Who is Actually the Biggest Fan of your Food Waste?

Anaerobic Digestion (AD) is a microbially mediated process by which organic matter (agricultural residues, food wastes, manure, etc.) is turned into biogas and biofertilizer in conditions that require the absence of oxygen (anoxic environment) (Kelleher et al., 2002). The advantages of this kind of waste decomposition over traditional waste management are obvious: low energy requirement, potential energy recovery, reduction of greenhouse gas released into the atmosphere, etc. However, because of the complexity of the AD process and poor knowledge of some of its components (mainly microbiological frame of the flow), the process stays too sensitive and difficult to control in many cases.

Comment by Arika Nyhus: Food waste is more appropriate than wastes Comment by Arika Nyhus: What do you mean when you say that the process is too sensitive? Is there a better word that could explain what you mean in a more effective way?

Anaerobic digesters have been mostly treated like a “black box”, which operation depends and based exceptionally on the chemical composition of the infeed, its physical state and internal temperature of the digester. Despite a large number of scientific papers describing the influence of environmental parameters on the microbial community structure, the reverse approach describing the influence of microbial community structure on AD function, its stability and productivity has been researched less. However, it becomes obvious now that the structure of the microbial community within AD systems is another main factor which governs operation efficiency (Demirel at al., 2008). Only recently the development and maintenance of the most efficient digester microbial community has become a research objective, mainly due to the development of tools able to describe a complex microbiological composition of a bioreactor as a whole.

Comment by Arika Nyhus: I think it would be beneficial to add references that have studied this Comment by Arika Nyhus: Are there scientific papers that have investigated the influence of microbial community structure on AD function? If so, I would put those references here and say (but see AUTHOR, YEAR). If there are no studies, then I would say that it hasn’t been studied at all. Comment by Arika Nyhus: Since there are only two authors, you would do (Demirel & Scherer, 2008).

Complex microbial alliance of the bioreactor that transforms organic waste into biogas requires four stages: hydrolysis, acidogenesis, acetogenesis and methanogenesis. This is only and possible only when several different microbial guilds of Bacteria and Archaea are present. At the beginning of the AD process, hydrolytic bacteria break down the complex carbohydrates, proteins and lipids in the infeed waste, converting them to more simple monomers and amino acids. The taxonomy of bacteria responsible for hydrolysis is dictated by the composition of the material. Acidogenic bacteria then ferment these substrates into volatile fatty acids (VFA). Typically the most common VFAs at that stage are acetate, propionate, and butyrate. Acetogenic Bacteria then convert propionate, butyrate, and formate to acetate via oxidation, producing H2 and CO2 in the process. At the final stage, Hydrogenotrophic Archaea process the H2 produced by bacteria during previous stages to produce methane.

Equally comfortable conditions for all of the alliances of bacteria involved and balance, between the biochemical activities between them is necessary to successfully digest organic waste and produce biogas. (Weiland, 2010). If the balance is disrupted and the digester is missing Bacteria and Archaea that can fill the niches of the AD process, the reduction of VFAs catabolization will occur and methane production will stop. , tTo avoid this, it is crucial to study and predict the composition and pathways of interaction between all the parties involved in the process. Comment by Arika Nyhus: I would stay consistent with your commas you use. For example, in the previous texts you haven’t been using commas before and but here you do. I would choose one or the other. Comment by Arika Nyhus: This sentence is a bit confusing to me. Also, remove the period after biogas. Comment by Arika Nyhus: I do not recognize this word. You could be right but want to discuss. Comment by Arika Nyhus: Period instead of comma and start a new sentence.

In order to be able to determine the structure of the microbial community of the AD process, several methods are available. In the past, researchers that were trying to characterize microorganisms that produce methane were only relying on culture techniques. With a traditional cultivation-based method, only a small part of the microbial composition of the bioreactor can be described, due to demanding culture requirements. In the case of syntrophic organisms that are present in AD process, reproduction of a pure culture for description is impossible. However, progress in DNA sequencing technology, have made analysis of AD microbial structures possible on all four stages of the process, giving comprehension of the changes and composition in bioreactors. (Levén et al., 2007). At this point, researchers have just begun to utilize the information pertaining to collective microbial genome structure to understand and to predict its influence on a digester performance. Comment by Arika Nyhus: Would be a good place to add references so readers could check out these older methods. Comment by Arika Nyhus: Remove comma Comment by Arika Nyhus: Since there is only three authors, you would write out all of their names. Then in subsequent texts (if you were to use the same reference again), you would write (Leven et al., 2007).

The collective genomes of microbes in the environmental sample is called the ‘metagenome’ and studies are called ‘metagenomics’ (Handelsman et al., 1998). Metagenomics main goal is to study uncultured organisms and to be able to understand the true diversity, functions and cooperation in the collective microbiological communities that can be found in soil, digestive system of animals, water, anaerobic digesters, etc. In the present, a sample can be obtained and directly sequenced from an AD environment. The entire communities are analyzed, without isolating and culturing distinct organisms with the power of genome analysis. Current research into increasing the output and capacity of anaerobic digesters is focused on microbial community characterization and monitoring and this kind of research is accelerating. (Vanwonterghem et al., 2014). Technologies such as Illumina/Solexa are now able to deliver now more sequenced data than ever before in comparison with the conventional Sanger sequencing, for example. (Zhou et al., 2011). Also, Illumina/Solexa technology does not require cloning of the DNA before sequencing, this way one of the main biases in this kind of sampling is avoided. (St-Pierre et al., 2013).

However there are several technologies that can be used, Illumina is currently the state of the art when it comes to 16S rRNA gene sequencing. It surpasses the previous 454 technology most notably due to its lower costs, higher accuracy and greater throughput (Sinclair et al. 2015) It is likely the most powerful and trustworthy tool for description of the metagenome of an anaerobic digester of any kind at this point. It will be a very useful tool to describe microbial composition of an anaerobic benchtop digesters in a laboratory setting that are mimicking all processes of an industrial size bioreactors and use this knowledge to maximize biogas outputs and stability of similar systems in the future. Comment by Arika Nyhus: Since there are five authors, list all of their names. Then use (Handelsman et al., 1998) after that. It is when you have six or more authors that you don’t have to introduce all of the names the first time you cite them.

Comment by Arika Nyhus: Remove period Comment by Arika Nyhus: Use all the authors Comment by Arika Nyhus: Avoiding one of the main biases in this kind of sampling (St-Pierre & Wright, 2013).What kind of sampling is it? Comment by Arika Nyhus: Use all the authors Comment by Arika Nyhus: Technologies Comment by Arika Nyhus: Use all the authors Comment by Arika Nyhus: Run on sentence. I would suggest breaking this into two sentences. OVERALL COMMENT: I think you did a good job at explaining AD, however, I am still a little confused at what your objectives are for your thesis. Additionally, it was unclear to me the three articles that you were summarizing. From what I understood from the project, we were suppose to introduce our thesis and then tie that in with three articles that have used genetic techniques to answer the questions we plan on answering for our projects (even if we don’t plan on using genetic techniques). What I would suggest for your final report is to shorten up your introduction to AD, correlate that to your main objectives for your thesis, and then summarize three articles that have used genetic techniques to answer similar questions.

References:

Demirel, B., and Scherer, P., 2008, The roles of acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of biomass to methane - a review. Environmental Science and Bio/Technology, v. 7, p. 173–190, doi: 10.1007/s11157-008-9131-1. Comment by Arika Nyhus: Do not bold the title of the articles. Do italicize the journal or book that the article came from

Handelsman, J., Rondon, M. R., Brady, S. G., Clardy, J., and Goodman, R., 1998, Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chemistry & Biology, 5:245–249. Comment by Arika Nyhus: Put year in (1998) for all references

Kelleher, B.P., Leahy, J.J., Henihan, a M., O’Dwyer, T.F., Sutton, D., and Leahy, M.J., 2002, Advances in poultry litter disposal technology - a review. Bioresource Technology, v. 83, p. 27–36, doi: 10.1016/S0960-8524(01)00133-X. Comment by Arika Nyhus: Use & instead of spelling out “and” for all references

Levén, L., Eriksson, A.R.B., and Schnürer, A., 2007, Effect of process temperature on bacterial and archaeal communities in two methanogenic bioreactors treating organic household waste. FEMS Microbiol. Ecol. 59(3): 683-693. doi: 10.1111/j.15746941.2006.00263.x.

Sinclair, L., Osman O.A., Bertilsson, S., Eiler, A., 2015, Microbial community composition and diversity via 16S rRNA gene amplicons: Evaluating the illumina platform. PLoS ONE. 10(2): e0116955. doi: 10.1371/journal.pone.0116955

St-Pierre, B., Wright, A.D.G., 2013, Metagenomic analysis of methanogen populations in three full- scale mesophilic anaerobic manure digesters operated on dairy farms in Vermont, USA. Bioresour. Technol., 138, 277–284.

[bookmark: bau010][bookmark: bau015][bookmark: bau020][bookmark: bau025]Treu, L., .Kougias, P. G., Campanaro, S., Bassani, I., Angelidaki I., 2016, Deeper insight into the structure of the anaerobic digestion microbial community; the biogas microbiome database is expanded with 157 new genomes. Bioresource Technology, 216, 260-266. doi: https://doi.org/10.1016/j.biortech.2016.05.081

Vanwonterghem, I., Jensen, P.D., Ho, D.P., Batstone, D.J., and Tyson, G.W., 2014, Linking microbial community structure, interactions and function in anaerobic digesters using new molecular techniques. Curr. Opin. Biotechnol. 27(0): 55-64. doi: http://dx.doi.org/10.1016/j.copbio.2013.11.004.

Weiland, P., 2010. Biogas production: Current state and perspectives. Appl. Microbiol. Biotechnol. 85(4): 849-860. doi: 10.1007/s00253-009-2246-7.

Zhou, M., McAllister, T.A., Guan, L.L., 2011, Molecular identification of rumen methanogen: Technologies, advances and prospects. Anim. Feed Sci. Technol., 166–167, 76–86.

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