Mesophilic Phase Research Articles

Diversity and abundance of ammonia oxidizing archaea in tropical compost systems

Composting is widely used to transform waste materials into valuable agricultural products. In the tropics, large quantities of agricultural wastes could be potentially useful in agriculture after composting. However, while microbiological processes of composts in general are well established, relatively little is known about microbial communities that may be unique to these in tropical systems, particularly nitrifiers. The recent discovery of ammonia oxidizing archaea (AOA) has changed the paradigm of nitrification being initiated solely by ammonia oxidizing bacteria. In the present study, AOA abundance and diversity was examined in composts produced from combinations of plant waste materials common in tropical agriculture (rice straw, sugar cane bagasse, and coffee hulls), which were mixed with either cow- or sheep-manure. The objective was to determine how AOA abundance and diversity varied as a function of compost system and time, the latter being a contrast between the start of the compost process (mesophilic phase) and the finished product (mature phase). The results showed that AOA were relatively abundant in composts of tropical agricultural wastes, and significantly more so than were the ammonia-oxidizing bacteria. Furthermore, while the AOA communities in the composts were predominatly group I.1b, the communities were diverse and exhibited structures that diverged between compost types and phases. These patterns could be taken as indicators of the ecophysiological diversity in the soil AOA (group I.1b), in that significantly different AOA communties developed when exposed to varying physico-chemical environments. Nitrification patterns and levels differed in the composts which, for the mature material, could have significant effects on its performance as a plant growth medium. Thus, it will also be important to determine the association of AOA (and diversity in their communities) with nitrification in these systems.

Archaeal community composition affects the function of anaerobic co-digesters in response to organic overload

Microbial community diversity in two thermophilic laboratory-scale and three full-scale anaerobic co-digesters was analysed by genetic profiling based on PCR-amplified partial 16S rRNA genes. In parallel operated laboratory reactors a stepwise increase of the organic loading rate (OLR) resulted in a decrease of methane production and an accumulation of volatile fatty acids (VFAs). However, almost threefold different OLRs were necessary to inhibit the gas production in the reactors. During stable reactor performance, no significant differences in the bacterial community structures were detected, except for in the archaeal communities. Sequencing of archaeal PCR products revealed a dominance of the acetoclastic methanogen Methanosarcina thermophila, while hydrogenotrophic methanogens were of minor importance and differed additionally in their abundance between reactors. As a consequence of the perturbation, changes in bacterial and archaeal populations were observed. After organic overload, hydrogenotrophic methanogens (Methanospirillum hungatei and Methanoculleus receptaculi) became more dominant, especially in the reactor attributed by a higher OLR capacity. In addition, aggregates composed of mineral and organic layers formed during organic overload and indicated tight spatial relationships between minerals and microbial processes that may support de-acidification processes in over-acidified sludge.Comparative analyses of mesophilic stationary phase full-scale reactors additionally indicated a correlation between the diversity of methanogens and the VFA concentration combined with the methane yield. This study demonstrates that the coexistence of two types of methanogens, i.e. hydrogenotrophic and acetoclastic methanogens is necessary to respond successfully to perturbation and leads to stable process performance.

Biodegradation of Bloodmeal-Based Thermoplastics in Green-Waste Composting

Polymers that are compostable and manufactured from renewable resources have gained significant importance in recent years. The objective of this work was to assess the biodegradability of bloodmeal-based thermoplastics in a commercial green-waste composting situation. Materials plasticised with tri-ethylene–glycol lost about 45% of their original mass after 12 weeks composting while unplasticised samples lost 35%. Degradation appeared to have been in two phases; an initial loss of soluble, low molecular compounds in the mesophilic phase followed by degradation of high molecular compounds as the temperature exceeded about 40 °C in the thermophilic phase. It was found that as degradation proceeded materials became more soluble. In addition, plasticised and unplasticized samples contained about 60 wt% moisture after 4 weeks of composting conditioning at 50% relative humidity resulted in approximately 8–10 wt% moisture, unaffected by the extent of degradation. FTIR revealed that proteins underwent hydrolytic cleavage resulting in the formation of primary amines. A significant reduction in combustion temperature was observed, indicative of a reduction in covalent bonding, likely due to shorter chains lengths or less cross-linking.

Changes in physicochemical and microbial community during co-composting of oil palm frond with palm oil mill effluent anaerobic sludge

The aims of this study were to investigate the physicochemical changes and microbial population during co-composting of 1 ton oil palm frond (OPF) with 1,000 L palm oil mill effluent (POME) anaerobic sludge. In the first 30 days of composting, the temperature of the composting piles was observed in the thermophilic phase, within a range of 50 - 56oC. Meanwhile, the oxygen level, moisture content, and pH profiles of the compost were maintained at 2.0 to 12%, 60 to 70%, and 7.9 to 8.5, respectively, throughout the composting process. The total bacteria count was estimated to be about 55 x 1010 CFU/mL in the mesophilic phase, and then it increased up to 66 x 1010 CFU/mL in the thermophilic phase, and finally decreased to 9.0x1010 CFU/mL in the curing phase. The initial C/N ratio, 64, decreased to 18 after 60 days of composting process, indicating the maturity of compost product from OPF-POME anaerobic sludge. The diversity of the bacterial community was investigated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. The results suggested that the co-composting process of OPF with POME anaerobic sludge was dominated by Pseudomonas sp.

Dynamics of bacterial communities during solid-state fermentation using agro-industrial wastes to produce poly-γ-glutamic acid, revealed by real-time PCR and denaturing gradient gel electrophoresis (DGGE)

The dynamics of bacterial communities play an important role in solid-state fermentation (SSF). Poly-γ-glutamic acid (γ-PGA) was produced by Bacillus amyloliquefaciens C1 in SSF using dairy manure compost and monosodium glutamate production residuals as basic substrates. The production of γ-PGA reached a maximum of 0.6% after 20 days fermentation. Real-time polymerase chain reaction showed the amount of total bacteria reached 3.95 × 10(9) 16S rDNA copies/g sample after 30 days, which was in good accordance with the 4.80 × 10(9) CFU/g obtained by plate counting. Denaturing gradient gel electrophoresis profile showed a reduction of microbial diversity during fermentation, while the inoculum, B. amyloliquefaciens C1, was detected as the dominant organism through the whole process. In the mesophilic phase of SSF, Proteobacteria was the dominant microbial, which was replaced by Firmicutes and Actinobacteria in the thermophilic phase. The molecular analysis of the bacterial diversity has significant potential for instructing the maturing process of SSF to produce γ-PGA at a large-scale level, which could be a benefit in the production of high quality and stable SSF products.

Biodegradation of Phenolic Compounds in Creosote Treated Wood Waste by a Composting Microbial Culture Augmented with the Fungus <i>Thermoascus aurantiacus</i>

Problem statement: Creosote is used as a wood preservative and water proof agent in railway sleepers, utility poles, buildings foundations and fences and garden furniture. It is a mixture of over 300 hydrocarbons which include 75% polycyclic aromatic hydrocarbons, 2-17% phenolic compounds and 10-18% heterocyclic organic compounds. Exposure to creosote may result in several health problems including damage to kidney, liver, eyes and skin. Potential contamination of soil and water exist from creosote treated wood from construction and demolition sites. Approach: The possibility of using an invessel composting process augmented with the ascomycetous fungus Thermoascus aurantiacus as a mesophilic/thermophilic bioremediation option for the degradation of phenolic compounds in creosote treated wood waste was evaluated. Results: The temperatures of bioremediation process reached thermophilic phase and the mesophilic and thermophilic lag phases were clearly identified. The moisture content decreased significantly indicating that the water produced by microbial respiration did not compensate for the water lost as vapor with the exhaust gases. Initial increases in pH due to the breakdown of organic nitrogen to ammonium and final drop in pH due to the formation of organic acids and the loss of ammonium with the exhaust gases in the latter stage were observed. Different degradation rates were observed in the mesophilic and thermophilic stages of composting. The control experiment achieved higher reductions of volatile solids, total carbon and TKN and higher degradation of phenolic compounds, cellulose and lignin, indicating a higher level of activity of microorganisms during the composting process compared with the inoculated experimental trial. The stability and maturity of the product of the control experiment were also better than those of the product from the inoculated experimental trial. Conclusion: The inoculation of the cellulolytic-thermophilic fungus Thermoascus aurantiacus did not accelerate the bioremediation process in degrading phenolic compounds and the fungus may have inhibited the growth and metabolic activities of composting organisms.

TEMPERATURE AND pH MONITORING IN WINDROWS FROM ANIMAL FACILITY WASTE COMPOSTING

The objective of this study was to monitor temperature and pH in composting windrows constituted of rabbit, guinea pig, mouse and hamster wastes. Wastes from these laboratory animals were mixed to cotton wastes to produce 721.65 kg of composts. There were constructed 6 composting windrows and they were monitored daily for temperature and pH. Composts were watered daily until humidity level was near 55%. The compost piles were turned every 15 days. All the windrows passed through a thermophilic phase followed by a mesophilic phase, and maximum temperature was near 60°C. The pH values ranged from 6 to 8 in most of the windrows. In conclusion, characteristics of the compost studied such as humidity, pH, color and absence of foul odors makes it suitable for agricultural use. Keywords: Laboratory animals, environmental pollution, waste management.

Putting microbes to work in sequence: Recent advances in temperature-phased anaerobic digestion processes

Methane biogas production through anaerobic digestion (or biomethanation) is one of the few technologies that both produce bioenergy and protect the environment. When the focus of anaerobic digestion (AD) is shifted from primarily wastewater treatment to bioenergy production, efficiency and process stability become critical to the economic viability of AD technologies. Temperature-phased anaerobic digestion (TPAD) is a promising process that can significantly enhance both digestion efficiency and process robustness. A TPAD system separates the conventional AD process into two phases, so both phases can be optimized according to their individual functional needs. In the first, thermophilic phase, the often rate-limiting hydrolysis step of polymeric feedstock is accelerated by elevated temperatures, while in the second, mesophilic phase, the fastidious syntrophic acetogens and methanogens are provided with permissive conditions where inhibitions to key guilds (e.g., syntrophic acetogens and methanogens) are attenuated. Although large-scale TPAD systems have not been applied widely, researchers have demonstrated the potential superiority of TPAD systems over single-stage digesters and other AD processes with enhanced VS (volatile solids) and pathogen removal; increased methane yield, process stability, OLR (organic loading rate); shorter HRT (hydraulic retention time); decreased foaming and short-chain fatty acids in effluent.

Bioconversion of rice straw and certain agro-industrial wastes to amendments for organic farming systems: 1. Composting, quality, stability and maturity indices

The microbiological and physicochemical parameters were monitored for 12weeks during composting of five piles (1.5×1.0×0.80m3) containing mainly rice straw, soybean residue and enriched with rock phosphate. Two treatments were inoculated with buffalo’s manure or composite inoculum, two were supplemented with vinasse and inoculated with either the composite inoculum or with both, the last one was served as a control. Four typical phases of composting were observed during the bioprocess: short initial mesophilic phase followed by, thermophilic, cooling and maturation phases. Physicochemical changes confirmed the succession of microbial populations depending on the temperature of each phase in all treatments. Intense microbial activities led to organic matter mineralization and simultaneously narrow C/N ratios. Inoculation of composting mixtures enhanced the biodegradation of recalcitrant substances. The duration of exposure to a temperature above 55°C for at least 16 consecutive days was quite enough to sanitize the produced composts. After 84days, all composts reached maturity as indicated by various parameters.

Microbial community structure changes during oyster mushroom substrate preparation

Although oyster mushroom (Pleurotus spp.) is a valuable food, cultivated worldwide on an industrial scale, still very little is known about the microbial dynamics during oyster mushroom substrate preparation. Therefore, the characterization of the microbial dynamics by chemical and biological tools was the objective of this study. During substrate preparation, enzymatic digestibility of the substrate improved by 77%, whereas the cellulose and hemicellulose to lignin ratios decreased by 9% and 19%, respectively. Fluorescein diacetate hydrolysis reached its minimum value at the temperature maximum of the process during the composting phase and exceeded the initial level at the end of the process. Fungal species played part in the initial mesophilic phase of the substrate preparation process, but they disappeared after pasteurization in tunnels at constant elevated temperatures. Changes in the microbiota showed a marked bacterial community succession during substrate preparation investigated by 16S ribosomal deoxyribonucleic acid-based terminal restriction fragment length polymorphism (T-RFLP). Mature samples represented the least variance, which indicated the effect of the standardized preparation protocol. The relation between mushroom yield and the bacterial community T-RFLP fingerprints was investigated, but the uniformity of mushroom yields did not support any significant correlation.

Two-phase (acidogenic–methanogenic) anaerobic thermophilic/mesophilic digestion system for producing Class A biosolids from municipal sludge

Two different arrangements of two-phase anaerobic sludge systems were operated treating a mixture of primary and secondary sludge. Two steady state periods were evaluated: the first acidogenic thermophilic phase was operated at hydraulic retention times of 3 and 2 days and the second methanogenic (mesophilic and thermophilic) phases at 13 and 10 days. The two-phase systems had an efficient removal of pathogens and parasites, achieving values lower than those specified for Class A biosolids, according to the Mexican Standard NOM-004-SEMARNAT-2002. The first thermophilic phase achieved almost complete destruction of pathogens and parasites by itself. During the second steady state period, volatile fatty acids accumulated in the second methanogenic phases (HRT of 10 days and an organic load of 3 kgVS/m 3d) indicating that the systems were overloaded, mainly the mesophilic digester. In this case, the accumulation of propionic acid may be related to a deficiency of micronutrients. The results show that the two-phase thermophilic/mesophilic anaerobic sludge digestion may be considered as an adequate option for the production of Class A biosolids.

Determination of fungal succession during municipal solid waste composting using a cloning-based analysis

The microbiota at industrial full-scale composting plants has earlier been fragmentarily studied with molecular methods. Here, fungal communities from different stages of a full-scale and a pilot-scale composting reactors were studied before and after wood ash amendment. The portion of fungal biomass, determined using phospholipid fatty acid analysis, varied between 6.3% and 38.5% in different composting phases. The fungal internal transcribed spacer (ITS) area was cloned and sequenced from 19 samples representing different stages of the composting processes. Altogether 2986 sequenced clones were grouped into 166 phylotypes from which 35% had a close match in the sequence databases. The fungal communities of the samples were related with the measured environmental variables in order to identify phylotypes typical of certain composting conditions. The fungal phylotypes could be grouped into those that dominated the mesophilic low pH initial phases (sequences similar to genera Candida, Pichia and Dipodascaceae) and those found mostly or exclusively in the thermophilic phase (sequences clustering to Thermomyces, Candida and Rhizomucor), but a few were also present throughout the whole process. The community composition was found to vary between suboptimally and optimally operating processes. In addition, there were differences in fungal communities between processes of industrial and pilot scale. The results of this study reveal the fungal diversity with molecular methods in industrial composting process. This is also one of the first studies conducted with samples from an industrial biowaste composting process.

Succession of Actinomycetes During Composting Proccess of Dairy-Farm Waste Investigated by Culture-Dependent and Independent Approaches

Mesophilic, thermophilic, and maturation phases were recognized in composting proccess. Temperature changes influence the microbial communities in compost within composting proccess. Actinomycetes account for a larger part of compost microbial population. The aim of this research was to study succession of actinomycetes community during composting of dairy-farm waste investigated by culture-dependent and independent approaches. In culture-independent method, the succession of actinomycetes community was analyzed by nestedpolymerase chain reaction of ribosomal intergenic spacer (nested-PCR RISA) using spesific primer F243 and primer R23S followed by a second PCR using primers F968 and R23S. In culture-dependent method actinomycetes from compost were isolated on selective media, starch-nitrate medium and humic-acid + vitamins medium. DNA of actinomycetes was extracted and amplified by repetitive sequence-based PCR (rep-PCR) using primer BOXA1R. The banding patterns were used to generate dendrograms by UPGMA clustering with NTSYS program. Microcosm containing sterile rice-straw and water which is inoculated with each actinomycetes isolates was used for examining the ability of each isolate in rice-straw degradation. The experiment results showed that succession of both bacteria and actinomycetes was occured within composting proccess of dairy-farm waste. Analysed by culture-independent method revealed that the highest community of compost’s bacteria was on mesophilic, thermophilic, and maturation phases, respectively. Whereas PCR-nested RISA resulted the highest population of actinomycetes was on thermophilic, maturation, and mesophilic phases, respectively. By culture-dependent method was obtained 29 actinomycetes isolates from mesophilic phase, 23 isolates from thermophilic phase, and 19 isolates from maturation phase. Genetic diversity analysis of the obtained isolates showed the presence of phylogenetic grouping on each phase of composting proccess. This result illustrated the occurance of succession of actinomycetes community in compost. The ability of each isolates in rice-straw degradation was different, and SnT9 isolate was found to be a promising rice-straw degrader. Keywords: succession, actinomycetes, composting, nested-PCR RISA, rep-PCR

Structural and functional studies on a mesophilic stationary phase survival protein (Sur E) from Salmonella typhimurium

SurE, the stationary-phase survival protein of Salmonella typhimurium, forms part of a stress survival operon regulated by the stationary-phase RNA polymerase alternative sigma factor. SurE is known to improve bacterial viability during stress conditions. It functions as a phosphatase specific to nucleoside monophosphates. In the present study we reported the X-ray crystal structure of SurE from Salmonella typhimurium. The protein crystallized in two forms: orthorhombic F222; and monoclinic C2. The two structures were determined to resolutions of 1.7 and 2.7 A, respectively. The protein exists as a domain-swapped dimer. The residue D230 is involved in several interactions that are probably crucial for domain swapping. A divalent metal ion is found at the active site of the enzyme, which is consistent with the divalent metal ion-dependent activity of the enzyme. Interactions of the conserved DD motif present at the N-terminus with the phosphate and the Mg(2+) present in the active site suggest that these residues play an important role in enzyme activity. The divalent metal ion specificity and the kinetic constants of SurE were determined using the generic phosphatase substrate para-nitrophenyl phosphate. The enzyme was inactive in the absence of divalent cations and was most active in the presence of Mg(2+). Thermal denaturation studies showed that S. typhimurium SurE is much less stable than its homologues and an attempt was made to understand the molecular basis of the lower thermal stability based on solvation free-energy. This is the first detailed crystal structure analysis of SurE from a mesophilic organism.

Degradation kinetics of di-(2-ethylhexyl) phthalate (DEHP) and organic matter of sewage sludge during composting

The potential degradation of di-(2-ethylhexyl) phthalate (DEHP) and organic matter of sewage sludge by composting was investigated using laboratory reactor at different operating conditions (E-1, E-2 and E-3). In all conditions, single stage thermophilic phase was observed within 2 days and almost, 60% of DEHP was degraded under this phase. At the end of composting, total DEHP degradation of more than 85% was observed in all conditions and total carbon reduction was 11.8% in E-1, 7.6% in E-2 and 10.8% in E-3. Similar trend was observed in the degradation of total nitrogen. The reduction of volatile solids (VS) in the composting reactors was 5.4% in E-1 (18 days), 5.5% in E-2 (12 days) and 4.3% in E-3 (18 days). The degradation kinetics of DEHP in thermophilic phase (including initial mesophilic phase) and the phase there after were determined by first order and fractional power kinetics, respectively. The significance of experimental parameters in DEHP degradation was assessed by Pearson correlation approach. Elevated temperature produced during composting was effective for the rapid degradation of DEHP from sewage sludge compared to mesophilic treatment.

High Rate Composting with Innovative Process Control

Despite the variety of composting plants present on the market, the design and operation of composting systems to improve the rate of the process and quality of end products are still an objective that has not been achieved. The composting process evolves through a thermophilic phase followed by a mesophilic one. The metabolism of microorganisms involved in the process is deeply disturbed by environmental changes like temperature, oxygen level and moisture. The aim of this study was to carry out a composting process in two separate reactors: the first, only in the thermophilic phase, with the purpose of pathogen destruction and decomposition by selected thermophilic microorganisms; the second, only in the mesophilic phase, to perform the main microbial transformations with a higher rate of volatile solids destruction. The results confirmed that maintaining constant the main parameters which affect the process, the microbial activity is enhanced. In the two-phase process, the evolution of parameters like moisture, organic carbon, humified organic matter, organic N, C/N and germination index (phytotoxicity) is more rapid relative to the single phase process. Also pathogen destruction is improved with the complete elimination of Faecal coliforms and Enterobacteriaceae and with a stronger reduction of Faecal streptococci.

Co-composting solid swine manure with pine sawdust as organic substrate

The main objectives of this work were to investigate the evolution of the principal physicochemical properties, i.e., bulk temperature, pH, electrical conductivity (EC), moisture content, total organic matter, total nitrogen and total phosphorus, in co-composting pine sawdust with increasing percentages of fresh solid swine manure, and thus to evaluate the most desirable manure proportion for producing organic substrates in consideration of the quality of the resulted compost. The composting was in four identical 100.5 l lab vessels, using 5% each tea leaves and herb residues as conditioners. The swine manure was added in the trials at 20%, 30%, 40%, respectively, and was substituted in the control with 30% lake sludge corrected by 0.5% urea. The initial humidity of each treatment was 60 ± 2%. While being aerated actively at approximately 0.3 m 3/min at intervals of 10 min/h, the mixture was composted for 29 days. The results indicated that N and P decomposition primarily occurred in the mesophilic phase, while organic carbon decomposed in the thermophilic phase and 30% swine manure with initial C/N ratio of about 40 was more desirable for composting organic substrates.

Composting of Source-Separated Household Organics At Different Oxygen Levels: Gaining an Understanding of the Emission Dynamics

Source-separated household organics were composted in a reactor at three oxygen levels, 16%, 2.5% and 1% in the compost gas. Short-chained fatty acids were initially present in the compost material, and were also produced during the mesophilic phase at all three oxygen levels. This indicated that partial anaerobic conditions existed. No NH3 emissions occurred during the mesophilic phase due to acidic conditions. Composting at 2.5% and 1% O2 concentrations prolonged the mesophilic phase and reduced the microbial activity as compared to 16% O2. This led to delayed and decreased emissions of NH3. Nitrous oxide was not formed during thermophilic conditions. Methane, which was measured at 2.5% and 1% O2, was only found during thermophilic conditions. The emission of methane indicates that anaerobic conditions occurred during the thermophilic phase. The main reactions regulating pH during composting were outlined involving the ion species VFA, NH4+/NH3 and CO2/HCO3−/CO32−.

Microbiological aspects of biowaste during composting in a monitored compost bin.

To determine the microbial succession of the dominating taxa and functional groups of microorganisms and the total microbial activity during the composting of biowaste in a monitored process. Biowaste (vegetable, fruit and garden waste) was composted in a monitored composting bin system. During the process, taxonomic and functional subpopulations of microorganisms were enumerated, and dominating colonies were isolated and identified. All counts decreased during the thermophilic phase of the composting, but increased again when the temperature declined. Total microbial activity, measured with an enzyme activity assay, decreased during the thermophilic phase, increased substantially thereafter, and decreased again during maturation. Bacteria dominated during the thermophilic phase while fungi, streptomycetes and yeasts were below the detection limit. Different bacterial populations were found in the thermophilic and mesophilic phases. In fresh wastes and during the peak-heating phase, all bacterial isolates were bacilli. During the cooling and maturation phase the bacterial diversity increased, including also other Gram-positive and Gram-negative bacteria. Among the fungi, Aspergillus spp. and Mucor spp. were predominant after the thermophilic phase. The microbial abundance, composition and activity changed substantially during composting and compost maturity was correlated with high microbial diversity and low activity. A more complete overview of the whole composting process of biowaste, based on microbial counts, species diversity and functional groups and abiotic parameters is presented, and the potential of a simple enzyme assay to measure total microbial activity was demonstrated.

Removal of fecal coliforms by thermophilic anaerobic digestion processes

Recent U.S. EPA regulations (40 CFR Section 503) specify maximum concentrations of pathogens and metals for Class A wastewater treatment plant sludges. The most common sludge process is mesophilic (35 degrees C) digestion which stabilizes the solids, produces a combustible gas but does not create an effluent that meets the 503 Class A pathogen requirements. This investigation was conducted to determine whether anaerobic digestion processes incorporating a thermophilic stage could achieve 503 Class A pathogen levels. The research reported here was a bench-scale screening study meant to identify the most promising process alternatives for further investigation. Fecal Coliform (FC) concentrations were used to assess disinfection efficiency. Digesters were 30 L capacity fed semi-continuously in draw-fill mode. Digester startup was rapid to produce true thermophiles. Temperature staging and pH were assessed in 3 sets of experiments: Set 1 were one stage ("acid phase"), Set 2 were one stage ("acid + methane phases") and Set 3 were two stage ("acid phase" then "methanogenic phase"). Feed was a 1:1 mixture of Thickened Waste Activated Sludge and Primary Sludge. The following anaerobic digestion configurations and operating parameters allowed the production of digested sludge with a mean FC concentration statistically less than 10(3) (the regulatory value for Class A sludge): thermophilic single stage acid phase at 52 and 62 degrees C; thermophilic single stage acid + methane phase at 48 degrees C, 52 degrees C and 62 degrees C; two-stage mesophilic acid phase followed by mesophilic methane phase; two stage mesophilic acid phase followed by thermophilic methane phase at 48 degrees C, 52 degrees C and 62 degrees C. If the maximum digested FC concentration must be below 10(3) MPN/g TS then the following digester configurations and operating conditions will be compliant: two stage mesophilic acid phase followed by thermophilic methane phase at 52 degrees C and 62 degrees C.