Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge: I. Enumeration1

Agar plating media containing solely activated sludge extracts yielded, in general, higher viable counts of activated sludge bacteria than any other culture medium tested. Activated sludge extracts made from different treatment plants varied in efficacy in evoking maximal viable counts. Frequently, homologous plating, i.e., plating inocula of activated sludges on extracts made from the same activated sludges, tended to yield lower counts than the heterologous platings tried in this investigation. The counts obtained by homologous plating of activated sludge were not significantly lower and sometimes were even significantly higher than the counts obtained on standard Nutrient Agar, which had been found by previous workers to be a good medium for counting activated sludge bacteria. The higher counts obtained with activated sludge extracts set objectives for formulating reproducible or defined culture media for the enumeration of activated sludge bacteria.     

Bacteriology and Enzymology of Fellmongery Activated Sludge Systems

The activities of certain hydrolytic and oxidative enzymes and the number of bacteria capable of degrading specific substrates during extended aeration of fellmongery effluent was investigated. An enzymic approach was shown to be a potentially useful method for the quantitative biological characterization of activated sludges. The bacterial population of fellmongery activated sludge mixed liquor was dominated by bacteria from the two genera Pseudomonas and Acinetobacter .     

Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge: V. Analysis of Population Structure and Activity

Two procedures, the confidence interval method and Mountford's index, were tested in analyses of the microbial populations of 11 laboratory activated sludges acclimated to aromatic compounds. The two methods gave somewhat different results but indicated that the populations were quite dissimilar. The activity of seven of the sludges correlated well with the population structure. Some considerations in analysis of microbial population structure are discussed.     

Members of the Family Comamonadaceae as Primary Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate)-Degrading Denitrifiers in Activated Sludge as Revealed by a Polyphasic Approach

The distribution and phylogenetic affiliations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria in activated sludge were studied by a polyphasic approach including culture-independent biomarker and molecular analyses as well as cultivation methods. A total of 23 strains of PHBV-degrading denitrifiers were isolated from activated sludges from different sewage treatment plants. 16S ribosomal DNA (rDNA) sequence comparisons showed that 20 of the isolates were identified as members of the family Comamonadaceae, a major group of β-Proteobacteria. When the sludges from different plants were acclimated with PHBV under denitrifying conditions in laboratory scale reactors, the nitrate removal rate increased linearly during the first 4 weeks and reached 20 mg NO₃⁻-N h⁻¹ g of dry sludge⁻¹ at the steady state. The bacterial-community change in the laboratory scale sludges during the acclimation was monitored by rRNA-targeted fluorescence in situ hybridization and quinone profiling. Both approaches showed that the population of β-Proteobacteria in the laboratory sludges increased sharply during acclimation regardless of their origins. 16S rDNA clone libraries were constructed from two different acclimated sludges, and a total of 37 clones from the libraries were phylogenetically analyzed. Most of the 16S rDNA clones were grouped with members of the family Comamonadaceae. The results of our polyphasic approach indicate that β-Proteobacteria, especially members of the family Comamonadaceae, are primary PHBV-degrading denitrifiers in activated sludge. Our data provide useful information for the development of a new nitrogen removal system with solid biopolymer as an electron donor.     

Studies on Induction and Repression in Activated Sludge Systems

Both repression and induction of substrate utilization have been the subject of many basic research investigations employing pure cultures. In this investigation these effects were studied using heterogeneous microbial populations prevalent in such biological treatment processes as activated sludge systems.Diauxic substrate removal by activated sludge was observed in a multicomponent medium consisting of glucose and sorbitol. The sludge was acclimated solely to sorbitol; however, the presence of glucose blocked sorbitol removal until glucose was completely utilized. Both diphasic and triphasic oxygen utilization was shown for activated sludges metabolizing multicomponent synthetic wastes consisting of glucose, melibiose, and lactose. It appears from these studies that melibiose utilization was suppressed by the presence of glucose and, although melibiose induced acclimation to lactose, the presence of melibose suppressed lactose utilization. Studies were also conducted using glycogen and starch systems in which it was found that acclimation to either compound conferred immediate acclimation to the other. It was also found that loss of acclimation to lactose was a passive phenomenon and its kinetics could be predicted on the basis of simple diluting out of the enzyme(s) responsible for such acclimation.     

Removal of Phthalate Esters by Activated Sludge Inoculated with a Strain of Nocardia erythropolis

Phthalate esters, such as di-2-ethyl hexyl phthalate (DEHP) and di-n-butyl phthalate (DBP), were efficiently removed from wastewater by inoculating viable cells of Nocardia erythropolis, a bacterium capable of rapidly degrading phthalate esters, in activated sludge. When the wastewater containing 1500 ppm of DEHP was treated with the activated sludge inoculated with Nocardia erythropolis, the DEHP was found to be removed at a rate of 98.2% in 1 day and to be gas-chromatographically free on and after the 3rd day. Activated sludges, in particular, when high concentration of substances was used, were efficiently prevented from deflocculation of sludge by inoculation of Nocardia erythropolis, and moreover, the deflocked sludge was restored and recovered by the addition of Nocardia erythropolis.     

On the Occurrence of Anoxic Microniches, Denitrification, and Sulfate Reduction in Aerated Activated Sludge

A combination of different methods was applied to investigate the occurrence of anaerobic processes in aerated activated sludge. Microsensor measurements (O₂, NO₂⁻, NO₃⁻, and H₂S) were performed on single sludge flocs to detect anoxic niches, nitrate reduction, or sulfate reduction on a microscale. Incubations of activated sludge with ¹⁵NO₃⁻ and ³⁵SO₄²⁻ were used to determine denitrification and sulfate reduction rates on a batch scale. In four of six investigated sludges, no anoxic zones developed during aeration, and consequently denitrification rates were very low. However, in two sludges anoxia in flocs coincided with significant denitrification rates. Sulfate reduction could not be detected in any sludge in either the microsensor or the batch investigation, not even under short-term anoxic conditions. In contrast, the presence of sulfate-reducing bacteria was shown by fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes and by PCR-based detection of genes coding for the dissimilatory sulfite reductase. A possible explanation for the absence of anoxia even in most of the larger flocs might be that oxygen transport is not only diffusional but enhanced by advection, i.e., facilitated by flow through pores and channels. This possibility is suggested by the irregularity of some oxygen profiles and by confocal laser scanning microscopy of the three-dimensional floc structures, which showed that flocs from the two sludges in which anoxic zones were found were apparently denser than flocs from the other sludges.     

Biochemical Studies on Accelerated Treatment of Thiocyanate by Activated Sludge using Growth Factors such as Pyruvate

S ummary . Micro-organisms in activated sludge oxidizing thiocyanate behave in a similar way to thiobacilli. Organic compounds such as pyruvate stimulate thiocyanate oxidation and in so doing become incorporated into cellular material; such metabolites are not oxidized to produce energy. The presence of thiocyanate in batch cultures accelerated pyruvate uptake. The carbon of thiocyanate is recovered mainly as CO₂; carbonate becomes incorporated into cellular protein, but less so in sludges previously treated with pyruvate. TCA cycle enzymes are not altered appreciably after pyruvate is added to activated sludge.     

Application of the Fluorescent-Antibody Technique for the Detection of Sphaerotilus natans in Activated Sludge

Sphaerotilus natans, one of the most widely reported causes of bulking in activated sludge, can exist both within and outside of a sheath. It can easily be confused with similar activated sludge bacteria and thus can be overlooked when present in low numbers. Fluorescent antiserum was successfully prepared against the nonfilamentous form and was shown to be highly specific, showing no reaction with either pure cultures of similar filamentous bacteria or entirely unrelated organisms. It did, however, show a lack of strain specificity since it reacted with S. natans isolates from the Federal Republic of Germany and the United States and with filamentous bacteria in South African activated sludges. Fluorescent antibody is capable of penetrating the filaments of S. natans to stain the cells individually. The use of fluorescent antiserum in the identification of S. natans filaments obscured by activated sludge flocs and other suspended matter was simple since the cells stained brightly and could be observed through the less dense matter, while the use of other microscope techniques would be hampered by these obstructions. The use of fluorescent antibody will facilitate ecological studies of S. natans in activated sludge and other aqueous environments.     

Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal

A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like β-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg²⁺, and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.     

Population Dynamics of Phenol-Degrading Bacteria in Activated Sludge Determined by gyrB-Targeted Quantitative PCR

A method for quantifying bacterial populations introduced into an activated-sludge microbial community is described. The method involves extraction of DNA from activated sludge, appropriate dilution of the extracted DNA with DNA extracted from nonintroduced activated sludge, PCR amplification of a gyrB gene fragment from the introduced strain with a set of strain-specific primers, and quantification of the electrophoresed PCR product by densitometry. The adequacy of the method was examined by analyzing the population dynamics of two phenol-degrading bacteria, Pseudomonas putida BH and Comamonas sp. strain E6, that had been introduced into phenol-digesting activated sludge. The density of each of the two populations determined by the PCR method immediately after the introduction was consistent with the density estimated from a plate count of the inoculum. This quantitative PCR method revealed different population dynamics for the two strains in the activated sludge under different phenol-loading conditions. The behavior of both of these strains in the activated sludge reflected the growth kinetics of the strains determined in laboratory axenic cultures.     

Aerobic Heterotrophic Bacterial Populations of Sewage and Activated Sludge: II. Method of Characterization of Activated Sludge Bacteria

The replica-plating technique and Lochhead's nutritional method were combined in exploratory experiments to test their feasibility as useful means for characterizing the aerobic heterotrophic flora of activated sludge and to minimize the burdensome process of isolation, purification, and testing of isolates. In the test run, the method was about 86% reliable at the 0.05 level of significance. About 40% of the total number of bacteria able to grow on an aqueous extract of activated sludge did not grow on media containing glucose, amino acids, growth factors, and inorganic salts. The requirement for activated sludge extract suggested the existence of a requirement for unidentified nutrients contained in the activated sludge extract.     

Members of the family Comamonadaceae as primary poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-degrading denitrifiers in activated sludge as revealed by a polyphasic approach

The distribution and phylogenetic affiliations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria in activated sludge were studied by a polyphasic approach including culture-independent biomarker and molecular analyses as well as cultivation methods. A total of 23 strains of PHBV-degrading denitrifiers were isolated from activated sludges from different sewage treatment plants. 16S ribosomal DNA (rDNA) sequence comparisons showed that 20 of the isolates were identified as members of the family Comamonadaceae, a major group of beta-Proteobacteria. When the sludges from different plants were acclimated with PHBV under denitrifying conditions in laboratory scale reactors, the nitrate removal rate increased linearly during the first 4 weeks and reached 20 mg NO(3)(-)-N h(-1) g of dry sludge(-1) at the steady state. The bacterial-community change in the laboratory scale sludges during the acclimation was monitored by rRNA-targeted fluorescence in situ hybridization and quinone profiling. Both approaches showed that the population of beta-Proteobacteria in the laboratory sludges increased sharply during acclimation regardless of their origins. 16S rDNA clone libraries were constructed from two different acclimated sludges, and a total of 37 clones from the libraries were phylogenetically analyzed. Most of the 16S rDNA clones were grouped with members of the family Comamonadaceae. The results of our polyphasic approach indicate that beta-Proteobacteria, especially members of the family Comamonadaceae, are primary PHBV-degrading denitrifiers in activated sludge. Our data provide useful information for the development of a new nitrogen removal system with solid biopolymer as an electron donor.     

Metabolic Factors Affecting Enhanced Phosphorus Uptake by Activated Sludge

Activated sludges obtained from the Rilling Road plant located at San Antonio, Tex., and from the Hyperion treatment plant located at Los Angeles, Calif., have the ability to remove all of the orthophosphate normally present in Tucson sewage within 3 hr after being added to the waste water. Phosphorus removal was independent of externally supplied sources of energy and ions, since orthophosphate and (32)P radioactivity were readily removed from tap water, glass-distilled water, and deionized water. Phosphorus uptake by Rilling sludge in the laboratory appears to be wholly biological, as it has an optimum pH range (7.7 to 9.7) and an optimum temperature range (24 to 37 C). It was inhibited by HgCl(2), iodoacetic acid, p-chloromercuribenzoic acid, NaN(3), and 2, 4-dinitrophenol (compounds that affect bacterial membrane permeability, sulfhydryl enzymes, and adenosine triphosphate synthesis). Uptake was inhibited by 1% NaCl but was not affected by 10(-3)m ethylenediaminetetraacetic acid disodium salt (a chelating agent for many metallic ions).     

Quinone profiling of bacterial communities in natural and synthetic sewage activated sludge for enhanced phosphate removal

Respiratory quinones were used as biomarkers to study bacterial community structures in activated sludge reactors used for enhanced biological phosphate removal (EBPR). We compared the quinone profiles of EBPR sludges and standard sludges, of natural sewage and synthetic sewage, and of plant scale and laboratory scale systems. Ubiquinone (Q) and menaquinone (MK) components were detected in all sludges tested at molar MK/Q ratios of 0.455 to 0.981. The differences in MK/Q ratios were much larger when we compared different wastewater sludges (i.e., raw sewage and synthetic sewage) than when we compared sludges from the EBPR and standard processes or plant scale and laboratory scale systems. In all sludges tested a Q with eight isoprene units (Q-8) was the most abundant quinone. In the MK fraction, either tetrahydrogenated MK-8 or MK-7 was the predominant type, and there was also a significant proportion of MK-6 to MK-8 in most cases. A numerical cluster analysis of the profiles showed that the sludges tested fell into two major clusters; one included all raw sewage sludges, and the other consisted of all synthetic sewage sludges, independent of the operational mode and scale of the reactors and the phosphate accumulation. These data suggested that Q-8-containing species belonging to the class Proteobacteria (i.e., species belonging to the beta subclass) were the major constituents of the bacterial populations in the EBPR sludge, as well as in standard activated sludge. Members of the class Actinobacteria (gram-positive bacteria with high DNA G+C contents) were the second most abundant group in both types of sludge. The bacterial community structures in activated sludge processes may be affected more by the nature of the influent wastewater than by the introduction of an anaerobic stage into the process or by the scale of the reactors.     

Activated sludge transformation in anoxic condition

Summary Substantial increase of methanogens and obligate hydrogen producing acetogens is occurs when activated sludges are allowed to remain under anoxic conditions at either 25°C and 35°C, without oxygen free atmosphere or any external addition of reductants. Acetate addition to activated sludge stimulates anoxic growth of all anaerobes except for butyrate users. During this period, the sludge volume index decrease.     

Dewatering of Extracellular Polymer and Activated Sludges by Thermochemical Treatment

Of the dewatering characteristics of activated sludges in our previous paper, the dewatering rate of sludge decreased in proportion to increasing amounts of extracellular polymer. As extracellular polymer in activated sludge was one of the important factors in the dewatering process, the change of the dewatering characteristics of thermochemically treated sludge (containing extracellular polymer) were compared with that of extracellular polymer extracted from sludge. The ultrafiltration rate of extracellular polymer flocculated by thermochemical treatment was much faster than that without treatment, indicating improved dewatering characteristics. Under the same treatment conditions, the dewatering characteristics of sludge were also much improved. The addition of the extracellular polymer treated thermochemically below pH 3 had no effect on the dewatering characteristics of the sludge. The addition of a flocculant to the thermochemically treated sludge was found to further improve the dewatering characteristics. The thermochemical treatment under low pH condition facilitated the flaking of the cake from the filter.     

Chlorine-Susceptible and Chlorine-Resistant Type 021N Bacteria Occurring in Bulking Activated Sludges

Two filamentous bacteria causing bulking in two activated sludges were examined. Investigations using morphological features, staining techniques, and fluorescent in situ hybridization identified both filaments as type 021N. However, an examination of the effect of chlorine on the sludges revealed a chlorine-susceptible type 021N in one sludge and a chlorine-resistant type 021N in the other.     

Microbial Ecology of Activated Sludge: I. Dominant Bacteria

Over 300 bacterial strains were isolated from seven samples of activated sludge by plating on sewage agar. Gram-negative bacteria of the genera Zoogloea and Comamonas predominated. Many isolates (51%) showed sudanophilic inclusions of poly-β-hydroxybutyric acid, whereas 34% accumulated iodophilic material on media containing starch. A large number required either vitamins or amino acids, or both, for growth. None of the isolates tested for their ability to bring about changes in autoclaved sewage produced an effluent comparable in quality to the activated sludge control, although the Zoogloea did produce activated sludgelike flocs. A study of 150 bacterial strains isolated from raw sewage revealed that they differed from the sludge isolates in several respects. Coliforms, which constitute nearly a quarter of the sewage isolates, were rarely encountered in sludge.