Elsevier

Bioresource Technology

Volume 265, October 2018, Pages 275-281
Bioresource Technology

Methanogenic community shifts during the transition from sewage mono-digestion to co-digestion of grass biomass

https://doi.org/10.1016/j.biortech.2018.06.005Get rights and content

Highlights

  • Microbial community during fed-batch anaerobic digestion of grass was analysed.

  • Community changes based on 16S-rRNA- and cofactor F420 profiles were detected.

  • Methanomethylovorans and Methanosarcina indicated increasing digester performance.

  • The increase of methanogens was similar in liquid and solid anaerobic fed batches.

  • Nanopore-based sequencers are well suited for surveillance of AD processes.

Abstract

In this work, liquid and solid fractions of grass biomass were used as co-substrates for anaerobic co-digestion of sewage sludge. The input of grass biomass was increased gradually, and the underlying methanogenic microbiome was assessed by means of microscopy-based cell counting and full-length 16S rRNA gene high-throughput sequencing, proving for the first time the suitability of nanopore-based portable sequencers as a monitoring tool for anaerobic digestion systems. In both cases co-fermentation resulted in an increased number of bacteria and methanogenic archaea. Interestingly, the microbial communities were highly different between solid and liquid-fed batches. Liquid-fed batches developed a more stable microbiome, enriched in Methanosarcina spp., and resulted in higher methanogenic yield. In contrast, solid-fed batches were highly unstable at higher substrate concentrations, and kept Methanosaeta spp. – typically associated to sewage sludge – as the majoritary methanogenic archaea.

Introduction

Anaerobic digestion is a well-known technology that allows microbial conversion of biomass into methane and carbon dioxide. Often the process is combined with physical, chemical or biological pretreatments, which facilitate the subsequent biomass degradation (Rani et al., 2012, Kannah et al., 2017, Kavitha et al., 2017). Basically, anaerobic fermentation consists of four phases (Bischofsberger et al., 2005): hydrolysis (biomass fragmentation), acidogenesis (formation of organic acids, alcohols, carbon dioxide, and hydrogen), acetogenesis (formation of acetic acid), and methanogenesis (last phase of the process, in which acetic acid, hydrogen, and carbon dioxide are the main substrates for the formation of methane). The key microorganisms in the methanogenesis phase are methanogenic archaea, whose composition depends on the operation conditions and strongly changes when co-digestion with additional substrates occurs (Sundberg et al., 2013). Mesophilic methanogens that can be found in especially high abundances belong to the genus Methanosaeta, Methanoculleus, and Methanosarcina (Abendroth et al., 2015, Abendroth et al., 2017a).

A strong microbial shift can be observed under thermophilic conditions, in which methanogens such as Methanothermobacter or Methanobacterium show an increased abundance (Maus et al., 2016, Lin et al., 2017, Xiao et al., 2018). Besides temperature, methanogens are also very sensitive to the organic loading rate. For example, under mesophilic conditions digestion processes with high amounts of chemical oxygen demand (COD) tend to have high amounts of Methanosarcina and Methanoculleus. On the other hand, sewage sludge, which has typically lower amounts of COD compared to typical industrial co-digesters, tends to have higher amounts of archaea corresponding to the genus Methanosaeta (Abendroth et al., 2015, Abendroth et al., 2017a).

Even though there is a basic understanding of the distribution of methanogenic genera under certain digestive conditions, there are still some gaps remaining. For example, the gradual increase of COD in sewage sludge by means of co-digestion with other substrates has not been sufficiently characterized. However, as the application of co-substrates in sewage mono digesters could lead to a substrate overload, it is important to characterize the transition of a typical sewage microbiome into a high-performance microbiome. According to the current state of art, the mentioned microbial transition is of high interest for the scientific community in this field, as this process is of crucial importance for an efficient transition from anaerobic sewage sludge mono-digestion to co-digestion. With aims to reach the climate objectives of the European Union for the 21th century, researchers are continuously investigating new technologies and methodologies that might help to build a green and self-sustainable economy (European Commission, 2007). In this context, a very promising approach is to increase the efficiency of wastewater treatment plants by using existing sewage sludge digesters, in which co-digestion is implemented. Such a technological upgrade would allow efficient and local usage of organic waste sources, which are produced by surrounding communities and industries. In addition, it would be a further step towards powering self-sufficient wastewater treatment plants. Based on this idea, a number of works is showing the possibility to upgrade anaerobic sewage sludge digesters by using co-substrates. The proposed co-substrates include grass biomass (Hidaka et al., 2016; Abendroth et al., 2017a, Abendroth et al., 2017b), food waste (Zahan et al., 2016), municipal solid waste (Cabbai et al., 2016), glycerol (Jensen et al., 2014), microalgae (Mahdy et al., 2015), or pear residues (Arhoun et al., 2013). To facilitate the transition from anaerobic sewage sludge mono-digestion to co-digestion, and to meet the high standards of wastewater treatment plants regarding process stability, a better understanding of the microbial changes occurring during the transition from typical sewage digestion to high-load digestion processes is still needed. This work aimed to investigate the impact of slowly increasing concentrations of COD on the underlying microbiome of sewage sludge digesters. The impact of different feeding strategies (feeding with liquids or solids) was also analysed. On the one hand, lignocellulose from fresh grass biomass was mechanically treated, separated from liquids, and used for the solid feeding strategy. On the other hand, grass liquor (after separation from the solid fraction), was used for the liquid feeding strategy.

A powerful tool to investigate microbiome changes is 16S rRNA gene amplicon high-throughput sequencing or shotgun metagenomic approaches (e.g. Vanwonterghem et al., 2014, Abendroth et al., 2017b), since they enable the detection of thousands of species in one single experiment, and can also yield information on the metabolic pathways underlying the biogas production process. Within the present work, it was aimed aimed to apply the recently developed ONT™MinION sequencing platform, as this technology could have the potential to become a suitable monitoring tool for anaerobic digestion plants. The use of metagenomic sequencing as a monitoring tool for industrial processes (i.e. fermentations) has not been sufficiently explored to date.

One of the main reasons for this is the economic investment needed to acquire a sequencer, as well as the technical complexity of the sequencing process and the ulterior bioinformatic analysis. This process is typically simplified by submitting samples to specialized sequencing facilities. Unfortunately, this makes the whole procedure significantly slower (results are typically obtained after some weeks). However, the launch of new generation, portable sequencers opens up a new scenario for real-life sequencing applications. Therefore, the features of this technology (user-friendly operation, real-time analysis, and portability) prove the unprecedented impact in the clinical (Quick et al., 2017), biosecurity (Pritchard et al., 2016), and environmental (Brown et al., 2017) fields. This work assesses for the first time the suitability of the ONT™MinION platform as a monitoring tool for anaerobic digestion systems, and uses this technology to follow up the changes in the archaeal methane-producing community at nearly real time.

Section snippets

Substrate

Fresh grass biomass was chosen to be used as substrate (Gramineae). In was collected from the front garden of the Robert Boyle Institute and stored at 0° after pretreatment. To separate lquids from solids a conventional juicer (Angel Juicer 8500 s, Angel Co. LTD., Korea) was used for pre-treatment of grass biomass. The solid fraction contained a COD of 366 mg O2 per g of substrate (according to the German guideline DIN 3814-S9), and the liquid fraction had a COD of 82 mg O2 per g of substrate

Reactor performance: liquid vs. solid feeding

Two different strategies for the repowering of sewage sludge involving co-digestion were compared: (1) using a liquid co-substrate with very low percentage of total solids (TS) and, therefore, with low amounts of lignocellulose (batch reactions B and C); and (2) using a solid co-substrate with a very high percentage of TS and, therefore, with high amounts of lignocellulose (batch reactions D and E). Both co-substrates were obtained from fresh grass (Graminidae) biomass. Additionally, a control

Conclusions

Following the presented results, the microbiome of liquid-fed reactors was re-shaped, changing from a Methanosaeta spp.-dominated community, to a Methanosarcina spp.-enriched community. For the first time in this field, 16S rRNA gene high-throughput sequencing was performed with an ONTTM MinION sequencer, allowing the tracking of changes in taxonomic data from full-length 16S rRNA gene sequencing. This work reports that the addition of liquid co-substrates resulted in a more effective

Acknowledgements

We are grateful for proofreading by Kristie Tanner. We are also thankful for funding of the work by the German Ministry of Economic Affairs and Energy (grant nos. 03KB110A, 16KN041331 and 16KN070128).

Conflict of interest

The authors declare no conflict of interest.

References (29)

  • C. Abendroth et al.

    Potential pitfalls of FISH microscopy as assessment method for anaerobic digesters

    bioRxiv

    (2016)
  • C. Abendroth et al.

    From grass to gas: microbiome dynamics of grass biomass acidification under mesophilic and thermophilic temperatures

    Biotechnol. Biofuels

    (2017)
  • C. Abendroth et al.

    Microbial communities involved in biogas production exhibit high resilience to heat shocks

    Bioresour. Technol.

    (2017)
  • W. Bischofsberger et al.
  • Cited by (17)

    • Ammonia removal during leach-bed acidification leads to optimized organic acid production from chicken manure

      2020, Renewable Energy
      Citation Excerpt :

      This is the first study in which this device is used to analyse the bacterial fraction of anaerobic digestion experiments. This technology was recently applied for the first time to the archaeal population of anaerobic digestion experiments [39], where MinION sequencing resulted in extra-long amplicon sequences, which were covering the entire 16S-rRNA gene. Due to this advantage, a large fraction of bacterial sequences could be assigned to the genus level.

    • Computational methods for 16S metabarcoding studies using Nanopore sequencing data

      2020, Computational and Structural Biotechnology Journal
      Citation Excerpt :

      The library is prepared by the addition of adapters in the amplicon sequences, and samples are sequenced directly with a flowcell gripped on the MinION device (Fig. 1 c). Authors have tried to standardize a different 16S-based amplicon barcoding protocol by using a two PCR step-based protocol, with the first process to amplify the 16S rRNA gene and a second one for the addition of adapters for the 16S amplicons sequencing [48,49]. Another strategy has been based on the use of an ONT 1D2 chemistry library preparation where both DNA strands are sequenced (similar to the paired-end sequencing of Illumina), improving the quality of the reads by sequencing both strands of the target DNA [50].

    • Shedding light on biogas: Phototrophic biofilms in anaerobic digesters hold potential for improved biogas production

      2020, Systematic and Applied Microbiology
      Citation Excerpt :

      Over the past few years, next-generation sequencing techniques, such as 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing, have been applied to study the structure and composition of microbial communities in different types of anaerobic digesters [1,2,3,23,24,43,62]. These studies have shown that each particular community is influenced by parameters such as the type of feedstock [62], temperature [5,11,19], retention time [19], salt content [17,6], viscosity [24], pH [75], or the loading rate [11,24]. Although the influence of many physicochemical parameters on microbial communities has been studied in anaerobic digesters, very little is known about the influence of light on the process [53,59,66], mainly because of the obvious fact that conventional AD systems operate in complete darkness.

    • Characterization of microbial evolution in high-solids methanogenic co-digestion of canned coffee processing wastewater and waste activated sludge by an anaerobic membrane bioreactor

      2019, Journal of Cleaner Production
      Citation Excerpt :

      Thus, CCPW is a potential net supplier of renewable energy by means of the AD process, which provides dual environmental benefits through improved wastewater treatment and sustainable bioenergy generation. According to previous studies, co-digestion with other substrates has been proven superior to mono-substrate digestion due to balanced nutrients, sufficient alkalinity, and etc. (Razaviarani and Buchanan, 2014; Zhu et al., 2011, 2011), While many operational and physicochemical parameters, such as feeding characteristics, organic loading, temperature, pH and among others, are greatly impacting the performance and stability of co-digestion systems (Hardegen et al., 2018; Siddique and Wahid, 2018; Syaichurrozi et al., 2018; Zhao et al., 2018). Moreover, as a syntrophic biological process, anaerobic co-digestion also relies on the activities and community structure of microbes.

    View all citing articles on Scopus
    View full text