Elsevier

Waste Management

Volume 56, October 2016, Pages 290-297
Waste Management

Effect of biomass concentration on methane oxidation activity using mature compost and graphite granules as substrata

https://doi.org/10.1016/j.wasman.2016.08.003Get rights and content

Highlights

  • Methane oxidation activity per cell reflects the suitability of culturing conditions.

  • Biomass normalised MOA is widely applied to natural systems such as lake sediments.

  • The MOA per cell for a culture is shown to be the same on two different substrata.

  • The MOA per cell in lab cultures is significantly less than in natural systems.

Abstract

Reported methane oxidation activity (MOA) varies widely for common landfill cover materials. Variation is expected due to differences in surface area, the composition of the substratum and culturing conditions. MOA per methanotrophic cell has been calculated in the study of natural systems such as lake sediments to examine the inherent conditions for methanotrophic activity. In this study, biomass normalised MOA (i.e., MOA per methanotophic cell) was measured on stabilised compost, a commonly used cover in landfills, and on graphite granules, an inert substratum widely used in microbial electrosynthesis studies. After initially enriching methanotrophs on both substrata, biomass normalised MOA was quantified under excess oxygen and limiting methane conditions in 160 ml serum vials on both substrata and blends of the substrata. Biomass concentration was measured using the bicinchoninic acid assay for microbial protein. The biomass normalised MOA was consistent across all compost-to-graphite granules blends, but varied with time, reflecting the growth phase of the microorganisms. The biomass normalised MOA ranged from 0.069 ± 0.006 μmol CH4/mg dry biomass/h during active growth, to 0.024 ± 0.001 μmol CH4/mg dry biomass/h for established biofilms regardless of the substrata employed, indicating the substrata were equally effective in terms of inherent composition. The correlation of MOA with biomass is consistent with studies on methanotrophic activity in natural systems, but biomass normalised MOA varies by over 5 orders of magnitude between studies. This is partially due to different methods being used to quantify biomass, such as pmoA gene quantification and the culture dependent Most Probable Number method, but also indicates that long term exposure of materials to a supply of methane in an aerobic environment, as can occur in natural systems, leads to the enrichment and adaptation of types suitable for those conditions.

Introduction

The waste sector accounts for 2.4% of total greenhouse gas (GHG) emissions in Australia and 80% of these emissions are from landfills (DCCEE, 2012). Despite the existence of biogas collection systems in landfills, a portion of biogas still diffuses through landfill slopes and covers, particularly in active cells where gas extraction does not commence until a viable methane concentration is reached, or in old landfills where extraction of combustible biogas is no longer viable due to deteriorating capping systems. Landfill covers can be designed to enhance the growth of methanotrophic bacteria to mitigate GHG emissions (Barlaz et al., 2004). So called biocovers have been intensively studied with the aim of optimizing their efficiency in mitigating methane emissions generated at landfills, particularly with the regard to various types of biocover material (He et al., 2008, Pedersen et al., 2011), landfill gas flux (Stern et al., 2007, Bogner et al., 2010) and environmental conditions such as pH, temperature and moisture content of the biocover (Mor et al., 2006, Stern et al., 2007, He et al., 2008, Wang et al., 2011). All of these factors will affect the required thickness of a biocover.

A range of materials, including soils (sand, loam and clay), or organic material such as biochar, compost from municipal solid waste, agricultural wastes (straw, sugar cane mulch and shredded wood) and shredded garden waste, have been investigated as substrata or co-substrata for enhanced methanotrophic activity within landfill covers (Huber-Humer et al., 2011, Reddy et al., 2014, Scheutz et al., 2009).

There are wide variations in the MOA reported for each these landfill cover materials, as summarised in Table 1 (Huber-Humer et al., 2011, Limbri et al., 2014, Scheutz et al., 2009). A range of MOA values is expected because the composition and colonisable surface area of broadly defined materials such as compost and soil will vary from one study to another. Furthermore, biofilm establishment may not be repeatable on the same material for a variety of reasons including gas and moisture channelling due to non-uniform packing; the blockage of pore space by biofilm; or different growth phases of microorganisms. The variability in reported rates therefore makes it difficult for the practitioner to select the most suitable biocover material, or to anticipate if the biocover will improve with time, or to decide whether additional moisture or nutrient might enhance performance.

In contrast, the MOA per methanotrophic cell would reflect the microbial genera present in the system, the degree to which the organisms have adapted to local conditions and the availability of nutrients and the presence of inhibitory factors. Inhibitory agents may be associated with the supporting substratum or the culturing media. As an example, a number of studies have demonstrated the inhibitory effect of various metabolites on MOA including organic acids and ethanol, nitrogen species (NH4+, NO3, and NO2) and H2S (Duan et al., 2013, Long et al., 2013, Wieczorek et al., 2011, Wilshusen et al., 2004). Nutrient deficiency could also occur with an inert substratum such as crushed rock, in which case essential nutrients and trace elements must be supplied with the growth media.

The use of biomass normalised activity assays is well established in environmental biotechnology. Biomass normalised assays are used in anaerobic digestion to examine the effect of inhibitory substances on digester performance (Soto et al., 1993, Angelidaki et al., 2009) and in a number of studies on methanotrophic activity in natural environments including temperate lakes (Sundh et al., 2005), lake sediments (Rahalkar et al., 2009), on the surfaces of plants that generate methane as a by-product from photosynthesis (Yoshida et al., 2014) and in soils over naturally occurring subterranean sources of methane (Bender and Conrad, 1992). Correlations between MOA and the amount of methanotophic biomass was noted in all of these studies, although the biomass normalised rate varied between studies. The aim of this study was to measure biomass normalised MOA on selected substrata and to compare the rates with those reported in the studies listed above.

Biomass normalised MOA was measured on two substrata in this study: stabilised compost which is an established landfill cover material, and graphite granules, a proven electrode applied widely in microbial electrosynthesis studies (Erable et al., 2009, Freguia et al., 2007, Rabaey and Verstraete, 2005). Although graphite granules are too expensive to be a viable landfill cover material, they are an effective inert biofilm substratum with a well characterised shape and size range and they can be readily picked out of blends with compost, as required in this study, as opposed to fine grained materials such as sand.

Section snippets

Substrata

Woodchip based mature compost (10 months old) was used as a representative biocover material. The fraction passing through an 8 mm sieve was used in the experiments. The sub-8 mm fraction had a moisture content of 55.5% and volatile solid content of 35.4% of total solids (TS). A property of interest for material acting as a substratum is surface area. The compost particles had a wide size distribution (Table 2) and varied in shape. The graphite granules were typically spherical with a diameter of

MOA on various blends of graphite granules and compost

Trends in methane depletion in all vials are shown in Fig. 1. Methane was fully depleted within 24 h in the 100% compost and the 75:25 compost/granule vials in response to the first methane injection. Methane depletion followed a first order trend in the other vials with the exception of the 100% granule vial, where a lag phase was evident. More frequent sampling after the second methane injection confirmed that methane depletion followed a first order trend for all compost:granule blends

Discussion

The biomass normalised MOA determined in this study decreased by over a factor of two over repeated injections of methane to the same cultures. Biomass normalised activity can change depending on whether the microorganisms are in a growth or maintenance phase (Price and Sowers, 2004). Although MOA per unit mass of substrata improved in response to the second biogas injection, biomass normalised MOA was higher in response to the first injection, suggesting a more active microbial growth phase.

Conclusions

The current study shows that methane oxidation activity is correlated to the amount of methanotrophic biomass, in agreement with studies of methanotrophic activity in natural systems. Although biomass normalised MOA partially depends on the method used to measure biomass, the variety of biomass normalised MOA obtained in this and other studies indicates some systems are inherently more conducive to methanotrophic activity. The biomass normalised MOA observed for the freshly cultured biomass in

Acknowledgements

The authors would like to thank Remondis Australia and the University of Queensland who jointly fund the Centre for Solid Waste Bioprocessing. This work was performed within a landfill biocover program with additional funds from the Australian Research Council (DP140104572) and the Queensland Government under the Smart Futures Research Partnership Program. The authors gratefully acknowledge Dr. Yang Lu for technical assistance in carrying out the microbial phylogenetic analysis and Ms Andrea

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