Secondary Digestion Research Articles

A novel approach to pathogen reduction in biosolids: the enzymic hydrolyser

Revision of the Sludge (Use in Agriculture) Regulations in the UK has resulted in the requirement of a final product standard in terms of E. coli per gram of dry solids. Conventional mesophilic digestion including 14-day secondary storage should normally provide adequate treatment to meet the Treated Sludge Standard. Any process capable of greater pathogen reduction would ensure more process security and compliance comfort. Such a process would be a welcome alternative to extra secondary storage where an existing works does not have sufficient capacity, particularly if the differences in costs between the options are small. Enzymic hydrolysis was found to be up to ten-fold more effective in E. coli reduction than conventional secondary digestion. A two-stage digestion process based on this technique has been developed by United Utilities and Montgomery Watson Harza (termed the enzymic hydrolyser, patent pending). Studies showed that the mean numbers of E. coli were significantly lower in the enzymic hydrolyser systems (P > 0.05; t = 13.19) compared to conventional digesters. Increased stability was a secondary benefit of the system (foam was eliminated or greatly reduced in the enzymic hydrolyser units). Another benefit of the system for retrofit to existing assets is the decreased tankage volumes required compared to secondary digestion to achieve more than twice the log kill of pathogens.

Steam pressure disruption of municipal solid waste enhances anaerobic digestion kinetics and biogas yield.

Biomass waste, including municipal solid waste (MSW), contains lignocellulosic-containing fiber components that are not readily available as substrates for anaerobic digestion due to the physical shielding of cellulose imparted by the nondigestible lignin. Consequently, a substantial portion of the potentially available carbon is not converted to methane and the incompletely digested residues from anaerobic digestion generally require additional processing prior to their return to the environment. We investigated and developed steam pressure disruption as a treatment step to render lignocellulosic-rich biomass more digestible and as a means for increasing methane energy recovery. The rapid depressurization after steam heating (240 degrees C, 5 min.) of the nondigested residues following a 30-day primary digestion of MSW caused a visible disruption of fibers and release of soluble organic components. The disrupted material, after reinoculation, provided a rapid burst in methane production at rates double those observed in the initial digestion. This secondary digestion proceeded without a lag phase in gas production, provided approximately 40% additional methane yields, and was accompanied by a approximately 40% increase in volatile solids reduction. The secondary digestate was found to be enriched in lignin and significantly depleted in cellulose and hemi-cellulose components when compared to primary digestate. Thus, steam pressure disruption treatment rendered lignocellulosic substrates readily accessible to anaerobic digestion bacteria and improved both the kinetics of biogas production and the overall methane yield from MSW. Steam pressure disruption is central to a new anaerobic digestion process approach including sequential digestion stages and integrated energy recovery, to improve process yields, provide cogenerated energy for process needs, and to provide effective reuse and recycling of waste biomass materials.

Two-Dimensional Gene Expression Fingerprinting

In the present study we have developed the two-dimensional Gene Expression Fingerprinting (2-D GEF) procedure suitable for gene expression analysis and new gene discovery. The procedure is based on the two-dimensional gel display of 3′-terminal cDNA restriction fragments produced by one primary (first dimension) and several sequential secondary restriction digestions. Many thousands of individual sequences per cDNA sample can be visualized using this approach, which is also characterized by a high reproducibility, predictable spatial location of cDNA fragments on 2-D gels, and the potential for identifying cDNA fragments solely on the basis of their two-dimensional coordinates. Using this 2-D GEF method, we analyzed and compared the gene expression patterns of two related primitive hematopoietic cell lines, Kg-1 and Kg-1a. A total of 25 candidate differentially expressed sequences were identified, and for 75% of them the presumed expression pattern was confirmed by Northern blotting or reverse transcription-polymerase chain reaction. We also demonstrated that for 70% of bands, correct prediction of their identity could be made on the basis of two-dimensional coordinates, whereas the major part of incorrect predictions was caused by insufficient database quality.

Phosphorylation status of the parvovirus minute virus of mice particle: mapping and biological relevance of the major phosphorylation sites.

The core of the VP-1 and VP-2 proteins forming the T=1 icosahedral capsid of the prototype strain of the parvovirus minute virus of mice (MVMp) share amino acids sequence and a common three-dimensional structure; however, the roles of these polypeptides in the virus infection cycle differ. To gain insights into this paradox, the nature, distribution, and biological significance of MVMp particle phosphorylation was investigated. The VP-1 and VP-2 proteins isolated from purified empty capsids and from virions containing DNA harbored phosphoserine and phosphothreonine amino acids, which in two-dimensional tryptic analysis resulted in complex patterns reproducibly composed by more than 15 unevenly phosphorylated peptides. Whereas secondary protease digestions and comigration of most weak peptides in the fingerprints revealed common phosphorylation sites in the VP-1 and VP-2 subunits assembled in capsids, the major tryptic phosphopeptides were remarkably characteristic of either polypeptide. The VP-2-specific peptide named B, containing the bulk of the (32)P label of the MVMp particle in the form of phosphoserine, was mapped to the structurally unordered N-terminal domain of this polypeptide. Mutations in any or all four serine residues present in peptide B showed that the VP-2 N-terminal domain is phosphorylated at multiple sites, even though none of them was essential for capsid assembly or virus formation. Chromatographic analysis of purified wild-type (wt) and mutant peptide B digested with a panel of specific proteases allowed us to identify the VP-2 residues Ser-2, Ser-6, and Ser-10 as the main phosphate acceptors for MVMp capsid during the natural viral infection. Phosphorylation at VP-2 N-terminal serines was not necessary for the externalization of this domain outside of the capsid shell in particles containing DNA. However, the plaque-forming capacity and plaque size of VP-2 N-terminal phosphorylation mutants were severely reduced, with the evolutionarily conserved Ser-2 determining most of the phenotypic effect. In addition, the phosphorylated amino acids were not required for infection initiation or for nuclear translocation of the expressed structural proteins, and thus a role at a late stage of MVMp life cycle is proposed. This study illustrates the complexity of posttranslational modification of icosahedral viral capsids and underscores phosphorylation as a versatile mechanism to modulate the biological functions of their protein subunits.

Development of Disulfide Peptide Mapping and Determination of Disulfide Structure of Recombinant Human Osteoprotegerin Chimera Produced in Escherichia coli

Recombinant human osteoprotegerin chimera is a 90-kDa protein containing a human IgG Fc domain fused to human osteoprotegerin. The molecule is a dimer linked by two intermolecular disulfide bonds and contains eleven intramolecular disulfide bonds per monomer. A cysteine-rich region in osteoprotegerin contains nine disulfide bridges homologous to the cysteine-rich signature structure of the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In this report, we have developed peptide mapping procedures suitable to generate disulfide-containing peptides for disulfide structure assignment of the fusion molecule. The methods employed included proteolytic digestion using endoproteinases Glu-C and Lys-C in combination followed by LC–MS analyses. Disulfide linkages of peptide fragments containing a single disulfide bond were assigned by sequence analysis via detection of (phenylthiohydantoinyl) cystine and/or by MS analysis. Disulfide bonds of a large, core fragment containing three peptide sequences linked by four disulfides were assigned after generation of smaller disulfide-linked peptides by a secondary thermolysin digestion. Disulfide structures of peptide fragments containing two disulfide bonds were assigned using matrix-assisted laser desorption ionization mass spectrometry with postsource decay. Both the inter- and intramolecular disulfide linkages of the chimeric dimer were confirmed.

Dual anaerobic co-digestion of sewage sludge and confectionery waste

Three configurations for a dual digestion system were examined. The units were based on three 5 l completely stirred tank reactors (CSTR). A first-stage thermophilic digester was used to provide the feed to each of the two second-stage mesophilic (35°C) digesters. Using a mixture of sewage sludge and strong confectionery waste, the thermophilic digester was operated at 55°C with a hydraulic retention time of 4 h. The mesophilic digesters were operated at hydraulic retention times of 8, 12 and 15 days. In terms of the reduction of volatile solids (VS), the three dual digestion configurations were similar but were more effective than the single-stage reactor which was used as a control. However, based on the specific methane yield (m 3 CH 4/kg VS removed), the configuration with a first stage operating at 55°C and a secondary digester at 35°C with a hydraulic retention time of 12 days was the most effective. This configuration also maintained a more stable pH, irrespective of the quality of the feed sludge.

A thermophilic/mesophilic dual digestion system for treating waste activated sludge

Five configurations for a dual digestion were examined. The units were based on three 5 dm3 completely stirred tank reactors (CSTR). A first-stage thermophilic digester was used to provided the feed to each of the two second-stage mesophilic (35 °C) digesters. The thermophilic digester was operated initially at 55 °C with a hydraulic retention time of 4 h and then, later, at 60 °C with a hydraulic retention time of 3 h. The mesophilic digesters were operated at hydraulic retention times of 9, 12 and 15 days. In terms of volatile solids’ (VS) reduction, the systems with a first stage at 60 °C gave the better performance. However, based on the specific methane yield (m3 CH4 per kg VS removed), the configuration with a first stage operating at 55 °C and a secondary digester with a hydraulic retention time of 12 days was the most effective. © 1999 Society of Chemical Industry

A thermophilic/mesophilic dual digestion system for treating waste activated sludge

Five configurations for a dual digestion were examined. The units were based on three 5 dm3 completely stirred tank reactors (CSTR). A first-stage thermophilic digester was used to provided the feed to each of the two second-stage mesophilic (35 °C) digesters. The thermophilic digester was operated initially at 55 °C with a hydraulic retention time of 4 h and then, later, at 60 °C with a hydraulic retention time of 3 h. The mesophilic digesters were operated at hydraulic retention times of 9, 12 and 15 days. In terms of volatile solids’ (VS) reduction, the systems with a first stage at 60 °C gave the better performance. However, based on the specific methane yield (m3 CH4 per kg VS removed), the configuration with a first stage operating at 55 °C and a secondary digester with a hydraulic retention time of 12 days was the most effective. © 1999 Society of Chemical Industry

Preventing Growth of Pathogens in Pasteurized Digester Solids

The goal of this research was to determine conditions that could promote or inhibit pathogen growth during storage or application of pasteurized digester solids. Growth of enterobacteriaceae and Salmonella after pasteurization (70 °C for 30 minutes) of mesophilic anaerobic digester solids was reported for numerous facilities in Switzerland but the cause was not known. However, our work found no growth of indigenous Salmonella or fecal coliform in well‐mixed pasteurized samples in bench‐scale conditions. Salmonella typhimurium was then added in batch tests to determine growth or die‐off at 20 and 35 °C incubation temperatures. In addition, pasteurized digester solids were fed for 3 months to a laboratory 6‐day solids retention time (SRT) secondary mesophilic digester to determine if pathogen growth could occur. Fecal coliform die‐off occurred in inoculated pasteurized solids under all conditions, but Salmonella growth readily occurred within 5 days and died off within 30 days of incubation. The Salmonella grew better at 20 than at 35 °C, and substrate addition did not improve growth. When the pasteurized solids were mixed and oxygenated, Salmonella die‐off instead of growth occurred. Experiments at 35 °C with both batch bottles and the secondary digester showed that the presence of other active anaerobic bacteria apparently caused Salmonella die‐off instead of growth. Pathogen growth can be prevented and Class A biosolids can be ensured by storing pasteurized digester solids in a secondary mesophilic digester with a 6‐day SRT.

The Treatment of Simulated Sewage by Alternating Anaerobic Double Filtration (AADF)

The concept of treating low strength municipal wastewater and the resultant sludge in a single unit was tested using a synthetic feed and two anaerobic filters. These two filters were started-up separately using a stepped incremental loading until an organic loading rate (OLR) of 4 kg COD m−3 d−1 was achieved. The integrated double filter unit (i.e. operated in series) was then operated at this OLR until steady state had been achieved. The process was then operated at OLRs of 3, 4, 6 and 8 kg COD m−3 d−1 and at each rate the sequence of primary and secondary filters was alternated. An examination of the specific methane yields showed that the secondary digesters produced more gas than the theoretical value based on COD. An examination of the biomass in the filters showed the secondary digester contained less biomass than the primary digester. Taken together these results suggested that sludge digestion was taking place in the secondary digester and that the concept of AADF merited further evaluation.

Folding of pyrimidine-enriched RNA fragments from the vicinity of the internal ribosomal entry site of hepatitis A virus.

Two RNA fragments from the region just upstream of the internal ribosome entry site of Hepatitis A virus (HAV) were studied, a 35mer (HAV-35), 5'U4C3U3C3U4C3U3C2UAU2C3U33(4), and a 23mer (HAV-23), 5(4)U4C3U3C3U4C3U33(4). Secondary structural predictions and nuclease digestion patterns obtained with genomic RNAs suggested that they link two stable Watson-Crick (WC) hairpins in the genomic RNA and do not form conventional WC secondary structure, but do fold to form a condensed, stacked 'domain'. To obtain more information, folding of HAV-23 and -35 RNA fragments was characterized using 1H nuclear magnetic resonance, in H2O as a function of pH and temperature, circular dichroism as a function of NaCl concentration, pH and temperature, and square-wave voltammetry as a function of pH. The results indicate that these oligo-nucleotides form intramolecular structures that contain transient U*U base pairs at pH 7 and moderate ionic strength (100 mM NaCl). This folded structure becomes destabilized and loses the U*U base pairs above and below neutral pH, especially at ionic strengths above 0.1. All of the cytidine protons exchange relatively rapidly with solvent protons (exchange lifetimes shorter than 1 ms), so the structure contains few if any C*CH+base pairs at neutral pH, but can apparently form them at pH values below 6. We present a series of possible models in which chain folding draws the strand termini closer together, possibly serving to pull the attached WC hairpin domains together and providing a functional advantage by nucleating reversible formation of a more viable RNA substrate.

Development of a novel, two-step process for treating municipal biosolids for beneficial reuse

Modern municipal sewage waste treatment plants use conventional mechanical and biological processes to reclaim wastewaters. This process has an overall effect of converting a water pollution problem into a solid waste disposal problem (sludges or biosolids). An estimated 10 million tons of biosolids, which require final disposal, are produced annually in the United States. Although numerous disposal options for biosolids are available, including land application, landfilling, and incineration, disposal costs have risen, partly because of increased federal and local environmental restrictions. A novel, thermomechanical biosolids pre-treatment process, which allows for a variety of potential value-added uses, was developed. This two-step process first employs thermal explosive decompression to inactivate or kill the microbial cells and viruses. This primary step also results in the rupture of a small amount of the microbial biomass and increases the intrinsic fluidity of the biosolids. The second step uses shear to effect a near-complete rupturing of the microbial biomass, and shears the nondigested organics, which increases the overall surface area. Pretreated biosolids may be subjected to a secondary anaerobic digestion process to produce additional fuel gas, and to provide for a high-quality, easily dewatered compost product. This novel biosolids pretreatment process was recently allowed a United States patent.

The effect of heavy metals on a thermophilic methanogenic upflow sludge blanket reactor

Copper, nickel, zinc and lead were added to the feed of the methanogenic stage of a two-phase thermophilic anaerobic digester, which had an overall hydraulic retention time of 10·5 h. Each metal was added for a period of 30 h. A period of 7 days was used between the metal dosing as a recovery phase. All the metals caused a reduction in the chemical oxygen demand removal which was reversed when the metal dosing ceased. Nickel and lead had the greatest impact. Lead also had the greatest effect on biogas production and, on the basis of the volatile fatty acid production, appeared to act immediately on acidogenesis and more slowly on the methanogenic bacteria. Metal speciation, using a sequential extraction technique, showed that sludges in reactors being directly dosed with metals fixed the ions at sites with relatively weak binding, whereas sludges in secondary digesters used sites which bound the metals strongly. Lead was an exception to this, always being bound in the relatively weak, adsorbed state.

Pilot and model studies of toxic organic compounds in municipal sludge digestion

The removal of selected toxic compounds during anaerobic and aerobic digestion of municipal sludge was investigated at pilot scale. The candidate compounds included a mixture of cresols, chlorophenols, heterocyclic nitrogenous compounds, phthalates, and polyaromatic hydrocarbons. A wide range of responses of the candidate compounds was observed in anaerobic digestion with seven compounds removed at efficiencies greater than 90% in primary digestion, four compounds demonstrating removals of greater than 80% after secondary digestion, and five compounds demonstrating overall removals of less than 70%. The secondary digester demonstrated a substantial capacity to attenuate toxics. In aerobic digestion, all of the compounds except the cresols, 2, 4, 5-trichlorophenol, and bis-2-ethylhexylphthalate were essentially eliminated. A dynamic model, developed to predict the fate of toxics during primary sludge digestion, was calibrated with data from the experiments. Biodegradation rate coefficients only could be estimated because of statistical redundancy of the sorption partitioning parameter during the nonlinear regression. The sorption partition coefficients were estimated using octanol–water partitioning coefficients from literature. Key words: anaerobic, aerobic, municipal sludge digestion, toxic organic compounds, biodegradation rate coefficients, model, dynamic.

Fate of polynuclear aromatic hydrocarbons during anaerobic digestion of municipal wastewater sludges

The fate of six polynuclear aromatic hydrocarbons during anaerobic digestion of municipal sludges was investigated in a two-stage pilot-scale digester system. Primary digester removal efficiencies ranged from 23% for acenaphthene to 62% for acenaphthylene ; secondary digester removal efficiencies ranged from 22% for acenaphthylene to 42% for phenanthrene. Most of the remaining mass of candidate compounds was present in the secondary digester waste sludge stream. Less than 2.5% of the candidate compounds entering the digestion system was present in the secondary digester supernatant. A dynamic model of the primary digester incorporating mixed first-order biodegradation kinetics was calibrated for each of the candidate compounds. The biodegradation rate coefficients that were estimated for the primary digester ranged from 0.004 L/g.d for acenaphthene to 0.16 L/g.d for acenaphthylene. The biodegradation rate coefficient for acenaphthene did not significantly differ from zero.

Fibrin degradation product (FnDP) assays: analysis of standardization issues and target antigens in plasma

The in vivo formation of fibrin and its subsequent secondary fibrinolytic digestion yields a variety of crosslinked fibrin degradation products (XL/FnDP). One of these is known as D-dimer and a variety of ELISA-type commercial kits using monoclonal antibodies to D-dimer have been developed. We have investigated the possibility of establishing a standard such that these various kits might indicate the same levels of D-dimer in the same samples. We have concluded that because it seems that each individual monoclonal antibody to D-dimer targets a unique and distinct epitope in the FnDP fraction the notion of introducing a standard D-dimer which will respond uniformly to each D-dimer monoclonal antibody is not feasible. Using various monoclonal and polyclonal antibodies in conjunction with gel exclusion chromatography we have examined human plasma derived from patients with disseminated intravascular coagulation (DIC) which contained high levels of fibrin degradation products (FnDP). The data suggested that the FnDP fraction in plasma contained mostly high molecular weight (> 2 x 10(6) daltons) crosslinked fragments which contain high levels of fibrinopeptide A. Many of these crosslinked fragments crossreact with antibodies to D-dimer because they each contain D-dimer as a structural component. On the basis of this data, a novel sequence of events is proposed which may occur during the aggregation of fibrin in vivo.

Experiences of Digested Sludge Thickening by Aercon

ABSTRACTIncreasing restrictions on sewage sludge disposal routes and the tightening of treatment requirements have led to an escalation of sludge‐disposal costs which are out of proportion to other sewage‐treatment costs: consequently reducing sludge volume is becoming increasingly cost‐effective. Consolidation of anaerobically digested sludge has traditionally been difficult and takes many weeks in secondary digesters or deep storage tanks. Pre‐aeration of the sludge using the Aercon process can enable consolidation to take place in days instead of weeks.This paper reports the results of a pilot‐scale plant designed to investigate further the mechanism by which aeration speeds up consolidation. It was concluded that the rate of consolidation depended on aeration intensity and cooling of the sludge. Inhibition of anaerobiosis was more important than gas stripping in enhancing consolidation.

Identification of phosphorylation sites unique to the B form of human progesterone receptor. In vitro phosphorylation by casein kinase II.

The human progesterone receptor (PR), a member of the steroid/thyroid receptor superfamily of ligand-activated transcription factors, is expressed in most tissues as two forms that exhibit differential transcriptional activation potentials, full-length PR-B and NH2-terminally truncated PR-A. In human breast cancer cells (T47D) both forms of PR are constitutively phosphorylated but phosphorylation is increased in response to hormone treatment, suggesting that this modification has a role in regulating the activation state of the receptor. To more directly define the functional role of phosphorylation in the action of A and B receptors requires knowledge of the phosphorylated amino acid residues and the protein kinase(s) involved. Toward this end we have developed a strategy that combines isolation of PR phosphotryptic peptides by reverse phase high performance liquid chromatography, secondary analytical protease digestion, manual Edman degradation, and release of 32P that resulted in identification of two major phosphorylation sites, Ser81 and Ser162. Both sites are located in the amino-terminal region unique to PR-B, and one of these sites (Ser81) is encompassed in a casein kinase II (CKII) consensus sequence. Although human PR contains 11 potential CKII consensus sequences, CKII in vitro phosphorylated purified PR-B only at Ser81 suggesting that this may be an authentic site for CKII in vivo.

Estimation of anaerobic biodegradation rates for toxic organic compounds in municipal sludge digestion

The removal of selected toxic compounds during anaerobic digestion of municipal sludge was investigated at pilot scale. In primary digestion, isoquinoline, p-cresol, 4-chlorophenol, 2,4,5-chlorophenol, diethyl phthalate, di- n-butyl phthalate and butylbenzyl phthalate were removed at efficiences greater than 90%. Bis-2-ethylhexyl phthalate, o-cresol and 2,5-dichlorophenol were removed at efficiencies of between 55 and 65%. Greater than 60% of the toxic compounds entering secondary digestion were degraded. The anaerobic biomass appeared to acclimate to biodegrade the chlorophenolic compounds during the experimental period. A dynamic model, developed to predict the fate of toxics during primary sludge digestion, was calibrated with data from the experiment. Biodegradation rate coefficients only could be estimated due to statistical redundancy of the sorption partitioning parameter during the non-linear regression. The sorption partition coefficients were estimated from literature octanol-water partitioning coefficients.

Decomposition of different organic materials in soils

Laboratory experiments were conducted to evaluate organic C mineralization of various organic materials added to soils. A soil sample was mixed with organic material to approximate a field application of 9 g organic C kg-1 soil (0.9% or 50 Mg ha-1). The organic materials used were four crop residues [corn (Zea mays L.), soybean (Glycine max L. Merr.), sorghum (Sorghum vulgare Pers.), and alfalfa (Medicago sativa L.)], four animal manures [chicken (Gallus domesticus), pig (Sus scrofa), horse (Equus caballus), and cow (Bos taurus)] and four sewage sludges [Correctionville (Imhoff tank), Charles City (holding tank), Davenport (secondary digester), and Keokuk (primary digester)]. The soil-organic material mixture was incubated under aerobic conditions at room temperature (20±2°C) for 30 days. The CO2 evolved was collected in standard KOH solution by continuously passing CO2-free air over the soil. Results showed that, in general, the amounts of CO2-C released mereased rapidly initially, but the pattern differed among the organic materials used. More than 50% of the total CO2 produced in 30 days of incubation was evolved in the first 6 days. Expressed as percentages of organic C added, the amounts of CO2 evolved ranged from 27% with corn to 58% with alfalfa. The corresponding percentages for animal manures ranged from 21 to 62% with horse and pig manures, respectively, and for sewage sludges they ranged from 10 to 39% for Charles City and Keokuk sludges. All CO2 evolution data conformed well to a first-order kinetic model. Potentially, readily mineralizable organic C values and first-order rate constants (k) of the organic matter-treated soils ranged from 1.422 g C kg-1 soil with ak value of 0.0784 day-1 to 6.253 g C kg-1 soil with ak value of 0.0300 day-1. The half-lives of the C remaining in soils ranged from 39 to 54 days for plant materials. The corresponding half-lives for the C remaining from animal manures and sewage sludges ranged from 37 to 169 days and from 39 to 330 days, respectively.