Elevation of antibiotic resistance genes at cold temperatures: implications for winter storage of sludge and biosolids.

UNLABELLED: Prior research suggests that cold temperatures may stimulate the proliferation of certain antibiotic resistance genes (ARGs) and gene transfer elements during storage of biosolids. This could have important implications on cold weather storage of biosolids, as often required in northern climates until a time suitable for land application. In this study, levels of an integron-associated gene (intI1) and an ARG (sul1) were monitored in biosolids subject to storage at 4, 10 and 20°C. Both intI1 and sul1 were observed to increase during short-term storage (<2 months), but the concentrations returned to background within 4 months. The increases in concentration were more pronounced at lower temperatures than ambient temperatures. Overall, the results suggest that cold stress may induce horizontal gene transfer of integron-associated ARGs and that biosolids storage conditions should be considered prior to land application. SIGNIFICANCE AND IMPACT OF THE STUDY: Wastewater treatment plants have been identified as the hot spots for the proliferation and dissemination of antibiotic resistance genes (ARGs) and antibiotic resistant bacteria (ARB) to the environment through discharge of treated effluent to water bodies as well as application of biosolids to land. Identifying critical control points within the treatment process may aid in the development of solutions for the reduction of ARGs and ARB and curbing the spread of antibiotic resistance. This study found increases in ARGs during biosolids storage and identifies changes in operational protocols that could help reduce ARG loading to the environment when biosolids are land-applied.

The effect of thermal hydrolysis pretreatment on the anaerobic degradation of nonylphenol and short-chain nonylphenol ethoxylates in digested biosolids.

The presence of micropollutants can be a concern for land application of biosolids. Of particular interest are nonylphenol diethoxylate (NP(2)EO), nonylphenol monoethoxylate (NP(1)EO), and nonylphenol (NP), collectively referred to as NPE, which accumulate in anaerobically digested biosolids and are subject to regulation based on the environmental risks associated with them. Because biosolids are a valuable nutrient resource, it is essential that we understand how various treatment processes impact the fate of NPE in biosolids. Thermal hydrolysis (TH) coupled with mesophilic anaerobic digestion (MAD) is an advanced digestion process that destroys pathogens in biosolids and increases methane yields and volatile solids destruction. We investigated the impact of thermal hydrolysis pretreatment on the subsequent biodegradation of NPE in digested biosolids. Biosolids were treated with TH, anaerobic digestion, and aerobic digestion in laboratory-scale reactors, and NPE were analyzed in the influent and effluent of the digesters. NP(2)EO and NP(1)EO have been observed to degrade to the more estrogenic NP under anaerobic conditions; therefore, changes in the ratio of NP:NPE were of interest. The increase in NP:NPE following MAD was 56%; the average increase of this ratio in four sets of TH-MAD samples, however, was only 24.6 ± 3.1%. In addition, TH experiments performed in pure water verified that, during TH, the high temperature and pressure alone did not directly destroy NPE; TH experiments with NP added to sludge also showed that NP was not destroyed by the high temperature and pressure of TH when in a more complex sludge matrix. The post-aerobic digestion phases removed NPE, regardless of whether TH pretreatment occurred. This research indicates that changes in biosolids processing can have impacts beyond just gas production and solids destruction.

The influence of sludge interchange times on the oxic-settling-anoxic process.

The oxic-settling-anoxic (OSA) system operated as a sequencing batch reactor was studied. Sludge was interchanged between the aerobic and anaerobic reactors, and the number of daily interchange times was changed from 1 to 4 during the experiments. When the sludge was interchanged 1 time/d, the reduction of excess sludge mass was only 52.8%. When the sludge was interchanged 4 times/d, the reduction of excess sludge mass was 77.4%; reductions in the total chemical oxygen demand, total solids, and total volatile solids of both the anaerobic sludge and the aerobic settled sludge were the greatest; and more soluble iron was released from the anaerobic sludge. The water quality of the final effluent in the two systems was close. The investigation showed that interchange times are important in improving sludge reduction efficiency, and the parameter should be regarded as an important operating factor in the OSA process.

The effect of temperature on the performance and stability of thermophilic anaerobic digestion.

Sustainable operation of an anaerobic sewage sludge digester requires the effective shuttling of carbon from complex organic material to methane gas. The accumulation of intermediates and metabolic products such as volatile fatty acids and hydrogen gas not only reveal inefficiency within the digestion process, but can be detrimental to reactor operation at sufficiently high levels. Eight anaerobic digesters (1 mesophilic and 7 thermophilic) were operated in order to determine the effect of steady-state digestion temperature on the operational stability and performance of the digestion process. Replicate reactors operated at 57.5 degrees C, the highest temperature studied, were prone to accumulation of volatile fatty acids (4052 and 3411 mg/L as acetate) and gaseous hydrogen. Reactors operated at or below 55 degrees C showed no such accumulation of intermediate metabolites. Overall methanogenesis was also greatly reduced at 57.5 degrees C (0.09 L CH4/g VS fed) versus optimal methane formation at 53 degrees C (0.40 L CH4/g VS fed). Microbial community assessment and free energy calculations suggest that the accumulation of fatty acids and hydrogen, and relatively poor methanogenic performance at 57.5 degrees C are likely due to temperature limitations of thermophilic aceticlastic methanogens.

The role of iron and aluminium in digestion and odor formation.

The role of iron and aluminium in determining volatile solids reduction and odors from anaerobically digested, dewatered sludge cakes was evaluated from data collected from a variety of wastewater treatment plants. It was found that volatile solids reduction generally increased as the iron content of the sludge increased. It was also observed that odors increased with increasing iron. No correlation with aluminium or divalent cations was found. Based on these data it appears that the volatile solids reduction by anaerobic digestion is not useful for predicting the odors from anaerobically digested sludges.

Aeration tank odour by dimethyl sulphoxide (DMSO) waste in sewage.

Sewage plants can experience dimethyl sulphide (DMS) odour problems by at least one mg/L dimethylsulphoxide (DMSO) waste residue in plant influent, through a DMSO/DMS reduction mechanism. This bench-scale batch study simulates in bottles the role of poor aeration in wastewater treatment on the DMSO/DMS and sulphate/H2S reduction. The study compares headspace concentrations of sulphide odorants developed by activated sludge (closed bottles, half full) after six hours under anoxic versus anaerobic conditions, with 0 versus 2 mg/L DMSO addition. Anoxic sludge (0.1 - 2 mg/L dissolved oxygen, DO) with DMSO resulted in about 50 ppmv DMS and no other sulphide, while DMSO-free sludge was free of detectable sulphides. Anaerobic sludge (no measurable DO to the point of sulphate reduction) with DMSO resulted in 22/4/37 ppmv of H2S/methanethiol (MT)/DMS, while DMSO-free sludge resulted in 44/8/2 ppmv of H2S/MT/DMS. It is concluded that common "anoxic" aeration tank zones with measurable DO in bulk water but immeasurable DO inside sludge flocs (nitrate reducing) experience DMSO reduction to DMS that is oxidation resistant and becomes the most important odorant. Under anaerobic conditions, H2S from sulphate reduction becomes an additional important odorant. A strategy is developed that allows operators to determine from the quantity of different sulphides whether the DMSO/DMS mechanism is important at their wastewater plant.

Effect of biopolymer on the dewatering characteristics of autothermal thermophilic aerobic digestion of sludges.

Autothermal thermophilic aerobic digestion of sludge is known to produce poorly dewatering sludges. Laboratory studies were conducted to investigate the reasons for the poor dewatering. It was found that, during digestion, proteins and polysaccharides were released into solution, and that these could be linked to the deterioration in dewatering. The biopolymer release was accompanied by an increase in the monovalent-to-divalent (M/D) cation ratio. The degree to which the M/D caused deterioration of the sludges depended on the presence of iron in sludge. When the iron content was high, the release of protein and polysaccharides was low. When iron was low, the release of protein and polysaccharides increased linearly with the M/D ratio. The dose of conditioning chemicals, cationic polymer or ferric chloride, was related to the amount of colloidal biopolymer present in solution. The findings suggest that the addition of iron during the digestion process has the potential to produce better dewatering sludges.

Chemical conditioning of sludge.

With all the advances made in understanding the structure and composition of sewage sludges, chemical conditioning remains a trial and error process, both with regard to the type and dose of conditioner needed. Recent studies at Virginia Tech have found that biological floc consists of two types of biopolymer, material associated with iron and aluminium and material associated with calcium and magnesium. These materials behave differently when sludges undergo digestion. This results in very different material being released into solution during digestion and very different conditioning requirements. This study shows that the primary materials released during anaerobic digestion are proteins and coagulation of the colloidal protein fraction in solution is the primary mechanism for conditioning. For aerobically digested sludges, both proteins and polysaccharides make up the colloid fraction, which interferes with dewatering. This research also shows that the effectiveness of the digestion process as characterized by volatile solids destruction is directly related to the chemical dose required for conditioning. That is, as the solids destruction increases, the conditioning chemical requirement also increases. Well digested sludges dewater more poorly and require more conditioning chemical than those with less volatile solids destruction.

Influence of cations on activated-sludge effluent quality.

Laboratory experiments and field tests were conducted to determine the effect of inorganic cations on effluent from activated-sludge systems. Laboratory experiments showed that monovalent cations tend to increase the concentration of solution biological polymers (biopolymers), whereas divalent cations tend to retain the biopolymers in the floc. Biopolymers in solution affect effluent chemical oxygen demand (COD). Coagulation tests were performed on the effluent with ferric chloride. Ferric hydroxide can coagulate protein through possible adsorptive interactions and may be responsible for some biopolymer retention in the flocs. In the field study, it was found that sodium ions in the influent wastewater caused an increase in proteins and polysaccharides in solution, thereby increasing the effluent COD concentration of the treated municipal wastewater. The attachment or release of these microbially derived organic biopolymers and recalcitrant influent substrate may depend on the monovalent-to-divalent cation ratio and the concentration of iron. Modeling of effluent organics in the activated-sludge process can be enhanced through incorporation of concepts that take into account the partitioning (between floc and solution) of microbial biopolymers and influent recalcitrant substrate.

Floc structure and the role of cations.

Cations have been found to influence the settling and dewatering characteristics of biological sludges. This research was directed at investigating the role of iron in floc stability of sensitivity to shear and in the response to anaerobic and aerobic digestion. Our data shows that iron may contribute to floc strength and it appears that the deterioration in sludge dewatering during anaerobic digestion is associated with the reduction and solubilization of iron. Further, it is the presence of proteins in solution that contributes to poor dewatering and to the demand for conditioning chemicals. The addition of iron for improving dewatering shows that iron may selectively coagulate solution proteins.

Monoclonal antibodies against human antithrombin III.

Three monoclonal antibodies identified as D8, B11 and C5 of different specificities have been produced against human antithrombin III (AT). The apparent dissociation constants (Kd app) of the AT-antibody interaction were determined by ELISA method: Kd app (D8) = 2.4 nmole, Kd app (B11) = 13 nmole, Kd app (C5) = 24 nmole. All three antibodies reacted with isolated AT on immunoblots obtained with "native" PAGE. The D8 antibody also reacted with plasma and serum AT while B11 antibody reacted with serum thrombin-antithrombin (TAT) complexes as well.

Three-dimensional reconstructions of nucleolus-organizing regions in PHA-stimulated human lymphocytes.

Ultrastructure and three-dimensional distribution of nucleolus-organizing regions have been studied on ultrathin serial sections of PHA-stimulated human lymphocytes. During the 48 hr of activation the size of fibrillar centers (FCs) decreased from 0.6-0.9 microns to 0.2-0.3 microns and the number of FCs increased rapidly from one to 75-107 per cell. The number of fibrillar complexes (i.e. associations of a different number of FCs connected by the dense fibrillar component) also increased but did not reach the maximum number of nucleolar organizers presented here. Three-dimensional computer reconstructions of fibrillar complexes showed that lymphocyte activation was accompanied by early (2-4 hr) changes in the shape of the primary fibrillar center. Invagination of the dense fibrillar component on its surface occurred and division into two or more smaller FCs followed. Gradually, the typical structure of the nucleolus with several fibrillar complexes and many FCs was formed. These results confirm the hypothesis of fibrillar complex-nucleolar organizer correlation published recently.

Effect of ribonucleases on cell-mediated lympholysis reaction and on GM-CFC colonies in bone marrow culture.

Natural dimer of bovine seminal ribonuclease (AS RNase) suppressed markedly DNA synthesis in allogeneic mixed lymphocyte culture (MLC) of normal human lymphocytes and simultaneously inhibited induction of cytotoxic effector cells within the sensitization phase of indirect cell-mediated lympholysis (CML) reaction. The last purification step of the AS RNase isolation procedure did not increase the suppressive activity of AS RNase compared to a less purified preparation (ZS RNase), thus, the later preparation was mostly used. ZS RNase (10 micrograms/ml) caused 50% inhibition of MLC reaction whereas pancreatic ribonuclease (A RNase) was 10 times less effective. The suppressive effect of RNases added in the beginning of the sensitization phase of the CML reaction correlated with that observed in the MLC reaction. The concentrations of ZS RNase (10 micrograms/ml), A RNase (100 micrograms/ml), and additionally tested cyclosporin A (0.5 microgram/ml) resulted in nearly total abrogation of cytolysis in CML. ZS RNase added after the sensitization of effector cells did not influence their cytolytic action on target cells within the destruction phase of CML. Natural killer and killer cell activities in normal peripheral lymphocytes were not inhibited by ZS RNase at the concentration of 330 micrograms/ml. ZS RNase (20 micrograms/ml), cocultivated 1 h with normal human bone marrow cells and then washed off, enhanced formation of GM-CFC colonies in semisolid agar culture up to 200%. Simultaneously tested antilymphocyte globulin increased the number of GM-CFC colonies at the average of 128%. This stimulating effect on colony formation appeared also in bone marrow culture of patients suffering with various hematological disorders. The possibility of utilizing the preparations gained from seminal plasma in clinical bone marrow transplantation is discussed.

Study on immunosuppressive activity of alpha-globulin fraction of human blood plasma.

Immunosuppressive activity of alpha-globulin fraction of human blood plasma (Cohn fraction IV) was studied to determine the possible clinical utilization of this fraction as such, purified, or as the starting material for isolation of the active factor. The ethanol fractions IV, isolated and waste in the production of blood derivatives were found to be directly inapplicable because of relatively low activity and poor solubility. Therefore purification experiments were done to remove the denatured proteins and lipids and to increase the specific activity, they were performed on laboratory scale using DEAE ionex chromatography, and on semipilot scale utilizing the principles of ethanol fractionation of plasma. From the viewpoint of technology, the latter procedure seems more feasible; partial purification yielded good solubility and somewhat higher specific activity. Under study are further techniques capable of eliminating the contaminating proteins with do not carry the immunosuppressively active component, as well as the effectivity of pasteurization at 60 degrees C/10 hrs., which seems indispensable with respect to inactivation of infectious hepatitis viruses and the potential clinical application. Experimental isolations of the active component have led immunosuppressively active low-molecular part, which, however, is chemically nonhomogeneous. Its subsequent subfractionation will be necessary.

Human lymphocyte culture in semi-solid medium.

We pointed out some sources of variations in the results obtained by the method of human peripheral lymphocyte culture in semi-solid medium. The influence of changes in five important parameters of the two-layer method was studied: agar concentration in the upper layer, cell concentration, mitogen dilution, type of culture medium and serum. We found that the optimal conditions for lymphocyte culture and for the evaluation of the results were 0.3% agar concentration in the upper layer, the concentration of 5 x 10(5) cells/ml, and a double dilution of phytohaemagglutinin. Of the culture media tested, MEM with 0.2% Bactotryptose supplemented with 20% inactivated mixed human serum proved most appropriate. Cells in lymphocyte colonies were characterized morphologically, cytochemically and by means of basic membrane features. The possibilities of using the method in clinical work are shown and the necessity of standardizing the culture technique is emphasized.