Supplementation of trace metals such as Co and Ni may improve anaerobic digestion of organic material for biogas formation. Which trace metals that are needed and the quantity to apply are, at least partly, related to metal speciation and bioavailability. According to the common perception, metals have to be dissolved to be available for microbial uptake. However, the impact of trace metal speciation on bioavailability is still unclear. The purpose of the present study was to investigate the effect of Fe-, Co- and Ni-addition on the biogas process performance of stillage-fed lab-scale biogas tank reactors. Metal speciation was determined by sequential extraction (SE), extraction of acid volatile sulfides (AVS) and continuously extracted metals (AVS-Me). Sulfur forms, which may be associated to metal speciation, were studied with S XANES (sulfur X-ray absorption near edge structure). The effect of different Co- and Ni-concentrations on process microflora composition was examined with quantitative PCR (qPCR) and 454-pyrosequencing. The results showed that Co- and Ni-supplementation stimulated and stabilized the biogas process performance by increasing methane production and substrate utilization and by establishing low concentrations of volatile fatty acids. 10-20% of the total Co-amount was found in the dissolved phase, which shows that Co was relatively available for microbial uptake. Nickel was entirely associated to organic matter/sulfides and AVS, and was therefore considered to be non-bioavailable. Nevertheless, Ni-supplementation had stimulatory effects on the biogas process performance. This implies that Ni was available for microbial uptake despite its extensive association to sulfides and that other mechanisms than solubility govern the availability of this trace metal. The microbial analyzes revealed that it was primarily the methane producers which were affected by the concentration of Co and Ni. At stimulatory Co- and Ni-concentrations, the archaeal methanogenic community was dominated by aceticlastic Methanosarcinales. At lower Co- or Ni-levels, when biogas process performance was poor, an increase in hydrogenotrophic Methanomicrobiales was observed. This indicates a shift in the methanogenic flora, from being dominated by acetate utilizers to increased importance of hydrogen utilizers, and that the former was more dependent on Co and Ni. / Tillsats av spårmetaller kan förbättra rötning av organiskt material till biogas. Typ och mängd av respektive spårmetall som behöver tillsättas för att uppnå stimulerande effekter, varierar mellan processer. Detta är delvis kopplat till specieringen och biotillgängligheten av metallerna. Endast fria metalljoner och vissa metallkomplex antas vara tillgängliga för mikrobiellt upptag. Det är dock i många fall oklart hur metallernas speciering påverkar biotillgängligheten. Syftet med föreliggande studie var därför att undersöka effekten av tillsats av Fe, Co och Ni för biogasproduktion från drank, en restprodukt i produktion av bioetanol från spannmål, samt att undersöka hur dessa metallers speciering påverkar deras biotillgänglighet. Effekten av tillsatserna av Fe, Co och Ni undersöktes på biogasreaktorer i lab-skala. Metallernas speciering bestämdes genom sekventiell extraktion (SE), extraktion av AVS (acid volatile sulfide) och kontinuerligt extraherade metaller (AVS-Me). Svavelformer med betydelse för metallspeciering studerades med S XANES (sulfur X-ray absorption near edge structure). Effekten av olika Co- och Ni-koncentrationer på processens mikroflora undersöktes molekylärbiologiskt med kvantitativ PCR (qPCR) och 454-pyrosekvensering. Resultaten visade att Co och Ni stimulerade och stabiliserade biogasprocessen genom ökad metanproduktion, ökad utrötningsgrad samt låga halter av flyktiga fettsyror i det studerade systemet. 10-20% av totala mängden Co återfanns i löst fas, vilket visar att Co var relativt lättillgängligt för mikroorganismerna. Nickel var däremot enbart bundet till organiskt material/sulfider och AVS och kunde alltså betraktas som otillgängligt. Trots detta hade även tillsatsen av Ni stimulerande effekter på biogasprocessen. Det innebär att mikroorganismerna har förmåga att komma åt Ni bundet i svårlösliga sulfidföreningar och att andra mekanismer än löslighet reglerar tillgängligheten av denna spårmetall. De molekylärbiologiska analyserna visade att framför allt de metanbildande mikroorganismerna påverkades av halten av Co och Ni. De halter, som gav välfungerande processer, dominerades helt av acetiklastiska Methanosarcinales. Vid lägre halter av Co eller Ni, då processerna gick sämre, tillkom vätgasutnyttjande metanogener. Det tyder på ett skift i bildningen av metan från att ha dominerats av acetatklyvning till att vätgasutnyttjarna fått större betydelse och att de förra är mer beroende av Co och Ni.

Methylmercury (MeHg) is a neurotoxic compound that threatens the well-being of humans and wildlife. It is formed through the methylation of inorganic mercury (HgII) under suboxic/anoxic conditions in soils, sediment and waters. The chemical speciation of HgII, including specific HgII species in aqueous and solid/adsorbed phases, plays a key role in MeHg formation. Chemical forms of HgII which have been reported to be available for uptake in methylating bacteria include neutral HgII–sulfide complexes, HgII complexes with specific low molecular mass (LMM) thiols, and nanoparticulate HgS(s). Accurate determination of the chemical speciation of HgII is thus crucial when elucidating the mechanism of MeHg formation. The concentration of HgII–LMM thiols complexes is predicted to be extremely low (sub fM range). Current analytical methods do not allow direct quantification of HgII complexes due to the very low concentration of these complexes, and therefore determination rely on thermodynamic modeling. Accurate stability constants for HgII–LMM thiols complexes and quantification of LMM thiol ligands in environments are thus required to precisely determine the concentration of such complexes. In this thesis, a novel analytical method was developed based on online pre-concentration coupled with liquid chromatography tandem mass spectrometry to determine the concentration of 16 LMM thiols (Paper I). This method was successful in detecting 8 LMM thiols in boreal wetland porewaters, with mercaptoacetic acid and cysteine being the most abundant. The total concentration of individual detected LMM thiols ranged from sub nM (LOD=0.1 nM) to 77 nM. Moreover, the stability constant (β2) for HgII complexes with 15 LMM thiols were directly determined for the first time by competing ligand exchange experiments combined with liquid chromatography ICPMS analysis (Paper II). Values of log β2 for the reaction Hg2+ + 2LMM-RS- = Hg(LMM-RS)2 ranged from 34.6 for. Based on the determined constants of Hg(LMM-RS)2 complexes and state-of-the-art constants from literature for other HgII complexes, we established comprehensive thermodynamic speciation models for MeHg and HgII in boreal wetlands (Paper III). The speciation of HgII was coupled with the HgII methylation rate constant (km) determined with different enriched Hg isotope tracers (Paper IV). There was a good correlation (R2=0.88) between the km determined by a HgII(aq) tracer added as Hg(NO3)2 with high bioavailability and a tracer where HgII was bond to thiol groups in natural organic matter (HgII-NOM(ads)) and has a lower bioavailability. The HgII(aq) tracer was consistently methylated at 5 times higher rate than the HgII-NOM(ads) tracer. A good correlation was observed between the concentration of biologically produced LMM thiols and km in the boreal wetlands. In a mesocosm study of estuarine sediment-brackish water systems, increased concentration of phytoplankton chlorophyll α due to macro nutrient additions led to an increase in HgII methylation rate of the HgII(aq) but not of the HgII-NOM(ads) tracer or ambient HgII species (Paper V). Furthermore, simulated newly deposited HgII species from atmospheric and terrestrial sources were exhibited significantly higher HgII methylation rates when compared with simulated aged sediment HgII pools. Through the development and adoption of novel analytical methods, this thesis reveals the significance of LMM thiols in Hg biogeochemistry by precise determination of HgII–LMM thiol complexes in natural environmental systems.