed in the host cells. The rate of the inhibition was found to be directly related to the concentration of agent. And, the most effective adsorption-inhibition was exhibited at the critical micelle concentration of SAA. The concentration as used in our experiments did not affect the viability of either phages or the host cells.

The results also indicated that the inhibition of phage-adsorption was due to the action of SAA on the surface of the bacterial cells rather than on the phage. This is supported by the observation that preincubation of phage with SAA did not affect either the subsequent adsorption rate or the plaque-forming ability of the phage. In contrast with above, a short-term exposure of bacterial cells to SAA caused an apparent change to the cell surface which was only partially restored by washing repeatedly. Moreover, the inhibitory effect of SAA on phage-adsorption appears quite specific in the phage-host system.     

Bacteriophages of l-Glutamic Acid-Producing Bacteria

Deoxyribonucleic acids were isolated from Brevibacterium lacto fermentum No. 2256 and its four representative phages belonging to different serological groups, i.e., P465 (group I), P 468 II (group II), Ap 85 III (group III) and P4 (group IV), by phenol extraction.

DNA’s isolated from the four phages contained only usual four bases, i.e., guanine, adenine, cytosine and thymine. The G-C content of the phage DNA was determined by thermal denaturation method (T_m); the values of P465, P 468 II, Ap 85 III and P4 were 54.0, 54.6, 56.6 and 55.3%, respectively. Sedimentation coefficient was measured by ultracentrifugal analysis using ultraviolet optics; s_20,w of the phage DNA’s were 15.5 to 31.8 s.

Unusual bases were not detected also in the host bacterial DNA. The G-C content of the bacterial DNA determined by paperchromatography was 55.1% which came very close to the G-C content derived from the T_m.

Morphological properties of the P- and Ap-series phages described in previous papers were examined by means of electron microscopy, analytical centrifugation, CsCl density-gradient centrifugation and ultrafiltration.

In view of the buoyant density of phage particles, fourteen Brevibacterium phages were classified into four groups, i.e., the phages under group I had buoyant density of 1.511 to 1.514g cm^?3, and those under groups II, III and IV, densities of 1.482, 1.492 and 1.508 g cm^?3, respectively; and above grouping corresponded to that by the serological chracteristics of the phages.

Electron microscopic observation by means of shadow-casting or negative staining recognized all the phages as tadpole-shaped; their particles having polyhedral head (40 ~ 70 mμ in diameter) and long tail (80 ~ 275 mμ in length).

In relation to particle sizes of the phages as estimated by all of above-mentioned methods, no significant differences were observed between the sizes calculated from ultrafiltration and those obtained directly from electron micrographs by negative staining.     

Bacteriophages of l-Glutamic Acid-Producing Bacteria

Some of the characteristics of the four phages, P 465 (group I), P 468 II (group II), Ap 85 III (group III) and P 4 (group IV), of Brevibacterium lacto fermentum, i.e., stability in salt solution, thermal and pH stabilities, inactivation by ultraviolet irradiation, stability on air-drying, and lytic characteristics of infected cells, are descrided. We have observed differences in these physico-chemical characteristics among these four phages selected from the respective serological groups. An addition of protein such as casein to diluent at a 0.01 ~ 1% concentration increased remarkably the stabilities on shaking of these phages except Ap 85 III. There were great differences in stabilities on air-drying among the four phages. Phage P 61 (gropu I) which was isolated from the first abnormal fermentation broth showed the lowest stability in the dry state, while phages, P 468 II and Ap 85 III, were very stable and were not inactivated by the relative humidity of 10% at 30°C, for the period of five months. In general, Ар-series phages isolated from air-borne particles at fermentation factory can be distinguished from the other P-series phages which were isolated from abnormal fermentation broth, by their considerably high resistance to drying.     

Bacteriophages of l-Glutamic Acid-Producing Bacteria

The growth characteristics of phages were investigated with the four phages, active on Brevibacterium lactofermentum, which were selected from the respective serological groups, namely, P465 (group I), P468II (group II), Ap85III (group III) and P4 (group IV).

The adsorption rate of the phages, P465 and P468II, on the host bacteria was low, whereas that of the phages, Ap85III and P4, was higher. The adsorption rate constants for the four phages were respectively calculated at 2.02 × 10^?10, 1.87 × 10^?10, 4.32 × 10^?10 and 3.15 × 10^?10 cm³ per minute, at 30°C in G₅B₂ medium. With reference to the ionic environment for adsorption, the phages, P465 and Ap85III, specifically required either for Ca⁺⁺ or Mg⁺⁺; the phage P468II, for both; and the phage P4, for neither.

The growth characteristics of these phages were examined by the one-step growth experiment. The latent periods of the phages were 50, 53, 57 and 47 minutes, respectively; and the corresponding average burst sizes were about 98, 31, 145 and 126. The growth of the phage P4 was completely suppressed at above 34°C, although the host bacteria and the other three phages were capable of the full growth at that temperature.     

Survival of Bacterial Indicator Species and Bacteriophages after Thermal Treatment of Sludge and Sewage

The inactivation of naturally occurring bacterial indicators and bacteriophages by thermal treatment of a dewatered sludge and raw sewage was studied. The sludge was heated at 80°C, and the sewage was heated at 60°C. In both cases phages were significantly more resistant to thermal inactivation than bacterial indicators, with the exception of spores of sulfite-reducing clostridia. Somatic coliphages and phages infecting Bacteroides fragilis were significantly more resistant than F-specific RNA phages. Similar trends were observed in sludge and sewage. The effects of thermal treatment on various phages belonging to the three groups mentioned above and on various enteroviruses added to sewage were also studied. The results revealed that the variability in the resistance of phages agreed with the data obtained with the naturally occurring populations and that the phages that were studied were more resistant to heat treatment than the enteroviruses that were studied. The phages survived significantly better than Salmonella choleraesuis, and the extents of inactivation indicated that naturally occurring bacteriophages can be used to monitor the inactivation of Escherichia coli and Salmonella.     

Enumeration of Airborne Bacteria in Hospital

An investigation of the “normal” bacterial content of the air of a large general hospital is described. In many different places within five different areas 70 to 200 settle-plate or slit-sampler bacterial counts were carried out. Average counts were most often of the same order as or lower than other published results and were proportional to human activity. The use of the logarithms of the counts showed no advantage, and conventional statistics should be applied with caution in evaluating such studies. Slitsampler and settle-plate counts of all bacteria showed no correlation, whereas those of Staph. aureus were correlated. There is a lack of parallelism between hospital infection and air bacteria counted by current methods, which are, therefore, not suitable for routine use.     

Isolation from Sewage Influent and Characterization of Novel Staphylococcus aureus Bacteriophages with Wide Host Ranges and Potent Lytic Capabilities

Staphylococcus aureus is a gram-positive pathogen that causes a variety of diseases, including bovine mastitis, which has severe economic consequences. Standard antibiotic treatment results in selection of resistant strains, leading to a need for alternative treatments, such as bacteriophage therapy. Forty-nine S. aureus isolates were obtained from the milk of mastitic cows for use in screening of staphylococcal phages. Fifteen isolates which were positive for both coagulase and hemolysin were assayed by PCR for variation in the X region and the immunoglobulin G-binding region of the protein A gene (spa) and in the carboxy terminus of the coagulase gene (coa) and for the presence of enterotoxin C, G, H, and I genes. The host ranges of 52 phages isolated from sewage influent were determined by performing spot tests with the 15 S. aureus isolates, and two phages were subsequently chosen for further analysis. ΦSA039 had the widest host range, producing clear plaques on 13 of the 15 isolates (87%), while ΦSA012 produced clear plaques on 8 isolates (53%) and was the only phage that produced a clear plaque on a nonmastitic S. aureus strain. Transmission electron microscopy revealed that the phages were similar sizes and belonged to the Myoviridae family. Measurement of optical densities during coculture with S. aureus isolates confirmed the breadth of the ΦSA039 host range and showed that ΦSA012 had potent lytic capability. ΦSA012-resistant bacteria did not appear for three of seven isolates tested (43%) after 65 h of incubation. These two phages are proposed as candidates for phage therapy of bovine mastitis.In the dairy industry, mastitis is a widespread problem responsible for important decreases in milk production. Economic losses of $100 million per year have been estimated for farms in Hokkaido, one of the largest milk-producing areas in Japan (28). Mastitis can be caused by over 150 different microorganisms, and one of the most important of these organisms is Staphylococcus aureus (22). After diagnosis of mastitis, the standard treatment regimen consists of isolating the diseased cow and treating it with antibiotics. However, this approach has drawbacks, such as its high cost and the eradication of harmless or beneficial organisms due to the lack of specificity of antibiotics. Additionally, the incidence of antibiotic-resistant bacteria has increased in recent years (4). As a result, there has been renewed interest in the use of other natural or engineered antimicrobial agents as an alternative or supplementary treatment for staphylococcal diseases such as mastitis (11, 21, 26). One group of alternatives with great potential involves bacteriophages (phages) and their derivatives, and a number of promising candidates have been described (2, 5, 7, 13, 17, 18, 27), notably bacteriophage K.One of the main obstacles to successful treatment of mastitis using phages is the fact that most phages are able to infect only a very narrow range of hosts. Given the plural etiology of many mastitis cases, it is desirable to find a phage-based treatment that targets a wide range of pathogens. One proposed method is a combination of two or more phages with different and broad host ranges in a cocktail (25, 29). This method has been shown to delay considerably the appearance of phage-resistant cells in Escherichia coli cultures, providing hope that similar success can be achieved using S. aureus phages.In this paper we describe the isolation of two novel phages, designated ΦSA012 and ΦSA039, that were found to have a lytic effect on a broad range of S. aureus isolates obtained from mastitic milk. ΦSA012 and ΦSA039 were selected after host range analysis from a total of 52 phages screened from sewage influent. These two phages were characterized morphologically by using electron microscopy, and their effects on representative strains of S. aureus were examined by coculture in milk.     

Enumeration of Bacteria in Sea Water

The choice of methods to be used for the enumeration of bacteria in sea water must depend on the objectives of the investigators. Comparisons of data based on different methods may be invalid. An operational definition is given for a colony-generating unit (CGU) which, under some conditions, may be a single viable bacterium. Such units can conveniently be enumerated by a modification of the “minimum probable number” method currently employed in aquatic bacteriology. Counts of “viable bacteria” (CGU) in surface sea water samples from the region of the North Pacific Gyre (28° N, 155°W) were estimated to range from 10,000 to 200,000 per litre.     

Fluctuations in Populations of 3 Vorticella Species from an Activated-Sludge Sewage Plant

SYNOPSIS. To evaluate Vorticella populations in an activated sludge sewage-disposal plant as a ) indicators and b ) contributors to purification, population behavior in the plant was compared with behavior in clonaI cultures maintained with mixed sludge microflora. In a 12-month survey of protozoa in the plant, of 37 spp recorded only 9—all ciliates—were indigenous. Ciliate populations fluctuated cyclically. Alternations in dominance of Vorticella convallaria (L.) with V. octava (Stokes) + V. aequitata (Kahl) apparently followed changes in bacterial activity as indicated by changes in reduction of biochemical oxygen demand and suspended solids of the wastes. This correlation of Vorticella and bacterial activities was also investigated with clonal populations of the vorticellas and their accompanying mixed microflora—the bacteria being isolated together with the vorticellas from sludge samples. The mixed microflora which consistently supported healthy vorticellas was predominantly proteolytic. Varying periods of dominance in Vorticella species thus indicated activity of their supporting microflora. Log-phase bacteria activity stimulated V. octava ; stationary and declining phases stimulated V. convallaria. Thus, V. octava appeared predatory and V. convallaria appeared to require bacterial conditioning of the medium. Bacterial activity was stimulated by V. octava predation. Vorticella activity may thus promote purification.     

Occurrence of Purple Sulfur Bacteria in a Sewage Treatment Lagoon

The ecology of purple sulfur bacteria in a sewage oxidation lagoon was investigated. Chemical changes in the lagoon were investigated by monitoring biochemical oxygen demand (BOD(5)), sulfide, sulfate, phosphate, total carbohydrates, volatile acids, alkalinity, and pH. Lagoon water temperatures were observed daily. Microbial ecological relationships were deduced by enumerating coliforms, total bacteria other than anaerobes [Tryptone Glucose Extract (TGE) agar], methane formers such as Methanobacterium formicicum, sulfate reducers, purple sulfur bacteria, and algae. Finally, two strains of purple sulfur bacteria were characterized. Two populations, purple sulfur bacteria and total bacteria (TGE agar), reached maximal concentrations in the warmest part of the 1967 summer. Purple sulfur bacteria reached maximal numbers as concentrations of sulfide and volatile acids were depleted, whereas carbohydrates and alkalinity remained unchanged. Low sulfate levels, which were not limiting for sulfate reducers, may be attributable to storage of sulfur within purple sulfur bacteria. No biological, chemical, or physical agent was linked to the removal of coliforms. The increase of algae in the late summer of 1967 may have been related to the low organic content of the lagoon during this period. Although lagoon pH (7.7 to 8.2) was favorable for purple sulfur bacterial growth, temperatures and sulfides were not optimal in the lagoon for these organisms. Chromatium vinosum and Thiocapsa floridana (the predominant lagoon purple sulfur organism in 1967 and 1968) utilized certain carbohydrates, amino acids, volatile acids, and Krebs cycle intermediates. Also purple sulfur bacteria lowered BOD levels as demonstrated by the growth of T. floridana in sterilized sewage.     

Treatment of Oil-containing Sewage Wastewater Using Immobilized Photosynthetic Bacteria

Summary Removal of cooking oil from the domestic wastewater was carried out by immobilized photosynthetic bacteria, Rhodobacter shaeroide S (S) and R. shaeroides NR-3 (NR-3). The microorganisms were immobilized in sodium alginate (2%) or agar (2%). We treated 50 g of cooking oil suspended in artificial sewage wastewater under anaerobic dark conditions in a 15 l acrylic vessel. Results show that after 6 days of batch treatment, 74.2, 58.2 and 15.8% of oil was removed with the alginate-immobilized S, NR-3, and control, respectively. Relatively larger accumulations of volatile fatty acids such as propionic and acetic acids were observed in the control experiment compared with that in the immobilized cell (alginate and agar) experiment. In addition, continuous treatment of oil-containing wastewater was carried out with agar-immobilized S at a fixed dilution rate of 0.4/day. These results indicate that 96% of the oil was removed from the wastewater, and the maximum removal rate of oil reached approximately 3.83 kg oil/m³/d.     

Enumeration of Viable Bacteria in the Marine Pelagic Environment

The low percentage of living bacteria commonly obtained when comparing viable counts with total direct counts in seawater could be due more to inappropriate techniques for appreciating the growth ability of living cells than to unadapted culture conditions. The most-probable-number counts in filtered seawater cultures and the microscopic counts of 4(prm1),6-diamidino-2-phenylindole (DAPI)-stained aggregate-forming units grown on black polycarbonate filters appeared significantly correlated to the direct counts. Both these techniques show that in the superficial and intermediate water masses, the living cells may constitute an important (frequently higher than 20%) but highly variable part of the total populations. These viable counts appear more realistic than the conventional CFU counts, which provide only 0.001 to 0.2% of the total counts.     

Sunlight Inactivation of Fecal Bacteriophages and Bacteria in Sewage-Polluted Seawater

Sunlight inactivation rates of somatic coliphages, F-specific RNA bacteriophages (F-RNA phages), and fecal coliforms were compared in seven summer and three winter survival experiments. Experiments were conducted outdoors, using 300-liter 2% (vol/vol) sewage-seawater mixtures held in open-top chambers. Dark inactivation rates (k_Ds), measured from exponential survival curves in enclosed (control) chambers, were higher in summer (temperature range: 14 to 20°C) than in winter (temperature range: 8 to 10°C). Winter k_Ds were highest for fecal coliforms and lowest for F-RNA phages but were the same or similar for all three indicators in summer. Sunlight inactivation rates (k_S), as a function of cumulative global solar radiation (insolation), were all higher than the k_Ds with a consistent k_S ranking (from greatest to least) as follows: fecal coliforms, F-RNA phages, and somatic coliphages. Phage inactivation was exponential, but bacterial curves typically exhibited a shoulder. Phages from raw sewage exhibited k_Ss similar to those from waste stabilization pond effluent, but raw sewage fecal coliforms were inactivated faster than pond effluent fecal coliforms. In an experiment which included F-DNA phages and Bacteroides fragilis phages, the k_S ranking (from greatest to least) was as follows: fecal coliforms, F-RNA phages, B. fragilis phages, F-DNA phages, and somatic coliphages. In a 2-day experiment which included enterococci, the initial concentration ranking (from greatest to least: fecal coliforms, enterococci, F-RNA phages, and somatic coliphages) was reversed during sunlight exposure, with only the phages remaining detectable by the end of day 2. Inactivation rates under different optical filters decreased with the increase in spectral cutoff wavelength (50% light transmission) and indicated that F-RNA phages and fecal coliforms are more susceptible than somatic coliphages to longer solar wavelengths, which predominate in seawater. The consistently superior survival of somatic coliphages in our experiments suggests that they warrant further consideration as fecal, and possibly viral, indicators in marine waters.     

Sand Beach Bacteria: Enumeration and Characterization

Bacteria in the water-saturated sand of a relatively unpolluted sand beach were enumerated by direct microscope and viable counting. The number of interstitial bacteria was estimated to be a significant fraction of the total number of bacteria present. Three hundred sixty-two strains were isolated and submitted to cultural and biochemical tests. Fermentational abilities and the production of indole suggested that a significant number of these bacteria were symbiotically associated with resident metazoans.