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

Abstract

Conventional anaerobic digesters intended for the production of biogas usually operate in complete darkness. Therefore, little is known about the effect of light on their microbial communities. In the present work, 16S rRNA gene amplicon Nanopore sequencing and shotgun metagenomic sequencing were used to study the taxonomic and functional structure of the microbial community forming a biofilm on the inner wall of a laboratory-scale transparent anaerobic biodigester illuminated with natural sunlight. The biofilm was composed of microorganisms involved in the four metabolic processes needed for biogas production, and it was surprisingly rich in Rhodopseudomonas faecalis, a versatile bacterium able to carry out photoautotrophic metabolism when grown under anaerobic conditions. The results suggested that this bacterium, which is able to fix carbon dioxide, could be considered for use in transparent biogas fermenters in order to contribute to the production of optimized biogas with a higher CH₄:CO₂ ratio than the biogas produced in regular, opaque digesters. To the best of our knowledge, this is the first study characterising the phototrophic biofilm associated with illuminated bioreactors.

Introduction

Anaerobic digestion (AD) of organic matter is a robust technology for biogas synthesis from different types of waste [6], and numerous studies have been conducted to optimise the synthesis of biogas and evaluate potential substrates [28]. Anaerobic digesters can be fed with a wide range of substrates, such as grass biomass [2], [3], sewage sludge from water treatment [24], microalgal biomass [18], and food waste [70], among others. The main goal of AD is the production of methane, a renewable energy source that can be used for heating and electricity, as well as many other operations that use combustion engines [39]. Biogas is a mixture of methane (CH₄; 55–70% of the total volume), carbon dioxide (CO₂; 30–40%) and traces of other gases, such as hydrogen sulphide (H₂S) [14], [54]. Whereas methane is a flammable gas over a relatively large range of concentrations in air at standard pressure (5.4–17%), carbon dioxide is an inert gas. Therefore, increasing the CH₄:CO₂ ratio is one of the keystones for the production of high-quality biogas. The CH₄:CO₂ ratio can vary depending on the digester type, the substrate composition, and other factors such as temperature, pH, the degradation rate and substrate concentration [21]. An appropriate design for an anaerobic digester is thus central for the production of optimized biogas.

The microbial communities operating in the digester are the final key players responsible for the quality of the biogas produced. The role of different microorganisms in the four metabolic steps carried out during the AD of organic matter (hydrolysis, acidogenesis, acetogenesis, and methanogenesis) has been widely studied [74]. A diverse number of Bacteria are known to be involved in the hydrolysis and further acidogenesis of complex polymers, whereas the oxidation of intermediate metabolites to acetate (acetogenesis) is performed by either hydrogen- or formate-producing acetogens [61]. Lastly, methane synthesis is mainly derived from acetate and H₂/CO₂ by acetoclastic and hydrogenoclastic methanogenic Archaea. Therefore, an improved understanding of the microbial communities and their metabolic roles during the four stages of AD may also help to optimize biogas production in terms of quantity (yield) and quality (CH₄:CO₂ ratio of the gas produced).

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. Interestingly, a previous study reported an increase of the relative concentration of methane when an anaerobic digester was operated under the influence of light [63]. However, the effect of light on the entire microbiome of anaerobic digesters has yet to be addressed.

Therefore, the aim of the present work was to analyse the effect of natural sunlight on the microbial community of a laboratory-scale anaerobic co-digester, in order to explore the possibility of inducing light-sensitive pathways, which might improve biogas quality due to carbon fixation. In order to reach this goal, full-length 16S rRNA gene amplicon Nanopore sequencing, shotgun metagenomic sequencing and a complete bioinformatics analysis were used to unveil the structure and composition of the microbial community growing as a red-coloured biofilm over the transparent wall of a specifically designed transparent leach-bed bioreactor.