Microbial Community Analysis of the University of Wisconsin Oshkosh Dry Biodigester

UNIVERSITY OF WISCONSIN SYSTEM SOLID WASTE RESEARCH PROGRAM Student Project Report

May 2015

Student Investigator: Jessi Zimmerman Advisor: Dr. Eric Matson

University of Wisconsin-Oshkosh

Abstract

In natural environments, heterotrophic bacteria and methanogenic Archaea assist in the degradation of organic waste, which results in the production of a mixture of carbon dioxide and methane gas, together known as biogas. In the UW-Oshkosh dry anaerobic biodigester, these same naturally occurring microbial processes are utilized to benefit the University and surrounding community. Organic waste produced by local farms, grocery stores, and residential households are collected and delivered to the biodigester facility rather than being added to landfills. These waste products serve as the feedstock for biogas production. The combustible biogas is captured and used as fuel for a combined heat and power generator, which meets about 10% of the campus electricity demand and helps to heat the digester and other nearby campus facilities. The process reduces the University’s operating costs while diverting some forms of municipal waste away from landfills and provides a cleaner alternative to fossil fuels.

The composition of the incoming feedstock is closely monitored as are the physicochemical conditions such as temperature, pH and hydration. These are measured at the beginning and throughout the cycle, because they can influence methane output. What is not measured (and not currently known) is how the growth of microbial species changes throughout the course of the digestion cycle. In particular, changes that may occur in methanogenic species richness and relative abundance are likely to influence biogas quality and output. Therefore, the aims of this study were to 1) extract and purify DNA samples suitable for genomic and metagenomic analysis from the microbial community at time points that span the duration of a digestion cycle and 2) measure community dynamics in some of these samples using high-throughput next- generation sequencing (NGS). Biodigester samples collected and sequenced from two digester bays showed that methanogenic Archaea populations change markedly over time and differ between separate digestion cycles. Through understanding how these populations are changing, this research may help to identify conditions that could stimulate rapid growth and

prolonged maintenance of methanogen populations and, thus, improve the efficiency of

methane production in the biodigester.

Statement of Objectives

The primary objective of this project was to obtain high-quality microbial community DNA samples from two fermentation chambers of the UW-Oshkosh biodigester over the course of a

28-day cycle. These samples will allow researchers to examine several aspects of the Archaeal

communities - now and in years to come. The sample collection will be used for species-based

assessments of community structure, such as in the current study and functional gene-based

studies in future research.

The secondary objective was to use universal PCR primers to amplify Archaeal SSU rRNA

genes from the microbial community DNA samples and to sequence the resulting amplicons.

The SSU rRNA sequencing data provides species identity data as well as frequency of

occurrence throughout the biodigester cycle. Analyses that reveal structural changes to the

community over the course of a cycle and between digester cycles will be compared to data on

physicochemical conditions in the biodigester bays. Together, these comparisons will show

how the microbial community may be responding to conditions within the digester throughout

the cycle.

Introduction

The biodigester on the UW-Oshkosh campus (Fig. 1) is the only commercial dry biodigester in

the Western hemisphere and has been in operation since the fall of 2011 (Innovations in

Sustainability and Renewable Energy, 2014). Because it is a “dry” biodigester, the input

material used has a moisture content of 75% or less (BIOFerm Energy Systems, 2011). The majority of the input consists of organic materials such as food, agriculture and yard waste from

3 campus, local community sources and additional area partners (Innovations in Sustainability and Renewable Energy, 2014).

Figure 1. UW-Oshkosh biodigester facility located at 755 Dempsey Trail, Oshkosh, WI.

The organic waste is loaded into individual fermentation chambers (Fig. 2) and liquid percolate, which is rich in microorganisms such as methanogenic Archaea, is sprayed over the feedstock to assist in decomposition and methane production (BIOFerm Energy Systems, 2011). The organic waste is broken down throughout a series of steps, collectively known as anaerobic digestion (AD), which results in the production of carbon dioxide and methane, or biogas. The biogas is captured and combusted to produce heat and electricity. This environmentally beneficial process provides clean, renewable energy while reducing and reusing organic wastes. The amount of solid waste generated is reduced by 40% at the end of the 28-day cycle.

Figure 2. The UW-Oshkosh anaerobic digester has four separate bays that operate continuously on staggered 28 day cycles. Arrows indicate the location of floor drains for fermenter bays 1 (left) and 2 (right).

The left over solid waste, known as digestate, is low in odor and rich in nutrients and serves two

purposes: approximately half of the digestate is reserved and mixed in with the incoming fresh

feedstock as a source of inoculum, while the remaining half is further composted off-site and

can be later used as fertilizer or a soil supplement. This increases the lifespan of landfills and

capacity of compost sites, while decreasing the energy consumption and costs involved with

moving waste (Innovation in Sustainability and Renewable Energy, 2014). Each week, one of

the four digester bays starts a new 28-day cycle, such that the facility on the whole is

maintained in a state of continuous operation. Once the mixing and loading of feedstock is

complete, the airtight fermenter doors close and the chamber air is evacuated. Aerobic bacteria

consume residual oxygen within the chamber, creating an anaerobic environment and thus

starting the AD process.

Anaerobic digestion is a series of biological processes in which microorganisms decompose

organic material in the absence of oxygen. The process involves four steps: hydrolysis,

acidogenesis, acetogenesis and methanogenesis, and is carried out by communities of

hydrolyzing, acid-producing and acetate-producing bacteria and methane-producing Archaea

(Fig. 3). During hydrolysis, hydrolytic bacteria convert complex insoluble polymers into simpler,

soluble monomers, making them available for other bacteria. For instance, carbohydrates are

converted to simple sugars, lipids to fatty acids and proteins to amino acids. In the next step,

acidogenesis, fermentative bacteria convert the soluble organic monomers into volatile fatty

acids, ketones, ethanol, hydrogen and carbon dioxide. Acetic acid, carbon dioxide and

hydrogen created at this step skip acetogenesis (Serna, 2009), as they can be used directly by hydrogen-consuming (hydrogenotrophic) methanogens to produce methane. The majority of methanogens are hydrogenotrophs (Sarmiento, et al., 2011), which use hydrogen, and

sometimes formate and carbon monoxide, as the electron donors to drive the reduction of

carbon dioxide (Ferry, 2010). During acetogenesis, hydrogen-producing acetogenic bacteria

convert volatile fatty acids and ethanol into acetic acid, carbon dioxide and hydrogen. The

majority of the methane (approximately 72%) is produced by hydrogen-producing aceticlastic

methanogens, which produce methane and carbon dioxide via the decarboxylation of acetate.

Most of the balance is produced by hydrogenotrophic methanogens (Kanhal, 2009) though

small amounts of methane are produced by the conversion of methylotrophic substrates, such

as methanol, methylamines and methyl sulfides as well (Ferry, 2010).

1) Hydrolysis (fermentative bacteria)

2) Acidogenesis (fermentative bacteria)

3) Acetogenesis (acetogenic bacteria)

4) Methanogenesis Hydrogenotrophic Acetoclastic (methanogenic Archaea)

Figure 3. Anaerobic digestion process. Microorganisms decompose organic material through a series of biological processes in the absence of oxygen, resulting in the production of biogas.

Biogas typically consists of 60-70% methane, 30-40% carbon dioxide (EPA, 2014), and trace amounts of other gases, such as hydrogen, nitrogen, carbon monoxide, oxygen and hydrogen

sulfide. This gas is collected, treated to remove impurities, such as hydrogen sulfide, and used

to generate energy, providing a cleaner alternative to traditional fossil fuels (BIOferm, 2011).

Materials and Methods

Sample collection. Being that the biodigester is a closed system, one cannot enter to collect samples. However, the percolate that flows through the biomass is accessible via floor drains in

front of the individual chambers. The floor drains allow access to microorganisms that are

washed out with the percolate from each fermenter and should provide an estimate of the

microorganisms within the biomass piles. These floor drains, immediately adjacent to the

fermenter bays, were used to individually sample bay 1 and bay 2 (F1 and F2) over the duration

of a 28-day cycle (Fig. 2 and Fig. 4a). Samples of percolate were also collected from the

percolate holding tank, which is a large, 120,000 gallon reservoir where the percolate from all of

the fermenters is mixed together and reused. These samples were collected from the

“percolate out” drain (Fig. 4b) in the pump room and provided a baseline for the liquid being

delivered to each of the four fermenter bays at their various stages in the digestion cycle.

a) b)

Figure 4. Sample collection points. a) Fermenter samples from floor drain and b) drain percolate from percolate out drain.

Percolate from the two fermenters and the percolate out drain was collected throughout the 28-

day cycle, daily at the beginning of the cycle and then every other day. Samples measuring

approximately 50 ml were collected from Fementer 1 from May 8 - June 2 (16 samples) and

from Fermenter 2 from May 15 - June 10 (16 samples). Each time a sample was retrieved from

a fermenter, a sample was also taken from the percolate out drain (18 samples from May 8 -

June 10). This resulted in a total of 49 separate samples. Triplicates of these samples were

made, resulting in a total of 147 samples (Table A1 in appendix).

Sample processing. Immediately after collection, the 50 ml samples were mixed and three 1.5

ml aliquots were placed in microcentrifuge tubes and further processed. These samples were

centrifuged for 10 minutes at 13,523 x g to collect cells and insoluble debris. The cell-free

supernatant was carefully discarded by decanting the liquid. The remaining solids, which

included the microbial cell fraction, were re-suspended in a buffer containing Tris base and

Ethylenediaminetetraacetic acid (TE buffer) to help inactivate DNAse enzymes that can degrade

nucleic acid. The samples were then frozen at -80°C (see Sample Collection and Processing

Protocol in appendix for expanded methods).

High quality microbial DNA samples were isolated from the 49 samples using the MO-BIO

PowerSoil DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA). This kit was chosen because it optimizes cell lysis in Gram negative and positive bacteria and Archaea; removes

PCR inhibitors, such as humic acids; and has a rapid protocol that enables prompt isolation of

high quality, pure DNA. A randomly selected subset of the 49 extracted samples was selected

and verified as being amplifiable via PCR using universal primers for Archaeal SSU rRNA genes

as a means of quality assurance prior to NG sequencing.

Sample analysis. Ten high quality microbial DNA samples were selected for further analysis.

Days 2, 3, 4, and 6 in the cycle (see Table A1 in appendix) were chosen as these days led up to

peak methane output (Fig. 5) in each of the fermenter bays. In addition, two percolate samples

were sequenced using samples collected on May 15th, 2014 (corresponding to Day 9 in the F1

9 cycle and Day 2 in the F2) and May19th, 2014 (corresponding to Day 13 in F1 cycle and Day 6 in F2).

The ten samples were sent to MR DNA Lab (Shallowater, TX) for SSU rRNA amplification and next generation high-throughput sequencing. The V4 variable region of the 16S rRNA gene was amplified via PCR using barcoded PCR primers 515F/806R, which are nearly universal for both Archaea and bacteria (Walters, 2011). The PCR program consisted of an initial denaturation step (94°C for 3 min) followed by 28 cycles of denaturation (94°C for 30 seconds), annealing (53°C for 40 seconds), and extension (72°C for 1 minute) and a final extension step

(72°C for 5 minute). Following amplification, the PCR products, or amplicons, were checked using agarose gel electrophoresis to determine the success of amplification and relative intensity of bands. Multiple samples were then pooled together based on their molecular weight and DNA concentration and purified using calibrated Ampure XP beads. The pooled and purified PCR product was used to prepare a DNA library for sequencing. MR DNA performed the sequencing using an Illumina MiSeq sequencer according to the manufacturer’s guidelines and processed the sequence data using a proprietary analysis pipeline. Operational taxonomic units (OTUs) were assigned to the most relevant taxonomic level based upon the percent of similarity to reference sequences in BLASTn. Sequences with greater than 97% similarity were classified to a species level, while sequences between 97% and 95% were classified to a genus level according to the MR DNA protocol.

In a second round of sequencing, additional F1 and F2 samples from the time period following the peak in methane production have been sent to MR DNA and are awaiting sequencing. This includes Days 8, 10, 15 and 27 in F1 and Days 8, 10, 16 and 26 in F2 (Table A1 in appendix).

Results

The sequence reads were assigned to 22 species-level OTUs within the domain Archaea (Table

A2 in appendix). OTUs that did not contribute to at least 2.5% of the population in at least one

of the samples were not included in this analysis, which excluded 16 OTUs. The six remaining

OTUs used for data analysis in this study included: Methanomicrobium spp., Methanobacterium

formicicum, Methanobrevibacter spp., Methanoculleus bourgensis, Methanosarcina spp. and

Thermoplasma spp. (Table A3 in appendix).

The sequencing data contained both “counts” and “percentage” files. The counts files contain

the actual number of sequences that were found for each species. This indicates the number of

times a particular sequence was recognized, or how many “hits” the sequence received.

Though this does not give an exact measurement of the number of cells per gram of material in

the fermenter feedstock, it can provide an indication of the relative species abundance (e.g.

increases in hits correlate with increases in the number of particular types of cells per unit of

material). The percentage files reveal what relative percentage of the sample maps to a

designated taxonomic classification. This is an indication of relative abundance, or how

common a species is relative to other species in the defined sample. In this case, it describes

the number of a particular species of Archaea as a percentage of the total Archaea population

present in the sample.

Sampling Days 2 and 6 were chosen for OTU comparisons because both fermenters were

sampled on those days. In addition, Day 2 was at the beginning of the cycle when methane

production was just beginning and Day 6 was at or near the methane production peak (Fig. 5).

While there is some variation between the two percolate samples, collected on May 15th, 2014 and May 19th, 2014, the relative abundance of the Archaeal species remains relatively steady

over these time points (Fig. 6). Methanoculleus bourgensis made up the largest portion of the

population in sample 1 and sample 2 (49.1% and 33.1%, respectively). Methanomicrobium spp. was approximately 20% of the population in both percolate samples (18.9% in sample 1 and

21.0% in sample 2). Methanosarcina spp. made up a similar proportion of the population with

21.7% in sample 1 and 25.8% in sample 2. Methanobacterium formicicum and

Methanobrevibacter spp. showed slight differences between the two percolate samples.

Methanobacterium formicicum was 1.8% of the population in sample 1 and 7.3% in sample 2.

Methanobrevibacter spp. made up 2.1% of the population in sample 1 and 6.2% in sample 2.

In Fermenter 1, the relative abundance (Fig.7) of Methanomicrobium spp. rose from 18.2% to

45.6% from Day 2 to Day 6. Methanoculleus bourgensis and Methanosarcina spp. decreased in

abundance (34.4% to 29.3% and 23.1% to 17.8%, respectively). Methanobacterium formicicum

also decrease from 7.4 % to 0.6%, while Methanobrevibacter spp. remained fairly stable (3.7%

to 2.5%). Thermoplasma spp. dropped in relative abundance (5.4% to 3.4%).

In Fermenter 2, the relative abundance (Fig. 7) of Methanomicrobium spp. rose from 13.0% to

42.6% from Day 2 to Day 6, while Methanobrevibacter spp. and Methanobacterium formicicum

decreased in abundance (16.8% to 1.6% and 25.2% to 1.9%, respectively). Methanosarcina

spp. remained fairly stable (9.9% to 11.3%), as did Methanoculleus bourgensis (32.1% to

33.9%). In F2, the relative abundance of Thermoplasma spp. rose from 0.8% to 7.8%.

The relative abundance of Methanomicrobium spp. increased daily as the cycle approached

peak methane output in F1; however it peaked at Day 4 in F2. (Fig. 8). The Methanoculleus

bourgensis population rose from Day 2 to 3 and then remained fairly stable in both fermenters.

Methanobacterium formicicum decreased in abundance in both of the fermenters although the

decrease is more prevalent in Fermenter 2 (Fig. 9). In both fermenters, the relative abundance

of Methanobrevibacter spp. rose daily until reaching Day 4, and then dropped. Methanosarcina

spp. increased in abundance in Fermenter 1 and remained fairly stable in Fermenter 2. The

abundance of Thermoplasma spp. slowly rose daily as the cycle approached peak methane

output in F2; however, it peaked in relative abundance at Day 4 in F1. Overall, the total amount of Archaea relative to other microbes present in the samples increased from Day 2 to Day 6, indicting the growth of methanogen populations.

At the biodigester facility, the feedstock composition, temperature, pH and methane production

are closely monitored. Feedstock data is recorded at the beginning and end of each cycle and

data on physicochemical conditions are recorded throughout the cycle.

Temperature. Temperature data for Fermenter 1 is unavailable, as the temperature probe

reported erroneous readings. In Fermenter 2, the temperature of the biomass ranged from

44.2-48.6°C (Fig. 10) throughout the cycle, other than the first and last days of the cycle where

the temperature drops to 36.7°C and 36.2°C, respectively, when the chamber doors were open.

The average temperature of the biomass was 44.1°C. The heated floor of both the chambers

ranged in temperature from approximately 40°C to 44°C and had an average temperature of

42°C.

pH. The pH levels (Fig. 11) dropped early in the cycle (day 3) in both fermenters though

Fermenter 1 did not drop as low as Fermenter 2 (7.22 versus 6.53). The drop in pH may

indicate the acidogenesis stage of anaerobic digestion. Following Day 3, the pH levels in both

fermenters rose until around Day 8 and then leveled off between pH 7.8 and 8.1. Samples of

the drain percolate were checked for pH following collection and levels stayed fairly consistent

throughout the cycle, the lowest value recorded being pH 7.92 and highest pH 8.03.

Composition of Feedstock. The overall composition of the feedstock of the two fermenters

was similar in nature in both actual weights and the percentage of the feedstock each substrate

made up (Table 1a and b). Both fermenters contained digestate from a former batch, bedding

waste from area farms, food waste and yard waste; however, Fermenter 2 received a larger

load of feedstock (400.89 tons versus 344.75 tons). Approximately 200 tons of digestate were added to both digesters. The bedding waste added to each fermenter was 87.2 tons in F1 and

81.81 tons in F2. Sanimax provided 53.07 tons of food waste to F1 and 55.37 tons to F2. Only

1.78 tons of yard waste were added to F1, while 49.52 tons were added to F2, which accounts

for the majority of the difference in weight. The City of Madison contributed an additional 18.53

tons of curbside organics and Advanced Disposal provided 4.31 tons of sawdust to F2 as well.

Total solids (TS) and volatile solids (VS) values provide information on incoming feedstock. The

total solids (TS) and volatile solids (VS) levels are measurements of moisture content and

combustibility, respectively. The total solid and volatile solid values were similar, in regard to

the digestate, bedding waste, food waste and yard waste; however, there were additional data

for F2 (curbside organics and saw dust). The totals for TS and VS were higher in F2.

Methane Production. Overall, Fermenter 2 produced more methane (399,348.9 ft3 /cycle

compared to 531,319.4 ft3/cycle). More feedstock would naturally allow for higher amounts of

biogas production so methane production levels were adjusted to account for the inconsistency

in feedstock amounts. The amount of methane produced (ft3/day) was divided by the amount of

feedstock (tons) so values reflect the amount of methane produced per ton of feedstock each

day (Fig. 5). The amount of methane produced peaked at Day 5 in F1 and Day 6 in F2 with

51.5 and 48.5 ft3 of methane produced per ton of feedstock per day, respectively. After the

methane peak in both fermenters, the amount of methane produced slowly decreased as the

cycle continued. The total amount of methane produced per ton of feedstock was higher in F1

than F2 (1541.2 versus 996.2 ft3/ton over the 28 day cycle).

Figure 5. Methane produced by Fermenters 1 and 2. The amount of methane produced (ft3/day) has been divided by the amount of feedstock (tons) to show the amount of methane produced per ton of feedstock each day.

Percolate Samples

Sample 1 Methanomicrobium spp. 18.9%

Methanobacterium formicicum 1.8%

Methanobrevibacter spp. 2.1%

Methanoculleus bourgensis 49.1%

Methanosarcina spp. 21.7%

Thermoplasma spp. 3.4%

Other 3.1%

Sample 2 Methanomicrobium spp. 21% Methanobacterium formicicum 7.3% Methanobrevibacter spp. 6.2% Methanoculleus bourgensis 33.1% Methanosarcina spp. 25.8% Thermoplasma spp. 2.7% Other 3.9%

Figure 6. Relative abundance of Archaea present in drain percolate samples. The relative abundance describes the number of a particular species of Archaea as a percentage of the total Archaea population present in the sample. 15

Fermenter Samples Fermenter 1 – Day 2

Methanomicrobium spp. 18.2%

Methanobacterium formicicum 7.4%

Methanobrevibacter spp. 3.7%

Methanoculleus bourgensis 34.4%

Methanosarcina spp. 23.1%

Thermoplasma spp. 5.4%

Other 7.9%

Fermenter 1 – Day 6 Methanomicrobium spp. 45.6% Methanobacterium formicicum 0.6%

Methanobrevibacter spp. 2.5% Methanoculleus bourgensis 29.3% Methanosarcina spp. 17.8% Thermoplasma spp. 3.4% Other 0.8%

Fermenter 2 – Day 2 Methanomicrobium spp. 13%

Methanobacterium formicicum 25.2%

Methanobrevibacter spp. 16.8%

Methanoculleus bourgensis 32.1%

Methanosarcina spp. 9.9%

Thermoplasma spp. 0.8%

Other 2.3%

Fermenter 2 – Day 6 Methanomicrobium spp. 42.6%

Methanobacterium formicicum 1.9%

Methanobrevibacter spp. 1.6%

Methanoculleus bourgensis 33.9%

Methanosarcina spp. 11.3%

Thermoplasma spp. 7.8%

Other 0.9%

Figure 7. Relative abundance of Archaea present in fermenter samples. The relative abundance describes the number of a particular16 species of Archaea as a percentage of the total Archaea population present in the sample.

Figure 8. Changes in the population of Archaea present in F1. The number of sequence hits indicates the number of times a particular sequence was recognized during sequencing.

Figure 9. Changes in the population of Archaea present in F2. The number of sequence hits indicates the number of times a particular sequence was recognized during sequencing.

Figure 10. A temperature probe within the biomass pile provides biomass temperatures measurements throughout biodigester cycle. *Temperature data for Fermenter 1 not available due to malfunction of temperature probe.

Figure 11. Percolate samples from Fermenters 1 and 2 and the percolate out drain were used to show changes in pH levels throughout the biodigester cycle.

Table 1a. Composition of feedstock loaded into Fermenter 1.

Actual Weight Actual % Total Solid Volatile Solid Location Feedstock (US Tons) Weight (TS) % (VS) % UWO Biodigester Solid Digestate 200.4 58.13 23.12 51.99 Omro Dairy Bedding Waste 9.54 2.77 46.92 76.87 Allen Farms Bedding Waste 77.66 22.53 27.23 83.58

Sanimax Food Waste 55.37 16.06 11.9 92.03

County of Oshkosh Yard Waste 1.78 0.52 37.27 79.64 Total 344.75 100.01 22.98 66.37

Table 1b. Composition of feedstock loaded into Fermenter 2.

Actual Actual Weight Total Solid Volatile Solid Location Feedstock Weight (US Tons) (TS) % (VS) % % UWO Biodigester Solid Digestate 193.65 48.31 24 55 Omro Dairy Bedding Waste 8.36 2.09 51.94 80.16 Allen Farms Bedding Waste 73.45 18.32 25.8 89.36 Sanimax Food Waste 53.07 13.24 16.08 93.56 City of Oshkosh Yard Waste 49.52 12.35 37.27 79.64 City of Madison Curbside Organics 18.53 4.62 68.53 82.66 Advanced Disposal Saw Dust 4.31 1.08 94.68 95.2 Total 400.89 100.01 28.32 71.68

Total solids (TS) represents the amount of solid material (or dry matter) remaining after moisture is removed and is expressed as a percentage of the as-received or wet weight of a sample. Volatile solids (VS) refers to the amount of combustible material in a sample.

Discussion

Archaea indentified. Archaea are a diverse group of organisms that are capable of living in a

wide range of environments, some of them extreme. All of the Archaea studied belong to the

phylum Euryarchaeota which includes methanogens and halophiles. The samples can be further classified taxonomically down to a species level (Fig. A1 in appendix). In the past, differentiating characteristics such as cell morphology, cell wall composition and Gram reaction helped in classifying Archaea. Today, 16S rRNA sequencing provides data on how species are related (Balch, et al., 1979).

Of the six Archaea, used for analysis, five of them (Methanomicrobium spp., Methanoculleus

bourgensis, Methanobacterium formicicum, Methanobrevibacter spp., and Methanosarcina spp.)

were methanogens (Table A3 in appendix). Methanogens are strict anaerobes that produce

methane through three different pathways: hydrogenotrophic, aceticlastic and, less commonly,

methylotrophic. All of the above methanogens are capable of using the hydrogenotrophic

pathway, where carbon dioxide is reduced to methane using electrons derived from hydrogen,

formate or carbon monoxide. However, only Methanosarcina spp. is capable of also using

aceticlastic and methylotrophic pathways, where methane is produced via the decarboxylation

of acetate and the conversion of methylotrophic substrates such as methanol, methylamines

and methyl sulfides, respectively (Ferry, 2010). Methanobacterium formicicum, Methanoculleus

bourgensis, Methanomicrobium spp. and Methanosarcina spp. are commonly found in sewer

sludge and anaerobic digesters. Methanomicrobium spp. and Methanosarcina spp, are also

found in the GI tract or feces of mammals and in marine sediment. Methanobrevibacter spp. is

found in the GI tract or feces of mammals as well. All of these methanogens have optimal

growth temperatures within the mesophillic range and have a pH optima near neutral.

Thermoplasma spp., on the other hand, thrives in hot, acidic environments (55-60°C, pH 1-2). It

is a facultative anaerobe that respires using sulfur and organic carbon and is often found in

sulfur-producing environments, such as solfataras (sulfur-releasing volcanic steam vents) and

heat-generating coal refuse sites (Rodgers, 2015).

Various changes in the relative abundance and the amount of individual species present were

seen between Days 2 and 6 in the biodigester cycle. An overall rise in the population of total

Archaea was also noted. A change in methanogenic communities was expected early in the

cycle as conditions within the digester bays became permissive for methanogenic growth. At

the beginning of the cycle, the fermenter still contains oxygen, so the amount of anaerobic

methanogens would be expected to be low. With the formation of the anaerobic environment and substrates produced by the hydrolyzing, acid-producing and acetate-producing bacteria, a rise in the number of methanogens would be expected. It is expected that the development of the heterotrophic bacterial communities responsible for some of the physicochemical conditioning within the biodigester influence methanogenic community development, since

bacteria play a role in the production of substrates necessary for the methanogen metabolism.

Two dominant species (Methanomicrobium spp. and Methanoculleus bourgensis,) were present

at the peak of methane production in both fermenters, which may indicate their importance in

methanogenesis. The one aceticlastic methanogen (Methanosarcina spp.) did not change

appreciably from the beginning of the cycle to peak methane production. This was unexpected,

as past research has indicated the majority of methane production occurs via the aceticlastic

pathway.

Though data are limited, research indicates that methanogen load and diversity are dependent

on feedstock characteristics and biodigester conditions (Traversi, et al., 2011). There are

several factors that can affect the operation of anaerobic digesters, such as, temperature, pH

and composition and hydration of feedstock. Each of these factors must be taken into account

to ensure the biodigester runs efficiently.

Temperature. Psychrophilic biodigesters (5°-25°C) produce methane at slower rates than their higher temperature counterparts, as methanogenesis is greatly limited when the surrounding temperature drops below 15°C. Mesophilic biodigesters, such as the UW-Oshkosh digester, are the most common type of biodigester. This temperature range (~30°-40°C), balances the cost of maintaining the temperature with methane production. Thermophilic (50°-60°C) biodigesters are capable of producing the most methane, but are most sensitive to environmental changes.

Many Archaea can survive these higher temperatures, however, there are fewer bacterial species capable of growth in this temperature range (Sahu, O. & Abatneh, Y, 2013). In the case of the UW-Oshkosh biodigester, heated floors help to maintain mesophillic conditions within the fermenter bays, which facilitates faster substrate decomposition. The majority of methanogens, including those involved in this study, thrive in mesophillic temperatures (Sarmiento, et al.,

2011). The temperature of the biomass remains fairly consistent throughout the biodigester cycle. Temperature data for Fermenter 1 is unavailable, as the temperature probe reported erroneous readings. The temperature probe is placed within the mass of feedstock, which tends to move as the material decays. If the mass moves too much, the wires can be pulled from the probe, resulting in erroneous reads (Johnson, 2014). In Fermenter 2, the temperature ranged from 44.22-48.61°C throughout the cycle, other than the first and last days of the cycle where the temperature drops to 36.67°C and 36.17°C, respectively, when the chamber doors were open (Fig. 10). The average temperature of the biomass was 44.11°C. The heated floor of both the chambers ranged in temperature from approximately 40°C to 44°C and had an average temperature of 42°C. All five of the methanogens in this study have optimal growth temperatures within the mesophillic range (Balch, et al., 1979) Thermoplasma spp. prefers thermophillic temperatures (55-60°C), but it is capable of growth at mesophillic temperatures as well (Bergy’s Manual of Determinative Bacteriology, 1994).

22 pH. Though some methanogens can live in extreme pH environments, the optimum pH conditions for the majority of methanogens remains close to neutral (pH 6.5 to 7.6). The optimal pH range for an anaerobic biodigester is 6.8-7.5 (Sahu, O. & Abatneh, Y., 2013). At this pH, methane producing activity eliminates inhibitory byproducts that can impede the AD process.

Feedstock composition can have an effect on pH levels with the biodigester. Woody substances, which are high in lignin, should not be used in a biodigester as they can result in the buildup of acid, inhibiting methanogenic growth and methanogenesis. On the other hand, organic material that is too rich in proteins or ammonia may cause the pH to rise above 7.5, negatively affecting biogas production. The pH levels in both fermenters dropped around Day 3, although F1 did not drop as low as F2 (7.22 versus 6.53). The drop in pH early in the cycle may be an indication of acidogenesis, as the production of acids would lead to a drop in pH. The variance in the amount the pH levels dropped may be due to the difference in feedstock between the two bays. Fermenter 2 received more feedstock, in particular, larger amounts of yard wastes and curbside organics, which may contain substances that lead to additional acid production. The methanogens studied have a pH optima near neutral. Thermoplasma spp. prefers acidic environments (pH 1-2) (Rodgers, 2015) and is only capable of growth between pH

1-4 (Bergy’s Manual of Determinate Bacteriology, 1994). The population of Thermoplasma spp. within the biodigester appears to slowly rise over the first 6 days in F2. In F1, the population of rises over the first 4 days and then drops at Day 6. The rise in population may indicate that

Thermoplasma spp. is capable of surviving in the biodigester. Perhaps an explanation for this observation is that there exist areas within the biodigester that have a lower pH than indicated by the percolate that flows through the pile.

Composition of Feedstock. There are many sources of organic material used in the biodigester including agricultural bedding waste, food waste, yard waste and curbside organics.

Food products have a higher energy potential than many other substrates, meaning they

23 produce more methane. One might consider loading the biodigester with more food waste.

However, a bedding material, such as straw, is necessary to provide structure to the pile. In addition, hydrolysis of food waste results in higher levels of acetic acid, which lowers the pH.

While acetic acid is necessary for the production of methane in the aceticlastic pathway, the lower pH adversely affects the growth of many methanogens in this environment, which do not typically grow well in levels much lower than neutral (Table A3 in appendix). If the feedstock contains organic material high in lignin or too many food products, the hydrolyzing and acid- producing bacteria might outgrow the methanogens. Excess acid is produced, causing a drop in pH. If the pH drops below 6.8, methanogen growth will be inhibited. On the other hand, biodigesting organic material too rich in proteins or ammonia may cause the pH to rise above

7.5, negatively affecting biogas production. The overall composition of the feedstock of the two fermenters was similar in nature (Table 1a and b); however, Fermenter 2 received a larger batch of feedstock (344.75 tons versus 400.89 tons). The larger amount of feedstock is most likely responsible for the increased amount of methane produced in F2. The majority of the difference in feedstock weight was additional yard waste (49.52 tons versus 1.78 tons). F2 also received an additional 18.53 tons of curbside organics and 4.31 tons of sawdust. The feedstock composition may have an effect on methane efficiency. Lignins are found in the cell walls of vascular plants, especially in wood and bark. Sawdust would contain lignins and it is possible that the yard waste and curbside organics would as well, which could attribute to the lower drop in pH in F2.

Total solids (TS) and volatile solids (VS) data may be used to describe incoming feedstock composition and to estimate projected methane gas production totals. Total solids represents the amount of solid material (or dry matter) remaining after moisture is removed and is typically expressed as a percentage of the as-received or wet weight of a sample. Volatile solids refers to the amount of combustible material in a sample. The value is typically reported as a

percentage of the TS. VS values give a rough approximation of the amount of organic matter

present in the feedstock and are used as an indicator of the biodegradability of a material

(Rapport, 2008). The TS and VS totals were higher in F2, which may be due to the additional

curbside organics and saw dust.

It is important to note the time period at which the samples were collected (May 8 – June 10).

The time of the year could impact the biodigester input, as in the spring and summer more lawn

clippings would be available as opposed to late autumn or winter, when in a northern climate,

there are little to no lawn clippings. Repeating this experiment at a different time of the year,

when different input is available, may provide different data.

Methane Production. Overall, Fermenter 2 produced more methane (531,319.4 ft3/cycle

versus 399,348.9 ft3 /cycle). More feedstock would naturally allow for higher amounts of biogas

production so methane production levels were adjusted to account for the inconsistency in

feedstock amounts. The amount of methane produced (ft3/day) was divided by the amount of

feedstock (tons) showing the amount of methane produced per ton of feedstock each day (Fig.

5). Methane produciton peaked earlier in F1 and at a higher level (Day 5 in F1 and Day 6 in F2

with 51.5 and 48.5 ft3 of methane produced per ton of feedstock per day, respectively). The total amount of methane produced per ton of feedstock was also higher in F1 than F2 (1541.2

versus 996.2 ft3/ton over the 28 day cycle). This may indicate that between these two cycles,

F1 produced methane more efficiently; however, ultimately, F2 produced more methane overall.

At the beginning of the biodigester cycle, there is residual oxygen in the fermenters bays, so

large amounts of methane production would not be expected. The aerobic bacteria must

consume residual oxygen in the environment before the growth of strict anaerobes, such as

methanogens, is alllowed. Substrates for the methanogens’ energy requirements must also be

produced by bacteria during the hydrolysis, acidogenesis, and acetogenesis stages of

anaerobic digestion. Although there is some methane production in the first days of the cycle, it appears to take 5-6 days to develop an environment that supports peak methane production.

Future research. Further analysis of these samples and the additional samples sent in for sequencing may reveal potential correlations between existing biodigester data and the

Archaeal communities present. Analysis of the bacteria in the samples may indicate their importance in stimulating methanogenesis as well. Additional research may also show how physicochemical factors affect the microbial community over the course of a cycle and between digester bays. This research may prove beneficial to our long-term goal: to identify conditions that could stimulate rapid growth and prolonged maintenance of methanogen populations and,

as a result, improve the efficiency of methane production in the biodigester.

Appendix

Sample Collection and Processing Protocol

At Biodigester:

1. Mix fermenter sample by moving the collection stick/cup up and down inside floor drain collection area. 2. Transfer approximately 50 ml of sample into sterile 50 ml Falcon tube. 3. Pour any fluid remaining in collection cup down central floor drain in mixing area. 4. Repeat with second fermenter, if necessary. 5. Clear “percolate out” pipe by running approximately 1 L into large plastic container. 6. Collect 100-200 ml in a second large plastic container. 7. Transfer about 50 ml into a 50 ml Falcon tube. 8. Discard left over drain percolate into the central floor drain in mixing area.

At Lab:

1. Label 1.5 ml tubes with date and fermenter (F1 or F2) or drain percolate (P). 2. Invert Falcon tubes to mix sample. 3. Add 1.5 ml of percolate sample to 1.5 ml microcentrifuge tubes (3 for each sample). 4. Centrifuge for 10 minutes at 12,000 rpm. 5. Decant the supernatant. 6. Use P100 pipette to remove additional liquid. 7. Aspirate sample to remove any remaining fluid. 8. Record weights of microcentrifuge tubes in chart. 9. Weigh tubes with pellets and record weight in chart. 10. Add 100 ul of TE. 11. Stir with pipette tip to bring into solution. 12. Vortex samples for about 10 seconds to ensure they are mixed. 13. Place samples in appropriate boxes in -80°C freezer. 14. Reserve 10 ml of each sample in 15 ml Falcon tubes, label and put into chest freezer. 15. Take pH of samples remaining in 50 ml Falcon tube and add values to chart.

Table A1. Sample bank1 of F1, F2 and drain percolate samples over digestion cycle.

Stabilized Cell Location Sampling Date Day in Cycle Purified DNA Fraction F1 5/8/2014 2 1 sample 2 samples F1 5/9/2014 3 1 sample 2 samples F1 5/10/2014 4 1 sample 2 samples F1 5/12/2014 6 1 sample 2 samples F1 5/14/2014 8 1 sample 2 samples F1 5/15/2014 9 1 sample 2 samples F1 5/16/2014 10 1 sample 2 samples F1 5/17/2014 11 1 sample 2 samples F1 5/19/2014 13 1 sample 2 samples F1 5/21/2014 15 1 sample 2 samples F1 5/23/2014 17 1 sample 2 samples F1 5/25/2014 19 1 sample 2 samples F1 5/27/2014 21 1 sample 2 samples F1 5/29/2014 23 1 sample 2 samples F1 5/31/2014 25 1 sample 2 samples F1 6/2/2014 27 1 sample 2 samples F2 5/14/2014 1 1 sample 2 samples F2 5/15/2014 2 1 sample 2 samples F2 5/16/2014 3 1 sample 2 samples F2 5/17/2014 4 1 sample 2 samples F2 5/19/2014 6 1 sample 2 samples F2 5/21/2014 8 1 sample 2 samples F2 5/23/2014 10 1 sample 2 samples F2 5/25/2014 12 1 sample 2 samples F2 5/27/2014 14 1 sample 2 samples F2 5/29/2014 16 1 sample 2 samples F2 5/31/2014 18 1 sample 2 samples F2 6/2/2014 20 1 sample 2 samples F2 6/4/2014 22 1 sample 2 samples F2 6/6/2014 24 1 sample 2 samples F2 6/8/2014 26 1 sample 2 samples F2 6/10/2014 28 1 sample 2 samples Drain Percolate 5/8/2014 NA 1 sample 2 samples Drain Percolate 5/9/2014 NA 1 sample 2 samples Drain Percolate 5/10/2014 NA 1 sample 2 samples Drain Percolate 5/12/2014 NA 1 sample 2 samples Drain Percolate 5/14/2014 NA 1 sample 2 samples Drain Percolate 5/15/2014 NA 1 sample 2 samples Drain Percolate 5/16/2014 NA 1 sample 2 samples Drain Percolate 5/17/2014 NA 1 sample 2 samples

Drain Percolate 5/19/2014 NA 1 sample 2 samples Drain Percolate 5/21/2014 NA 1 sample 2 samples Drain Percolate 5/23/2014 NA 1 sample 2 samples Drain Percolate 5/25/2014 NA 1 sample 2 samples Drain Percolate 5/27/2014 NA 1 sample 2 samples Drain Percolate 5/29/2014 NA 1 sample 2 samples Drain Percolate 5/31/2014 NA 1 sample 2 samples Drain Percolate 6/2/2014 NA 1 sample 2 samples Drain Percolate 6/4/2014 NA 1 sample 2 samples Drain Percolate 6/6/2014 NA 1 sample 2 samples Drain Percolate 6/8/2014 NA 1 sample 2 samples Drain Percolate 6/10/2014 NA 1 sample 2 samples 1 Samples kept frozen at -80°C

Table A2. Archaea operational taxonomic units (OTUs) isolated from biodigester samples.

Archaea Species Candidatus nitrososphaera Candidatus nitrosocaldus spp. Candidatus nitrosopumilus spp. Halobacterium spp. Methanimicrococcus spp. Methanobacterium aarhusense Methanobacterium formicicum Methanobacterium sp. Methanobacterium spp. Methanobrevibacter arboriphilus Methanobrevibacter spp. Methanocorpusculum labreanum Methanoculleus bourgensis Methanoculleus marisnigri Methanoculleus spp. Methanofollis Methanomicrobium spp. Methanosarcina mazei Methanosarcina spp. Methanosphaera spp. Methanothermobacter thermautotrophicus Thermoplasma spp.

OTUs were assigned to the most relevant taxonomic level based upon the percent of similarity to reference sequences in the BLASTn database. Sequences with greater than 97% similarity were classified to a species level, while sequences between 97% and 95% were classified to a genus level.

Table A3. Summary of major features associated with Archaea identified in this study.

Methanogenic Archaea Cell Morphology Optimum Optimum Pathway Where Found Species / Characteristics Temp pH (Substrate Used) Slender, straight to irregularly crooked long rods often 37°C - Methanobacterium Hydrogenotrophic Sewer sludge, occurring in 45°C 6.6 - 6.8 formicicum (H2, formate) digesters filaments Mesophilic (0.7 x 1.5-2.0 µm) Nonmotile Lancet-shaped cocci or short rods which form pairs, Hydrogenotrophic Methanobrevibacter chains, or irregular 37-39°C GI or feces of 6-8* (H2 or H2, spp. clumps Mesophilic mammals formate) (0.5-1.0 µm width) Nonmotile to poorly motile Irregular cocci Methanoculleus 37-40°C Hydrogenotrophic Sewer sludge, (1–2 µm diameter) 6.7 bourgensis Mesophilic (H2, formate) digesters Nonmotile GI tracts of Short straight to animals, marine Methanomicrobium slightly curved rods 38-40°C Hydrogenotrophic sediment, soil, 6.1-6.9 spp. (0.7 by 1.5-2.0 µm) Mesophilic (H2) anaerobic Highly motile sewage digesters Rumen and feces of ungulates and the large Pleomorphic, Hydrogenotrophic, intestine of ranging from 25-55°C* Acetoclastic, humans, Methanosarcina coccoid Mesophilic Methylotrophic 6.5-7.8 freshwater and spp. (0.1-5 µm diameter) to (H2, acetate, marine to filaments thermophilic methanol, sediments, Nonmotile methylamine) decaying leaves, garden soils, oil wells, lagoons, and digesters Hot, acidic environments, Filamentous, sulfur-producing coccoid, disc or environments - club. Irregular form 55-60°C solfataras Thermoplasma spp. 1–2 NA when placed in Themophilic (sulfur-releasing water. (~1 µm) volcanic steam Motile vents) and heat- generating coal refuse sites

Figure A1. Taxonomic groupings of the six most populous Archaea identified in this study.

Kingdom: Archaea Phylum: Euryarchaeota

Class: Methanobacteria

Order: Methanobacteriales

Family: Methanobacteriaceae

Genus/Species: Methanobacterium formicicum

Genus/Species: Methanobrevibacter spp.

Class: Methanomicrobia

Order: Methanomicrobiales

Family: Methanomicrobiaceae

Genus/Species: Methanoculleus bourgensis

Genus/Species: Methanomicrobium spp.

Order: Methanosarcinales

Family: Methanosarcinaceae

Genus/Species: Methanosarcina spp.

Class: Thermoplasmata

Order: Thermoplasmatales

Family: Thermoplasmataceae

Genus/Species: Thermoplasma spp.

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