Performances and microbial composition of mesophilic and thermophilic anaerobic sludge digestion processes
Author(s)
Gagliano, Maria Cristina
Date Issued
June 5, 2014
Type
Doctoral Thesis
Abstract
The anaerobic digestion (AD) of organic wastes as sewage sludge still gathers a great interest due to the global needs for recycling and renewable energy production. The need of solutions for sustainable sludge disposal is increasing and anaerobic sludge treatment can be applied as cost-effective strategy, because it also allow to energy production in the form of biogas. AD of biological solids like sludge requires the hydrolysis of particulate matter to soluble substrates, rate-limiting step of the whole process. A pretreatment, mechanical or thermal, can enhance digestibility of sludge by disintegration, facilitating the release of organic matter in solution. Moreover, the performances of the AD process are primarily linked to the structure of the microbial community present in the system. However, knowledge of the microbial community structure – function relationships is limited.
This thesis presents results from microbiological investigations (mainly fluorescence in situ hybridization (FISH) and 16S rRNA gene clonal analysis) of three advanced sludge stabilization systems. The different research activities were conducted in the framework of the EU project “ROUTES” (Novel processing routes for effective sewage sludge management). Three different lab-scale AD processes were operated: 1) a thermophilic anaerobic digestion (TAD) of untreated and thermal pretreated waste activated sludge (WAS), 2) an innovative two stage mesophilic/thermophilic anaerobic digestion of untreated and ultrasound pretreated WAS, both in semi-continuous mode, and 3) batch mesophilic anaerobic digestion of untreated and ultrasound pretreated WAS. Since the dominant bacteria identified in thermophilic reactors by clonal analysis of 16S rDNA sequences belonged the genus Coprothermobacter, and no specific probe for in situ biomonitoring of this genus is available so far, a new oligonucleotide FISH probe was designed, and its application was optimized on thermophilic sludge samples trough the design of specific helper probes.
Thermophilic AD (TAD): The structure and dynamics of the microbial communities selected in two semi-continuous anaerobic digesters fed with untreated and thermal pretreated sludge were investigated. The systems operated for 250 days at different organic loading rate. 16S rRNA gene clonal analysis and fluorescence in situ hybridization (FISH) analysis allowed to identify the majority of bacterial and archaeal populations. Proteolytic Coprothermobacter spp. and hydrogenotrophic Methanothermobacter spp. living in strict syntrophic association were found to dominate in TAD process. The availability of a readily biodegradable substrate due to pretreatment
allowed to significant sCOD removals (more than 55%) corresponding to higher biogas production in the reactor fed with thermal pretreated sludge. Microbial population dynamics analyzed by FISH showed that Coprothermobacter and Methanothermobacter immediately established a stable syntrophic association in the reactor fed with pretreated sludge in line with the overall improved TAD performances observed under these conditions.
Innovative two-stage AD: The new approach was based on the concept to sub-divide the AD process into three different stages: 1) an ultrasounds pretreatment to improve hydrolysis, 2) a short mesophilic stage to improve volatile fatty acids formation and 3) a final thermophilic stage to convert these intermediates into methane and contemporarily assure the complete hygienization of the digested sludge. The biodiversity of microbial populations was evaluated in both systems by FISH analysis. Shannon–Weaver diversity (H’) and eveness (E) indices were calculated using FISH data. The increase of digestion temperature drastically affected the microbial composition and selected specialized biomass. Species richness was lower under thermophilic conditions compared to the values estimated in mesophilic samples and it was flanked by similar trend of the evenness indicating that thermophilic communities may be therefore more susceptible to sudden changes and less prompt to adapting to operative variations. HRT of the mesophilic stage was a crucial parameter to improve the performance of the following thermophilic stage. Shortening the HRT, a shift from methane to VFA production was observed, in particular by pretreating the sludge with ultrasounds. This lead to a dominance of syntrophic acetate oxidation during thermophilic stage, due to the high concentrations of VFA conveyed from mesophilic reactors.
Mesophilic batch AD: Mesophilic batch anaerobic tests were carried out using two different inocula, in order to evaluate the effect of abundance and physiological conditions of inoculum archaeal cells on ultimate methane yields of sludge anaerobic process with untreated and sonicated sludge. Differences in microbial composition of the startup inocula strongly influenced the overall performances. In particular, the reactors with an acclimated archaeal community obtained an higher specific methane production. FISH analysis of inocula showed that the simultaneous presence of acetotrophic Methanosarcina and Methanosaeta in the archaeal biomass, the higher initial archaeal cells relative abundance and their occurrence in the aggregated forms were the main factors positively affecting the conversion of organic matter into methane. This better physiological state of methanogens allowed to appreciate the effect of hydrolysis improvement by ultrasounds pretreatment in terms of biogas daily production.
Design of a FISH probe targeting the genus Coprothermobacter: A specific 16S rRNA-targeted oligonucleotide probe for detecting members of Coprothermobacter genus was designed on the basis of the 16S rDNA sequences obtained by a clonal analysis of thermophilic sludge. The newly
designed CTH485 probe and helpers probes hCTH429 and hCTH439 were optimized for use in fluorescence in situ hybridization on thermophilic anaerobic sludge samples. In situ probing revealed that thermo-adaptive mechanisms shaping the 16S rRNA could generally affect the in situ identification of thermophilic microrganisms, despite do not adversely affect the analysis. The novel developed FISH probe extends the possibility to study the widespread thermophilic syntrophic interaction of Coprothermobacter spp. with hydrogenotrophic methanogenic archaea, whose establishment is a great benefit for the whole anaerobic system.
Additional information
Dottorato di ricerca in Scienze ambientali
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