Research paperEffect of humic acids on the activity of pure and mixed methanogenic cultures
Introduction
Methanogens are strictly anaerobic archaea that have diverse morphology and phylogeny. Their ecological niches are widely distributed. They can be found in aquatic sediments (marshes and swamps), stagnant soil (peat bogs and rice fields), marine geothermal vents, the digestive tract of animals (ruminants and termites) and in engineered anaerobic digesters [1]. Methanogens are sensitive to environmental factors. Wide range of organic compounds, such as long chain fatty acids, aromatic compounds, xenobiotics, and inorganic compounds such as ammonia and heavy metals have been described to affect the methanogenic activity [2].
Humic acids (HA) are charged polyelectrolyte complexes due to the presence of carboxylic, phenolic, ketonic, aromatic and aliphatic groups and interact with both living and non-living matter [3]. They can function as electron shuttles in anaerobic environments for fermentive, iron-reducing and sulphate-reducing bacteria, as well as for methanogenic archaea [4], [5], [6], [7], [8].
Although the role of HA in natural environments is known, their abundance, composition and effect in engineered systems (e.g. in anaerobic digesters) are not defined well in the literature. In an anaerobic digester environment, abundance and composition of HA mainly depend on the type of the feed [9]. HA concentrations can reach up to mass fraction of 1.5% of total solids in the treatment sludge and agricultural waste, such as manure and maize [9], [10], [11]. Abundance of HA in anaerobic digesters may negatively affect the overall conversion processes. Indeed, the negative effect of HA on hydrolysis step of anaerobic digestion was shown [9], [12], [13], [14]. In addition, a decrease in methanogenic activity was observed in the presence of HA [12], [13], [14], [15]. However, from these experiments it was not evident whether the methanogens were affected and if so, which physiological groups/phylotypes of methanogens were most vulnerable to HA inhibition. Thus, it is important to determine the physiological response of different methanogenic groups to get more information about the methane production in the anaerobic digesters, having higher HA concentrations.
In this study, important acetoclastic and hydrogenotrophic methanogenic groups, belonging to Methanosaetaceae, Methanosarcinaceae, Methanospirillaceae, and Methanobacteriaceae, were selected to test their methanogenic activity in the presence and absence of HA. These methanogenic groups were selected due to their high abundance in most of the anaerobic digesters [16]. The methanogenic activity of pure cultures was compared to anaerobic crushed methanogenic granular sludge from a full scale UASB (Upflow Anaerobic Sludge Blanket) reactor treating paper mill wastewater. In this scope, batch tests were set-up in identical conditions for both pure and mixed cultures. During the batch experiments, methanogenic activity of each experimental group was monitored with gas and organic acid measurements.
Section snippets
Experimental set-up
The effect of humic acid (CAS Number 68131-04-4, Sigma-Aldrich, Zwijndrecht, The Netherlands) on mesophilic methanogens was investigated in batch tests. Crushed mesophilic anaerobic granular sludge and pure cultures of methanogens were tested. Batch incubations were performed in 120-cm3 bottles with 50 cm3 bicarbonate buffered mineral salts medium, supplemented with cysteine (0.96 kg m−3), trace elements and a vitamin mixture. Additionally, 0.12 kg m−3 acetate was added to the hydrogenotrophic
Effect of humic acid on methanogenic cultures
For all methanogenic pure cultures used in this study, the recovery of reducing equivalents in the form of CH4, produced from H2/CO2, acetate and formate, was always higher than 85%.
Conclusions
The effect of HA on methanogenic activity was demonstrated using pure cultures and mixed cultures. With the exception of Methanospirillum hungatei, all pure cultures of hydrogenotrophic methanogens tested were severely affected by addition of HA. Of the acetoclastic methanogens tested, Methanosaeta concilii was not affected by HA, whereas Methanosarcina barkeri was severely affected by HA. Anaerobic sludge was less affected by the addition of HA. However, a clear gap in the reducing equivalent
Acknowledgments
This research is supported by the Dutch Technology Foundation STW (STW-11612), which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs.
The authors thank Vicente T. Sedano Núñez for providing cultures of Methanobacterium formicicum and Methanospirillum hungatei, and Marjan J. Smeulders for proofreading the manuscript.
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Both authors contributed equally.