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Evaluation of the fungal-bacterial synergism in

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 Evaluation of the fungal-bacterial synergism in methane abatement
biotechnologies
Juan Carlos Lpez1, Raquel Lebrero1, Iiro Lehtinen1, Rebeca Prez1, Guillermo Quijano1,2, Ral
Muoz1
1 Department of Chemical Engineering and Environmental Technology, University of Valladolid, Valladolid, Spain
(jclneila@iq.uva.es; raquel.lebrero@iq.uva.es; iiro.leh@gmail.com; rebeca.perez@iq.uva.es;
gquijano@iq.uva.es; mutora@iq.uva.es)
2 Agrarian Engineering School, University of Valladolid Soria Campus, Soria, Spain

Keywords: fungal-bacterial biofilter; Graphium sp.; irrigation rate; methane biofiltration.

INTRODUCTION
Methane (CH4) is the 2nd most important greenhouse gas (GHG) and accounts for 10-16% of the greenhouse
impact in a 100-year horizon. Its atmospheric concentration increase of 150% over the past millennium seriously
compromises the maintenance of the average Earths temperature in this century (IPCC 2014). Among the CH4
anthropogenic emissions derived from intensive livestock farms, coal mines or landfills, 55% of them present CH4
concentrations below 5% v/v (Lpez et al. 2013).
Biotechnologies have become a feasible alternative to treat those diluted CH4 emissions with lower operating
costs and environmental impacts compared to their physical-chemical counterparts. However, the low mass
transfer of CH4 to the aqueous phase still compromises their full-scale implementation. In this context, fungal
biofilters usually exhibit a superior performance for hydrophobic VOCs since fungi provide larger surface areas
for gas mass transfer compared to bacterial biotechnologies (Kennes and Veiga 2004). Unfortunately, no
previous studies have investigated the performance of fungal biofilters for CH4 abatement, and the potential of
fungi to degrade CH4 is still unclear.
The aim of this work was to evaluate the potential of a mixed bacterial-fungal biofilter to abate CH4 at low
concentrations under different inlet loads and irrigation rates. Batch biodegradation tests were performed to
assess the capability of the fungi Graphium sp. to oxidize CH4. Further molecular biology analysis was carried
out to identify the most significant bacterial/fungal communities present in the system.

MATERIAL AND METHODS

Biofilter set-up and operation mode
A PVC biofilter (BF) with a working volume of 4 L was filled with compost as a packing material. An air-diluted
CH4 stream (inlet concentration ~14 g m-3, 2% v/v) was continuously fed at average temperature and humidity of
25C and 82%, respectively. The BF was operated for 2 days under abiotic conditions in order to rule out any
CH4 removal derived from photolysis or adsorption and then inoculated with the fungi Graphium sp. The BF was
operated at an inlet load (IL) of 20 0.8 g m-3 h-1 (empty bed residence time (EBRT) of 40 min) during the first 17
days. The IL was doubled from days 17-25, though initial IL values had to be restored due to an inhibition in the
system. Finally, IL was again doubled from day 54 onwards. The mineral salt medium (MSM), prepared
according to Lebrero et al. 2014, was supplemented with chloramphenicol (0.02 g L-1) at pH 4 to prevent
bacterial growth. Different irrigation rates were tested: no irrigation from days 0-7, 0.017 LMSM L-1packing bed d-1 from
days 8-32, 0.025 LMSM L-1packing bed d-1 from days 33-66, and 0.05 LMSM L-1packing bed d-1 from days 67-87. CH4, O2 and
CO2 concentrations at the BF inlet/outlet were periodically measured by GC-TCD.
Biodegradation tests
A pure strain of Graphium sp. was cultured batchwise (120 mL-bottles) during 33 days to assess its capability
to degrade CH4. Bottles were filled with 30 mL MSM, and CH4 at 20% v/v (diluted with air). Potato starch (1 mL,
24 g L-1) and methanol (100-200 mg L-1) were added on days 8 and 25-29, respectively.

RESULTS AND DISCUSSION

The BF did not show any CH4 degradation until the start-up of irrigation on day 8 (Fig. 1). The CH4 elimination
capacity (EC) rapidly rose up to 20 g m-3 h-1 (removal efficiency (RE) of ~100%). An EC of 44.6 g m-3 h-1 was
recorded by day 18 after increasing the IL, though a subsequent deterioration of the system performance
occurred. Thus, IL was restored to initial values and fluctuations in the EC were observed until an irrigation rate
of 0.025 LMSM L-1packing bed d-1 was applied. Once the performance was restored, IL was again doubled at day 53,
and a significant decrease in the EC and CO2 production rate (PCO2) was observed. The irrigation rate was
doubled by day 67, and increases in the EC and PCO2 up to 36.6 0.7 and 82.2 2.9 g m-3 h-1 were recorded
under steady-state conditions, respectively. To the best of our knowledge, the ECs and REs here shown rank
among the highest achieved for CH4 abatement under similar operational conditions (Lpez et al. 2013).
Moreover, despite excess irrigation of the packing material results in compaction and, thus, operational failures
(Lebrero et al. 2014), the irrigation up to 0.05 LMSM L-1packing bed d-1 was crucial for inhibitory metabolites washing-
out and improving removals.
CH4 degradation by Graphium sp. was not observed until methanol addition (Table 1), which has been
demonstrated to be a CH4-oxidizing promoter (Jesen et al. 1998). To the best of the authors knowledge, this is
the first work assessing the capability of fungi to degrade this GHG.

Fig. 1. Time course of the EC (), IL (continuous line), PCO2 () and humidity (dashed line). Vertical lines represent
the shifts in the irrigation rate.

Table 1. Batch biodegradation test for Graphium sp.

CH4 CO2 production/
Culture Condition
biodegradation O2 consumption
CH4 + O2 - -
CH4 + O2 + potato starch - +
CH4 + O2 + methanol + +

CONCLUSIONS
A BF treating CH4 at 2% v/v achieved maximum ECs of 36.6 0.7 g m-3 h-1 (RE of ~90%) at the lowest
residence time (20 min) and under an optimized irrigation rate of 0.05 LMSM L-1packing bed d-1. The capability of the
fungus Graphium sp. to co-metabolically oxidize CH4 together with methanol was here reported for the first time.

ACKNOWLEDGEMENTS
This research was supported by the Spanish Ministry of Economy and Competitiveness (BES-2013-063922
grant and CTQ2012-34949 project) and the European Union through the FEDER Funding Program.

REFERENCES
Intergovernmental Panel on Climate Change (2014). Fifth assessment report: Climate change 2014, Synthesis
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Kennes, C., Veiga, M.C. (2004) Fungal biocatalysts in the biofiltration of VOC-polluted air. Journal of Biotechnology
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Lebrero, R., Hernndez, M., Quijano, G., Muoz, R. (2014) Hexane biodegradation in two-phase biofilters operated
with hydrophobic biomass: effect of the organic phase-packing media ratio and the irrigation rate. Chemical
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Lpez, J.C., Quijano, G., Souza, T.S.O., Estrada, J.M., Lebrero, R., Muoz, R. (2013) Biotechnologies for
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