Anaerobic digestion (AD) is an important technology for achieving sustainability, but it faces challenges in meeting rising production demands while remaining economically profitable. One difficulty is the lack of a comprehensive understanding of the many interactions within anaerobic digesters, which makes it challenging to fully optimize them. This is particularly notable when considering the interlinked dynamics between micronutrient availability and fluid behavior. This study addresses this gap by focusing on key operational parameters affecting the efficiency of the process in continuous stirred-tank biogas reactors, which are the most used AD technology today. It does so by proposing and evaluating a novel conceptual model of the mechanisms behind how different parts of AD processes interact upon disturbance, highlighting strategies for preventing process failure. This article aims to improve our understanding of the complexity of AD biotechnology and to provide a starting point for developing advanced strategies for operational optimization.

Trace elements (TEs) are vital for anaerobic digestion (AD), due to their role as cofactors in many key enzymes. The aim of this study was to evaluate the effects of specific TE deficiencies on mixed microbial communities during AD of soluble polymer-free substrates, thus focusing on AD after hydrolysis. Three mesophilic (37 degrees C) continuous stirred-tank biogas reactors were depleted either of Co, Ni, or a combination of Se and W, respectively, by discontinuing their supplementation. Ni and Se/W depletion led to changes in methane kinetics, linked to progressive volatile fatty acid (VFA) accumulation, eventually resulting in process failure. No significant changes occurred in the Co-depleted reactor, indicating that the amount of Co present in the substrate in absence of supplementation was sufficient to maintain process stability. Archaeal communities remained fairly stable independent of TE concentrations, while bacterial communities gradually changed with VFA accumulation in Ni- and Se-/W-depleted reactors. Despite this, the communities remained relatively similar between these two reactors, suggesting that the major shifts in composition likely occurred due to the accumulating VFAs. Overall, the results indicate that Ni and Se/W depletion primarily lead to slower metabolic activities of methanogenic archaea and their syntrophic partners, which then has a ripple effect throughout the microbial community due to a gradual accumulation of intermediate fermentation products.

Anaerobic digestion (AD) is an established process for integrating waste management with renewable energy and nutrient recovery. Much of the research in this field focuses on the utilisation of new substrates, yet their effects on operational aspects such as fluid behaviour and power requirement for mixing are commonly overlooked, despite their importance for process optimisation. This study analysed rheological characteristics of samples from 21 laboratory-scale continuous stirred-tank biogas reactors (CSTBRs) digesting a range of substrates, in order to evaluate substrate effect on mixing efficiency and power demand through computational fluid dynamics (CFD). The results show that substrate and process parameters, such as solids content and organic loading, all have a significant effect on CSTBR fluid rheology. The correlation levels between rheological and process parameters were different across substrates, while no specific fluid behaviour patterns could be associated with substrate choice. Substrate should thus be considered an equally important rheology effector as process parameters. Additional substrate-related parameters should be identified to explain the differences in correlations between rheological and process parameters across substrate groups. The CFD modelling revealed that the rheology differences among the AD processes have significant implications for mixing efficiency and power demand of the CSTBRs, highlighting the importance of considering the substrate-induced effects on CSTBR rheology before including a new substrate.

As the anthropogenic greenhouse gas emissions continue imposing stress on our environment, it is becoming increasingly important to identify and implement new renewable technologies. Biogas production through anaerobic digestion has a great potential, since it links waste treatment with extraction of renewable energy, enabling circular bio-economies that are vital for a sustainable future.

For biogas to have an important role as a renewable energy carrier in society, the scale of its production will need to be increased substantially. New substrates need to be introduced along with raising organic loading rates of the reactors to increase the rate of biogas production. This contributes to challenges in maintaining process stability, thus increasing the risk for process disturbances, including problems that were not commonly encountered before. These difficulties may be particularly pronounced when a broad range of new, largely untested substrates are introduced, leading to an increased heterogeneity of organic material entering the reactors. In the case of currently the most common reactor type; the continuous stirred-tank biogas reactor (CSTBR); such problems may include shifts in rheology (i.e. fluid behaviour) of the anaerobic digester sludge. This may lead to increased energy consumption and decreased digester mixing efficiencies, which in turn may lead to inefficient biogas processes, ultimately decreasing the economic and environmental viability of biogas production. Much is still unknown regarding how rheology shifts happen in biogas reactors, particularly when it comes to what role the substrate plays in rheological dynamics, as compared to the microbial community during varying levels of biogas process stability.

This thesis elucidates the interactions between substrate type, microbial community and its metabolic activity, and anaerobic sludge rheology. A number of sludge samples from mesophilic and thermophilic CSTBRs digesting a broad range of substrates was analysed for their rheology. The specific effects of individual substrate types on CSTBR sludge rheology and the resulting implications for stirring power requirements and mixing efficiency were investigated. In order to also asses to which extent the microbial metabolism affects rheology at different levels of process disturbance, an experiment with a trace-element-induced inhibition of specific metabolic pathways under mesophilic reactor conditions was performed. This was used to identify the sequence of different interactions that occur in the reactor after the process begins to fail, and to evaluate how these interactions link to changes in digester sludge rheology. Finally, a case study of a disturbed thermophilic anaerobic digestion process was performed, including the monitoring of the response of rheology in relation to process stability, which was modified by changing trace element concentrations. The use of artificial substrate without polymeric compounds in both cases allowed for an evaluation of effects of the microbial community and its metabolic products on rheology without including the effects of complex substrates.

The results showed that substrate type has a large effect on how different process parameters correlate with fluid behaviour. This was particularly apparent in the case of total solids and total volatile solids, which correlated well with rheological parameters for samples from reactors digesting agricultural waste, sewage sludge, paper mill waste, or food waste, but not for mesophilic co-digesters. Among the different substrates investigated, food waste was generally observed to lead to the highest limit viscosities (i.e. apparent viscosities at high shear rates, where it becomes linear and constant) of the anaerobic sludge, while digestion of paper mill waste and thermophilic co-digestion led to some of the lowest. No fluid type could be clearly coupled to a specific substrate, but it could be observed that increased solids content could generally be associated with more complex, non-Newtonian rheological behaviour. The differences in fluid characteristics between reactors corresponded to large differences in modelled stirring power requirements and mixing efficiency. The results indicated that fluids with high values of rheological parameters, such as the consistency index (K) or yield stress (τ₀), would likely require more power or an adapted stirring system to achieve complete mixing. The substrates generally contributed more to the rheology characteristics of the anaerobic sludge than microbial cells on their own. Trace element-induced process disturbance initially led to the inhibition of specific microbial groups among methanogenic archaea or their syntrophic partners, which later escalated to broader inhibition of many microbial groups due to the accumulation of fermentation products. This resulted in microbial cell washout with a corresponding decrease of the contribution of the cells to anaerobic sludge rheology. A recovery of the thermophilic anaerobic digestion process was possible after the supplementation of selenium and tungsten was increased, resulting in increased propionate turnover rates, growing cell densities, and higher viscosity. Major shifts in the methanogenic community were observed, corresponding to the level of process stability. It could be concluded based on these experiments that the specific effect of microbial cells and their activity on sludge rheology were linked to cell density, which corresponded to process stability.

A conceptual scheme was developed based on the studies in this thesis, defining complex interactions between substrate, microbial metabolism, and anaerobic sludge rheology in biogas processes. The possible causes of rheology shifts are visualised and discussed.

Med anledning av att antropogena utsläpp av växthusgaser fortsätter att påverka vår miljö negativt, blir det allt viktigare att identifiera och implementera förnybara teknologier. Biogasproduktion, genom anaerob rötning, bidrar till att sammanlänka avfallshantering med förnybar energiomvandling samt möjliggör för cirkulära bioekonomier, avgörande för en hållbar framtid.

För att biogas ska utgöra en central roll som förnybar energibärare i samhället måste omfattningen av dess produktion ökas avsevärt. Nya substrat behöver introduceras, samtidigt som den organiska belastningen i existerande biogasreaktorer ökas, med syfte att öka produktionshastigheten. Detta bidrar emellertid till utmaningar avseende att upprätthålla processtabilitet, med ökad risk för processtörningar, inklusive nya typer av problem. Dessa svårigheter kan vara särskilt uttalade när nya, i stort sett otestade substrat, introduceras, vilket leder till ökad heterogenitet av organiskt material till reaktorerna. I tankreaktorer med omrörning (CSTBR), som i nuläget är den vanligast förekommande reaktortypen, kan sådana processförändringar innebära förändringar i reologiska egenskaper hos rötvätskor. Detta kan i sin tur leda till ökad energiförbrukning och lägre omrörningseffektivitet, vilket kan försämra biogasprocessens effektivitet samt bidra till minskad ekonomisk avkastning och minskade miljömässiga vinster. Det råder fortfarande oklarheter gällande reologiska förändringar av rötkammarmaterial i biogasreaktorer, särskilt med avseende på val av substrat i jämförelse med mikroorganismernas roll under varierade betingelser av processtabilitet.

Denna avhandling belyser interaktioner mellan typ av substrat, mikrobiella samhällen och deras metaboliska aktivitet och reologiska egenskaper av rötslam. Reologisk karaktärisering av rötvätskor från mesofila och termofila CSTBRs, som rötade ett brett spektrum av substrat, genomfördes. De specifika effekterna av individuella substrattyper på korresponderande rötvätskors reologiska egenskaper och dess implikationer för omrörning undersöktes. För att även kunna bedöma i vilken utsträckning den mikrobiella metabolismen inverkar på rötvätskors reologi, vid olika nivåer av processtörning, genomfördes en fallstudie med spårelement-inducerad inhibering av specifika metaboliska vägar, under mesofila reaktorbetingelser. Denna studie användes för att identifiera sekvensen av olika interaktioner som uppträder i reaktorn när processtörningar uppstår och för att utvärdera hur dessa interaktioner kopplar till förändringar i slamreologi. Slutligen genomfördes en fallstudie av en termofil biogasprocess innefattande karaktärisering av reaktormaterialets reologi i respons till förändringar i processtabilitet orsakad av förändrade spårelementkoncentrationer. I båda dessa fallstudier, möjliggjorde användningen av ett artificiellt substrat utan komplexa polymerer, att mikrobiella effekter på förändringar i rötvätskans reologi kunde studeras frånkopplat effekter av komplexa substrat.

Resultaten visade att substrattyp har stor inverkan på hur olika processparametrar korrelerar med rötvätskors reologiska egenskaper. Detta var särskilt tydligt med avseende på andelen totala respektive organiska fasta ämnen, vilka korrelerade väl med reologiska parametrar för rötvätskor från reaktorer som rötade jordbruksrester, avloppsslam, avfall från pappersbruk, respektive matavfall, men ej för rötvätskor från mesofila samrötningsanläggningar. Av de undersökta substrattyperna bidrog rötning av matavfall generellt till anaerobt slam med högst uppmätta värdena för gränsviskositet (dvs när viskositeten blir linjär och konstant vid ökade skjuvhastigheter), medan rötning av avfall från pappersbruk respektive termofil samrötning av olika substrat bidrog till de lägsta värdena för gränsviskoistet. Ingen reologisk vätsketyp kunde tydligt kopplas till en specifik substrattyp, men ökad mängd torrsubstans i rötvästkan kunde generellt associeras med komplexa, icke-newtonska, flödesegenskaper. Skillnaderna i flödesegenskaper motsvarade stora skillnader i behov av omrörningskraft och omrörningseffektivitet. Resultaten indikerade att rötvätskor med höga reologiska värden, exempelvis för konsistensindex (K) eller flytgräns (τ₀), sannolikt kräver mer energi eller ett anpassat system för effektivare omrörning. Generellt bidrog substratet mer till rötvätskans reologiska egenskaper än de mikrobiella cellerna på egen hand. Inducerad spårämnesbrist i genomfört reaktorförök ledde i början till en hämning av specifika mikrobiella grupper inom metanogena arkéer och deras syntrofa partners, vilket i sin tur bidrog till en bredare hämning av flera mikrobiella grupper orsakad av ackumulering av olika fermentationsprodukter. Detta resulterade i ursköljning av cellbiomassa vilket i sin tur minskade deras effekt på slammets reologiska egenskaper. En återhämtning av den termofila biogasprocessen var möjlig efter ökade tillsatser av spårelementen selen och volfram, vilket resulterade i snabbare omsättning av propionsyra, förhöjd celldensitet och viskositet. Stora förändringar observerades samtidigt inom det metanogena samhället, vilka var kopplade till olika nivåer av processtabilitet. Den specifika effekten av mikroorganismerna och deras aktivitet med avseende på slammens reologiska egenskaper var kopplad till celldensitet, vilket motsvarade processtabiliteten.

Ett konceptuellt schema utvecklades baserat på resultat av beskrivna studier för att visualisera komplexa interaktioner mellan substrat, mikrobiell metabolism, och reologi av anaerobt slam i biogasprocesser. De möjliga orsakerna till reologiska förändringar visualiseras och diskuteras.

Background

Waste lipids are attractive substrates for co-digestion with primary and activated sewage sludge (PASS) to improve biogas production at wastewater treatment plants. However, slow conversion rates of long-chain fatty acids (LCFA), produced during anaerobic digestion (AD), limit the applicability of waste lipids as co-substrates for PASS. Previous observations indicate that the sulfide level in PASS digesters affects the capacity of microbial communities to convert LCFA to biogas. This study assessed the microbial community response to LCFA loads in relation to sulfide level during AD of PASS by investigating process performance and microbial community dynamics upon addition of oleate (C18:1) and stearate (C18:0) to PASS digesters at ambient and elevated sulfide levels.

Results

Conversion of LCFA to biogas was limited (30% of theoretical biogas potential) during continuous co-digestion with PASS, which resulted in further LCFA accumulation. However, the accumulated LCFA were converted to biogas (up to 66% of theoretical biogas potential) during subsequent batch-mode digestion, performed without additional substrate load. Elevated sulfide level stimulated oleate (but not stearate) conversion to acetate, but oleate and sulfide imposed a synergistic limiting effect on acetoclastic methanogenesis and biogas formation. Next-generation sequencing of 16S rRNA gene amplicons of bacteria and archaea showed that differences in sulfide level and LCFA type resulted in microbial community alterations with distinctly different patterns. Taxonomic profiling of the sequencing data revealed that the phylum Cloacimonetes is likely a key group during LCFA degradation in PASS digesters, where different members take part in degradation of saturated and unsaturated LCFA; genus W5 (family Cloacimonadaceae) and family W27 (order Cloacimonadales), respectively. In addition, LCFA-degrading Syntrophomonas, which is commonly present in lipid-fed digesters, increased in relative abundance after addition of oleate at elevated sulfide level, but not without sulfide or after stearate addition. Stearate conversion to biogas was instead associated with increasing abundance of hydrogen-producing Smithella and hydrogenotrophic Methanobacterium.

Conclusions

Long-chain fatty acid chain saturation and sulfide level are selective drivers for establishment of LCFA-degrading microbial communities in municipal sludge digesters.

Loading macroalgae into existing anaerobic digestion (AD) plants allows us to overcome challenges such as low digestion efficiencies, trace elements limitation, excessive salinity levels and accumulation of volatile fatty acids (VFAs), observed while digesting algae as a single substrate. In this work, the co-digestion of the brown macroalgae Saccharina latissima with mixed municipal wastewater sludge (WWS) was investigated in mesophilic and thermophilic conditions. The hydraulic retention time (HRT) and the organic loading rate (OLR) were fixed at 19 days and 2.1 g l-1 d-1of volatile solids (VS), respectively. Initially, WWS was digested alone. Subsequently, a percentage of the total OLR (20%, 50% and finally 80%) was replaced by S. latissima biomass. Optimal digestion conditions were observed at medium-low algae loading (=50% of total OLR) with an average methane yield close to [Formula: see text] and [Formula: see text] in mesophilic and thermophilic conditions, respectively. The conductivity values increased with the algae loading without inhibiting the digestion process. The viscosities of the reactor sludges revealed decreasing values with reduced WWS loading at both temperatures, enhancing mixing properties.