The aim of this study was to investigate the effect of trace element supplementation on operation of wheat stillage-fed biogas tank reactors. The stillage used was a residue from bio-ethanol production, containing high levels of sulfate. In biogas production, high sulfate content has been associated with poor process stability in terms of low methane production and accumulation of process intermediates. However, the results of the present study show that this problem can be overcome by trace element supplementations. Four lab-scale wheat stillage-fed biogas tank reactors were operated for 345 days at a hydraulic retention time of 20 days (37 degrees C). It was concluded that daily supplementation with Co (0.5 mg L(-1)), Ni (0.2 mg L(-1)) and Fe (0.5 g L(-1)) were required for maintaining process stability at the organic loading rate of 4.0 g volatile solids L(-1) day(-1).

From previous studies in our laboratory it was concluded that Co- and Ni-amendment was necessary for stable biogas process operation during anaerobic digestion of grain stillage. In the present study, shifts in microbial community structure were investigated in relation to omission of Co or Ni from the stable biogas processes. The first effect of the stopped Co- or Ni-additions was an increase in volatile fatty acid (VFA) concentrations. Eventually, the methane production ceased in the reactor without Niaddition. Quantitative polymerase chain reaction (qPCR) revealed that Methanosarcinales was the dominating order of methanogens during stable process performance (both Co and Ni supplied) while Methanomicrobiales increased with increasing VFA-concentrations at both Co- and Ni-deficiency. The increase was however more pronounced at Co-limitation. The qPCR results agreed with sequencing data obtained by 454-pyrosequencing, where the dominating sequences belonged to Methanosaeta sp (order Methanosarcinales) at stable conditions, while the proportion of sequences belonging to Methanoculleus sp. (order Methanomicrobiales) increased at reactor instability as a result of decreasing concentration of Co or Ni.

Several previous studies report stimulatory effects on biogas process performance after trace element supplementation. However, the regulation of the bioavailability in relation to chemical speciation (e.g. the role of sulfide) is not fully understood. The objective of the present study was to determine the effect of sulfide on the chemical speciation and bioavailability of Co and Ni in lab-scale semi-continuously fed biogas tank reactors, digesting grain stillage. The chemical forms and potential bioavailability of Co and Ni in the reactors were determined by sequential extraction (SE), and analysis of acid volatile sulfide (AVS) together with simultaneously extracted metals (AVS-Me). The results for metal speciation analysis demonstrated that Ni was completely associated to the organic  matter/sulfide fraction and AVS, suggesting low potential Ni-bioavailability. Cobalt was predominantly associated to organic matter/sulfide and AVS, but also to more soluble fractions which are considered to be more bioavailable. Process performance data showed that both Co and Ni were available for microbial uptake. Although the actual bioavailability of Co could be explained by association to more bioavailable chemical fractions as determined by SE, AVS and AVS-Me analysis, the complete association of Ni with organic matter/sulfides and AVS shows that Ni was taken up despite its expected low bioavailability. Thus, the results of the present study imply that Ni-sulfide precipitation does not prevent microbial uptake in the studied biogas reactors.

The effect of sequential extraction of trace metals on sulphur (S) speciation in anoxic sludge samples from two lab-scale biogas reactors augmented with Fe was investigated. Analyses of sulphur K-edge X-ray absorption near edge structure (S XANES) spectroscopy and acid volatile sulphide (AVS) were conducted on the residues from each step of the sequential extraction. The S speciation in sludge samples after AVS analysis was also determined by S XANES. Sulphur was mainly present as FeS (~60% of total S) and reduced organic S (~30% of total S), such as organic sulphide and thiol groups, in the anoxic solid phase. Sulphur XANES and AVS analyses showed that during first step of the extraction procedure (the. removal of exchangeable cations), a part of the FeS fraction corresponding to 20% of total S was transformed to zero-valent S, whereas Fe was not released into the solution during this transformation. After the last extraction step (organic/sulphide fraction) a secondary Fe phase was formed. The change in chemical speciation of S and Fe occurring during sequential extraction procedure suggests indirect effects on trace metals associated to the FeS fraction that may lead to incorrect results. Furthermore, by S XANES it was verified that the AVS analysis effectively removed the FeS fraction. The present results identified critical limitations for the application of sequential extraction for trace metal speciation analysis outside the framework for which the methods were developed.