Elsevier

Bioresource Technology

Volume 148, November 2013, Pages 517-524
Bioresource Technology

Substrate induced emergence of different active bacterial and archaeal assemblages during biomethane production

https://doi.org/10.1016/j.biortech.2013.09.017Get rights and content

Highlights

  • The structure and function of methane-producing microbial communities were studied.

  • The active bacterial and archaeal populations were analyzed with pyrosequencing.

  • Analyzing RNA more clearly revealed the composition of microbial communities.

  • Different organic substrates promoted the emergence of different active populations.

Abstract

This study analyzed the composition of a methane-generating microbial community and the corresponding active members during the transformation of three target substrates (food waste, cellulose or xylan) by barcoded 454 pyrosequencing of the bacterial and archaeal 16S rRNA genes in the DNA and RNA. The number of operational taxonomic units at 97% similarity for bacteria and archaea ranged from 162–261 and 31–166, respectively. Principal coordinates analysis and Venn diagram revealed that there were significant differences in the microbial community structure between the active members transforming each substrate and the inoculum. The active bacterial populations detected were those required for the hydrolysis of the amended substrate. The active archaeal populations were methanogens but the ratio of Methanosarcinales and Methanomicrobiales varied between the cultures. Overall, results of this study showed that a subset of the populations became active and altered in relative abundance during methane production according to the amended substrate.

Introduction

Food crisis and the increasing demand for energy have motivated industries to produce next generation biofuels from alternative feedstocks including cellulosic biomass and waste organic matters to replace first generation biofuels from food crops (Zaks et al., 2011). Methane gas generated by anaerobic digestion (AD) of organic matters is considered a cost-effective alternative to produce biofuels for heat and power purposes as well as waste management (Weiland, 2010). Hence, use of AD is likely to increase in the near future for these purposes. However, due to the complexity of its biochemistry and the lack of comprehensive phylogenetic and metabolic data on the microbes involved in its catalysis, AD has mostly been regarded as a “black box” consisting of diverse microorganisms affiliated with the Bacteria and Archaea domains (Nelson et al., 2011). Molecular techniques such as PCR followed by cloning/sequencing, quantitative real-time PCR, terminal restriction fragment length polymorphism, and fluorescence in situ hybridization have previously provided qualitative and quantitative assessments of the microorganisms in AD systems (Godon et al., 1997, Rittmann et al., 2008). Because these molecular methods tend to be low-throughput, they are suited in revealing the identity and distribution of the predominant populations (Angenent et al., 2002, Godon et al., 1997) and may not reflect the true diversity of a bioreactor sample. Therefore, the microbial community structure of AD systems has yet to be fully elucidated, especially the functional roles of both the predominant as well as minor members and when certain players are metabolically active.

In order to overcome the drawbacks of the traditional culture-independent tools, high-throughput amplicon pyrosequencing techniques with increased sensitivity have gained popularity in investigating the microbial community structure in various natural environments and engineered bioreactors such as human guts, seawater, soils, and sewage treatment plants (Nam et al., 2011, Werner et al., 2011). Thus far, a number of full-scale anaerobic digesters have been investigated to characterize the bacterial and archaeal communities (Chouari et al., 2005, Godon et al., 1997, Kröber et al., 2009). Many of these studies have focused on analyzing DNA only but not RNA. While DNA is useful in identifying members of the total community, it does not necessarily indicate which subpopulations may be active at a given time or under specific environmental conditions, making it challenging to fully understand the biology of the system to optimize its performance. This drawback is particularly problematic in the analysis of closed systems where the metabolically-inactive and non-growing cells are not washed out like in continuous systems. Conversely, RNA allows active members in a microbial community to be effectively identified, as metabolic activity is generally correlated with rRNA content (Kerkhof and Kemp, 1999). Hence, analysis of bacterial and archaeal 16S rRNA by means of reverse transcription-PCR is more appropriate in investigating the active microbial diversity of complex environmental systems (Brettar et al., 2012).

In this study, barcoded 454 amplicon pyrosequencing targeting the bacterial and archaeal 16S rRNA genes was applied to investigate a methane-producing microbial community in the laboratory. The objective was to examine and compare the diversity, structure and function of active assemblages within the microbial community during the catalytic transformation of three different organic substrates (food waste, cellulose or xylan) to methane in microcosm experiments. These substrates represent organic materials commonly found in waste streams that are suitable for bioenergy production. The insights gained into the metabolically-active populations should improve operation of biomethane systems.

Section snippets

Sampling of an anaerobic digester

The AD sludge samples used in this study were collected from a full-scale anaerobic digester located at the Shatin wastewater treatment plant in Hong Kong (22°24′23.44″, 114°12′49.36″). This plant treats municipal wastewater that is saline (∼5 g/L of chloride) as seawater is used for toilet flushing. The average total solids concentration of the AD sludge was 3.1% and volatile solids concentration was 51%. Multiple samples were collected from the mid-section of the tank when the system was in

Biomethane generation with different substrates

The performances of the laboratory cultures amended with food waste, cellulose or xylan were evaluated based on the total volume of methane produced (Fig. 1). Although the methane yield and lag period varied between the cultures, the microbial community was able to utilize all three of the amended organic substrates. The methane yield was 644 ± 32 mL/g of VS in food waste, 548 ± 27 mL/g of cellulose, 391 ± 20 mL/g of xylan, and the control with no amended substrate produced negligible amount of methane (

Conclusion

In this study, the bacterial and archaeal populations that were present and metabolically active during the conversion of organic substrates to methane in the inoculum were analyzed. Overall, the prevalence of different members within a particular microbial population has been shown to be dependent on the amended substrate. Thus, microbial population that is lower in abundance under one condition may emerge to become active and increase in prevalence under different conditions. Therefore, the

Acknowledgements

This research was supported by the Research Grants Council of Hong Kong through project #116111. The authors thank the Hong Kong Drainage Services Department for sampling assistance at the wastewater treatment plant. X.Y.L. thanks the City University of Hong Kong for a post-doctoral fellowship.

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