looking for syntrophic · 4). glass slides were also added to bottles #5 and 6 to promote biofilm...

Looking for syntrophic acetogen/methanogen interactions

Caroline Van Steendam

Final Report

Course Directors: Dr. Jared Leadbetter and dr. Dianne Newman

Microbial Diversity Course 2015

Caroline Van Steendam – Looking for syntrophic acetogen/methanogen interactions

2

1 Introduction Many natural environments (e.g., soil sediments) and engineered systems (e.g., rice paddy soils and

wastewater treatment) are known to produce methane when anaerobically degrading organic matter.

Uncontrolled release of methane in natural environments should be minimized as methane is 23 times

more potent at retaining heat in the atmosphere than CO2. In contrast, methane production is often

maximized in engineered systems because of its high calorific value and therefore its suitability as an

(alternative) energy source. Even though the anaerobic degradation process of organic matter has been

studied in detail for decades, new discoveries are still made.

Fermenter acetogens and methanogens work together to convert intermediate degradation products, i.e.,

volatile fatty acids (VFAs), into methane. The fermentative conversion of VFAs is thermodynamically

unfavorable under standard conditions (1 M concentration, or 105 Pa for gases) and requires a continuous

removal of its reaction products by methanogens to overcome this energy barrier. On top of the well-

known syntrophic interactions including interspecies H2, formate, and acetate transfer, it has recently

been discovered that electrons can also be exchanged directly between the fermenter acetogen and

methanogen (direct interspecies electron transfer, DIET) (Chen et al., 2014; Liu et al., 2012; Nagarajan et

al., 2013; Shrestha et al., 2014; Shrestha et al., 2013; Summers et al., 2010; Zhao et al., 2015).

The goal of this miniproject is to isolate different types of methanogens from Cedar Swamp and to identify

what syntrophic interactions they are capable of. The first stage of the project consists of setting up

different culture enrichments with inoculum from Cedar Swamp, to select for (i) hydrogenotrophic

(utilizing H2 and CO2) and aceticlastic (utilizing acetate) methanogens, (ii) butyrate fermenting acetogens,

and (iii) co-cultures of such acetogens and methanogens. Two bench-scale microbial fuel cells (MFCs),

inoculated with a sample from Cedar Swamp, are set-up in parallel to select for acetogens and

methanogens that are capable of directly exchanging electrons. A potentiostat is used in the final stage of

the project to study the DIET capabilities of an enriched methanogenic culture.

2 Methods

2.1 Anaerobic Culturing A total of seven bottles were inoculated with Cedar Swamp samples, all of which containing the same basic

liquid media (see Appendix A) but varying in gaseous headspace and acetate, butyrate, and bicarb

concentrations (see Figure 1, liquid volume of 25 mL, gaseous volume of 132 mL). Table 1 shows the

reactions related to the different types of metabolism targeted in these bottles (Dong et al., 1994).

Table 1: Different reactions expected in cultures

Aceticlastic methanogenesis

𝐶𝐻3𝐶𝑂𝑂− + 𝐻2𝑂 ↔ 𝐻𝐶𝑂3− + 𝐶𝐻4 Reaction 1

Hydrogenotrophic methanogenesis

𝐶𝑂2 + 4𝐻2 ↔ 𝐶𝐻4 + 2𝐻2𝑂 Reaction 2

Butyrate fermentation (1)

𝐶4𝐻7𝑂2− + 2 𝐻𝐶𝑂3

− ↔ 2𝐶𝐻3𝐶𝑂𝑂− + 𝐻+ + 2 𝐻𝐶𝑂𝑂− Reaction 3

Butyrate fermentation (2)

𝐶4𝐻7𝑂2− + 2𝐻2𝑂 ↔ 2𝐶𝐻3𝐶𝑂𝑂− + 𝐻+ + 2 𝐻2 Reaction 4


3

The first bottle was given a 100% N2 headspace and was supplemented with 2 mM acetate, in order to

enrich for Methanosaeta, an aceticlastic methanogen (Reaction 1). The second bottle also had 100% N2 as

headspace but contained a higher acetate concentration for the more copiotrophic Methanosarcina, an

aceticlastic methanogen often dominating in systems with an acetate concentration above 4 mM (Conklin

et al., 2006). The third and fourth bottle had a headspace of 80% H2 and 20% CO2 to enrich for

hydrogenotrophic methanogens (Reaction 2), while only the third contained 1 mM bicarbonate (in case

some of the methanogens are heterotrophs and need an additional carbon source). A co-culture of a

butyrate degrader and an aceticlastic methanogen was enriched for in the 5th bottle by adding 2 mM

butyrate, 1 mM bicarb, and 0.01 mM acetate to the basic medium while supplying 100% N2 in the

headspace (optimal thermodynamic concentration of acetate, as shown in Figure 2, and the relevant

reactions are Reaction 1 and Reaction 3). The 6th bottle aimed at enriching a co-culture of a butyrate

degrader and a hydrogenotrophic methanogen, by only supplying 2 mM butyrate (Reaction 2 and Reaction

4). Glass slides were also added to bottles #5 and 6 to promote biofilm growth and give the acetogens and

methanogens the possibility to grow closer together. A butyrate fermenter was targeted in the last bottle,

by supplying 2 mM butyrate and a 100% N2 headspace.

Figure 1: Different cultures. 1,2: aceticlastic methanogens, 3, 4: hydrogenotrophic methanogens, 5: coculture of butyrate fermenter / aceticlastic methanogen, 6: coculture butyrate fermenter/hydrogenotrophic methanogen, 7: butyrate fermenter.

2.2 Microbial fuel cells Two MFCs were built using available lab equipment, by connection two glass jars (referred to as chambers

in the rest of the text) with a salt bridge for proton transfer and a copper wire for electron transfer (see

Figure 3). All four chambers were inoculated with Cedar Swamp sample and had a headspace of 100% N2,

however, different electron donors were supplied (total liquid volume of 50 mL). Both acetogenic

chambers were supplemented with 2 mM butyrate, while one methanogenic chamber just consisted of

Figure 2: Input (lefthand side) and output (righthand side) for thermodynamic excel sheet


4

the basic methanogenic medium and the other methanogenic chamber had an additional 0.01 mM of

acetate and 69 mM bicarb. Each chamber contained a submerged graphene rod attached to a wire with a

resistor (470 kΩ) in the middle, allowing for electron transport. The connection of both chambers via a salt

bridge, made by hardening a hot solution containing 2% agar and 1M KCl into a non-gas-permeable tube,

allowed for proton transport between both chambers. Finally, some sampling ports were added and the

chambers were connected to a N2-filled bottle to enhance the ability of the system to cope with pressure

increase.

Figure 3: Set-up of a microbial fuel cell

2.3 Potentiostat Another methanogenic chamber, containing the hyphomicrobium medium (see Appendix B) and a 100%

N2 headspace, was inoculated with a culture enriched in methylotrophic methanosarcina (see Figure 4).

Two graphene rods connected to the working and auxiliary electrode of a potentiostat were submerged

in the chamber, as well as a reference electrode. The potentiostat was set-up in such a manner that the

voltage through the working electrode equaled -0.5V vs standard hydrogen electrode. The chamber was,

just as with the microbial fuel cells, connected to a N2-filled bottle to prevent gas leakage by providing

more flexibility towards increased system pressure.

Figure 4: Set-up of chamber connected to the potentiostat


5

2.4 Chemical analyses The methane production in each bottle was quantified using a gas chromatograph (GC) connected to a

flame ionization detector. A gas-tight syringe was flushed with N2 before it was used to inject 250 µL of

sample into the GC. N2 was used as a carrier gas. A 12.7% methane standard was made by adding 20 ml

of CH4 to a N2-flushed vial of 157 ml, and 17 ml was added to a 69.56 ml vial to obtain a 24.44% standard

Both standards and a blank were run each time samples were analyzed, and all samples were run in

triplicates.

The acetate and butyrate concentrations were measured using a high pressure liquid chromatography

(HPLC) connected to a UV detector. Butyrate standards, ranging from 20, 10, 5, 2.5, to 1.25 mM, were run

once and used for the calibration of all samples. HPLC samples were prepared by centrifuging 900 µL of a

sample together with 100 µL of a 5N H2SO4 solution, and adding 550 µL of the supernatant to a HPLC vial.

2.5 Molecular analyses Two attempts at creating a clone library were made. In the first try the genomic DNA was obtained by

boiling the samples in ALP reagent (in order to take a sample from the liquid cultures, 750 µL of each

sample was centrifuged and the pellet was picked with a toothpick) after which PCR amplification was

performed with a mastermix solution (25 µL of GoTag green, 1 µL fwd primer, 1 µL rev primer 1341R, 20

µL NF H2O, 3 µL of the boiled template) and controlled with a gel extraction (1x TAE, 1% agarose LE, over

120 V). The PCR products were cloned into a plasmid vector (according to the pGEM T-easy cloning

procedure) and then transformed into E. coli (utilizing the transformation procedure) and plated onto LB

plates with ampicillin/IPTG/X-Gal.

The second attempt included using a DNA extraction kit (‘Wizard Genomic DNA Purification Kit’) instead

of performing boiling in an ALP solution. PCR amplification was performed with the same mastermix

solution, after which the PCR products were extracted with a gel.

2.6 Microscopy The available Zeiss microscope was used to visualize liquid mounts with a 100X objective while applying

phase contrast, while the measured excitation when transmitting Lucifer Yellow light was used to identify

methanogens.

The cultured samples were analyzed with CARD-FISH once, in order to identify and/or determine the

relative abundance of the present microorganisms. After fixing these planktonic cells on a filter using a 2%

PFA solution, the microorganisms were embedded with 0.1% agarose, and permeabilized using both a 1

mg/mL lysozyme solution and a fresh 15 µg/mL proteinase K solution. The endogenous peroxidases were

inactivated using a 0.01 M HCl solution (3% H2O2) and the samples were hybridized using a hybridization

buffer of the correct formamide concentration (e.g., probes ARCH 915 requires 35% formamide). The

filters were washed and the final CARD protocol was performed according to the manual (a detailed

rendition of these protocols can be found in Appendix C). The probes ARCH 915, NON338, EUB338 I-III,

MSMX860, and LGC354a were applied to stain Archaea, nothing (negative control), Bacteria,

Methanosarcinales, Firmicutes. The ZEIS microscope was used to visualize the fluorescence.

Mono-FISH was performed afterwards, where the fixed cells are directly hybridized after fixation using the

protocol shown in Appendix D. The ZEIS microscope was used to visualize the fluorescence.


6

3 Results and discussion

3.1 No methane production After setting up the seven cultures, the methane production and substrate removal was monitored using

GC and HPLC. Figure 5 shows the final GC results (the areas outputted by the program are converted to

moles using a standard calibration curves). Although distinct methane peaks can be seen on the GC, these

do not translate to large amounts of methane due to the steep slope of the standard calibration curves.

None of the cultures is, over the entire time period, capable of producing enough methane to surpass the

blank. As the maximum amount of methane that can be produced for, e.g., the 2 mM Acetate culture is

50 mmoles (>> the blank), the absence of methane in the chromatographs is not related to a too high

detection limit of the GC. Even though the outputted methane productions always fall below the employed

calibration curve (12.7 and 24.44% CH4), the expected amount is higher (as can be seen on the right hand

side of Figure 5). The low amounts of methane are thus also not caused by an extrapolation error.

As the low production of methane persisted, the somewhat turbid cultures (i.e., 1mM Ac, 2mM Ac, and

0mM Ac) were re-passaged on the 7th day of the experiment (now called P1 1mM Ac, P1 2mM Ac, and P1

0mM Ac respectively). These cultures showed a similar methane production trend. In a last attempt to

spike the methane production, each culture is re-inoculated with 2ml of the supernatant of Trunk River

sediment sample on the 14th day, again followed by no apparent increase in methane production.

Figure 5: (Left hand side) summary of GC data over entire culturing period, (right hand side) plot of the standard calibration values together with the actual and maximum methane production of the 2mM Acetate methanogenic culture on 8/16/2015

3.2 Variable trends in acetate concentrations and slight butyrate degradation The HPLC data of the methanogenic cultures, as shown in Figure 6.a, shows varying acetate concentration

trends. The 2mM Ac and 10 mM Ac bottles, for instance, show a small decrease in acetate concentration.

The decrease from 10 mM acetate to 8.145 mM acetate should have resulted in the production of 0.0464

moles of methane, which is about two orders of magnitude higher than the measured value (0.00025 mol).

It is not clear what the acetate was converted into, neither the HPLC nor the GC picked up increasing

concentrations of other organics. Based on the low increase in turbidity it can be excluded that the

organics were used for anabolic growth. The P1 1mM Ac and P1 2mM Ac bottles did not show any change

in acetate concentration, while an increase in acetate concentrations is observed in the 1mM Ac, 0mMAc,

and P1 0mM Ac bottles. These bottles were supplied with H2 and CO2 as a headspace, which are both

substrates for homoacetogens (4 𝐻2 + 2 𝐶𝑂2 ↔ 𝐶𝐻3𝐶𝑂𝑂𝐻 + 2 𝐻2𝑂). Even though homoacetogenesis is

0 2 4 6 8 10 12 14 160

1

2

3

4

5

6x 10

-4

Time [days]

Me

tha

ne

Pro

du

ctio

n [m

ol]

Methanogens

Blank

1mM Acetate

P1 1mM Acetate

2 mM Acetate

P1 2mM Acetate

10mM Acetate

0mM Acetate

P1 0mM Acetate

Acetogen

Coculture 0.01 mM Acetate

Coculture 0mM Acetate

10-7

10-6

10-5

10-4

105

106

107

108

109

1010

Amount of Methane [Mol]

Are

a [/]

Standard and Samples on day 16

Standard day 16

2 mMAc day 16

Maximum for 2mMAc


7

thermodynamically more unfavorable than hydrogenotrophic methanogesis, biological reasons can cause

the homoacetogens to dominate (e.g., faster growth). Re-inoculation of the bottles on the 14th day of the

experiment, caused the acetate trend to invert in the P1 0mM Ac bottle and resulted in a decreasing

acetate concentration. Since the experiment was stopped on the 16th day, however, this is just a

preliminary observation.

Figure 6.b shows that, even though the culture remained non-turbid throughout the experiment,

the butyrate concentration did decrease from 2 mM to 1.522 mM while the acetate concentration only

increased with 0.118 mM. The variable formate concentration, combined with the fact that this culture

never produced any gas, illustrates that butyrate was mostly degraded via Reaction 3. Given the low

thermodynamic favorability of butyrate degradation, it would have been better to replenish the medium

daily to keep the concentrations of the reaction products low and that of the reactants high. The addition

of Trunk River inoculum on the 14th day did not result in an extra decrease of butyrate concentration,

although a decreasing formate concentration is observed.

Figure 6.c and d show an initial decrease in butyrate concentration, and again this decrease is not

accompanied by a stoichiometrically correct increase in formate/acetate. The acetate and formate

concentrations, however, are seen to decrease after being accumulated for a couple of days, illustrating

that there are some formate and acetate consumers present that grow slower than the butyrate

degraders. To restore the butyrate concentration in the 0.01mM Ac co-culture and to decrease the amount

of acetate accumulated in the 0mM Ac co-culture, the medium is replenished on the 10th day of the

experiment. Both the replenishment of the medium and the addition of Trunk River inoculum on the 14th

day did not result in an extra decrease of butyrate concentration.

0 2 4 6 8 10 12 14 160

0.0625

0.125

0.1875

0.25

0.3125

0.375

0.4375

0.5

Time [days]

Ace

tate

/fo

rma

te c

on

ce

ntr

atio

n [m

M]

Coculture 0mM Acetate, 2mM Butyrate

0 2 4 6 8 10 12 14 160

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Bu

tyra

te c

on

ce

ntr

atio

n [m

M]

Acetate

Formate

Butyrate

0 2 4 6 8 10 12 14 160

0.0625

0.125

0.1875

0.25

0.3125

0.375

0.4375

0.5

Time [days]

Ace

tate

/fo

rma

te c

on

ce

ntr

atio

n [m

M]

Coculture 0.01mM Acetate, 2mM Butyrate

0 2 4 6 8 10 12 14 160

0.5

1

1.5

2

2.5

3

3.5

4

Bu

tyra

te c

on

ce

ntr

atio

n [m

M]

Acetate

Formate

Butyrate

0 2 4 6 8 10 12 14 160

0.125

0.25

0.375

0.5

0.625

0.75

0.875

1

Time [days]

Ace

tate

/fo

rma

te c

on

ce

ntr

atio

n [m

M]

Acetogen, 2mM Butyrate

0 2 4 6 8 10 12 14 160

0.5

1

1.5

2

2.5

3

3.5

4

Bu

tyra

te c

on

ce

ntr

atio

n [m

M]

Acetate

Formate

Butyrate

0 2 4 6 8 10 12 14 160

1

2

3

4

5

6

7

8

9

10

11

Time [days]

Ace

tate

co

nce

ntr

atio

n [m

M]

Methanogens

1mM Acetate

P1 1mM Acetate

2 mM Acetate

P1 2mM Acetate

10mM Acetate

0mM Acetate

P1 0mM Acetate

a. b.

c. d.

Figure 6: (a) Acetate concentrations in methanogenic cultures over entire culturing period, (b) Acetate and butyrate concentrations in acetogenic culture over entire culturing period, (c) Acetate and butyrate concentrations in co-culture supplemented with 0.01 mM Ac over entire culturing period, and (d) Acetate and butyrate concentrations in co-culture (no acetate) over entire culturing period


8

3.3 No direct electron transfer observed The microbial fuel cell did not produce any biogas nor was there any current/voltage measured along the

copper wires. Fu et al. (2015) noticed enhanced methane production as soon as a culture previously

enriched for DIET capabilities was entered in a potentiostat (-0.5V vs standard hydrogen electrode), with

a 6-fold increase over 160 hours. DIET in a coculture of G. metallireducens and G. sulfurreducens,

furthermore, was stimulated within two days upon addition of a conductive carbon cloth (Chen et al.,

2014). Although these results are promising, the examples started with microorganisms already capable

of DIET which can explain for the difficult start-up of the microbial fuel cells inoculated with a Cedar Swamp

sample. In order to facilitate DIET in the MFCs, it would have been desirable to re-inoculate the MFC-

chambers with a growing acetogenic and methanogenic culture.

A potentiostat consisting of a methanogenic chamber was employed to test the DIET capabilities of a

methanogenic culture. Several groups in the Microbial Diversity Course had been able to grow some

hydrogenotrophic methanogens, while one culture with a methylotrophic Methanosarcina was available.

As it is known that some Methanosarcina strains are capable of DIET (Rotaru et al., 2014), it would be

interesting to see if our specific strain was as well. The methylotrophic Methanosarcina was enriched in a

hyphomicrobium medium (a simple medium containing methanol and nitrate), and its identification was

solely based on microscopy images. Figure 7 shows autofluorescence with Lucifer Yellow fluorescence,

pointing towards the presence of methanogens (the autofluorescent cofactor F420 is a signature for

methanogens, (Solera et al., 2001)). The distinct tetrade structure of these methanogens, furthermore, is

typical for Methanosarcina species (Domingues et al., 2002). And finally, as the culture is growing on

methanol, it is known that there are methylotrophic Methanosarcina present.

In contrast to the set-up of the MFC, methanol was added as an electron donor to the potentiostat in order

to give the Methanosarcina some time to adjust its metabolisms towards favoring of DIET. After growing

for 5 days, no current was observed in the working electrode. It would have been desirable to measure

the methanol concentration over time in the system to see if at least the methanol was being utilized.

However, with the available GC and HPLC set-ups, this was impossible. The absence of current in the

working electrode could be caused by the slow adaptation of the present Methanosarcina species, or the

microorganisms might have been too comfortable with the readily available methanol in the medium.

Furthermore, it was not clear if an anaerobic atmosphere was maintained at all times.

A future revenue for isolating acetogens/methanogens capable of DIET in the future, might be to select

for microorganisms containing the required genetic material (e.g. cytochromes).

Figure 7: Autofluorescence with Lucifer Yellow fluorescence of (?) methylotrophic Methanosarcinas


9

3.4 Identification of microorganisms in cultures The same technique as described above with the methylotrophic Methanosarcina was successfully utilized

to detect the presence of methanogens in the cultures. In addition to normal microscopy, two attempts

were made to create a clone library of the cultures. Both attempts, of which the procedure is explained in

the Methods section, were unsuccessful. The first attempt resulted in no E. Coli growth, whereas the

second did not render any bands when checking the PCR products on a gel. Thirdly, one attempt at utilizing

CARD-FISH to identify and determine the relative abundance of Bacteria, Archaea, Methanosarcinales, and

Firmicutes failed. No fluorescence was discernible in the final result, which was probably due to failing of

the permeabilization step. This step often needs to be optimized for a specific sample, while there was not

enough time to do this at the end of the experiment. A final try at identification was performed with mono-

FISH, this time not with a Methanosarcinales probe as there was no mono-FISH probe available. The

resulting slides showed fluorescence for each of the probes (but not for the none probe), and provided

further confirmation of the presence of Archaea in the growing cultures (see Figure 8). There was not

enough time to study the amount of images necessary for a statistically correct relative abundance

quantification.

Figure 8: A dapi stained (left hand side) and Archaeal probe stained (right hand side) image of a sample of the 0.01 mM Ac co-culture.


10

4 Conclusion The goal of this miniproject was to isolate different types of methanogens from Cedar Swamp and to

identify what syntrophic interactions they are capable of. Isolation of methanogens proved to be difficult

(none of the ‘enriched’ cultures produced methane), although mono-FISH confirmed the presence of

Archaea in the bottles while microscopic analysis with Lucifer Yellow autofluorescence confirmed the

presence of methanogens. The addition of new inoculum from Trunk River did not result in enhanced

methanogenesis (over the course of two days). Homoacetogens were found in the bottles with a H2/CO2

headspace, as the acetate concentration was shown to increase over time. Acetate consumption was

observed in two bottles (fed with 2mM Ac and 10mM Ac), however, it was not clear what the carbon was

converted into. As these microorganisms degraded acetate, they would have been capable of

syntrophically interacting with a fermenter acetogen by exchanging acetate. The degradation of butyrate

and the production and consumption of acetate and formate in the bottles enriching for an

acetogenic/methanogenic co-culture illustrated the presence of a co-culture that syntrophically

transferred H2 and formate.

In contrast to acetate, H2, and formate interspecies transfer, no DIET was observed using the MFC and

potentiostat set-up. It is unclear, however, if this is due to biological reasons (e.g., more time might be

needed for the environmental sample to adapt its metabolism) or engineering problems (e.g., occasional

O2 leaks). It would have been informative to test the MFC/potentiostat set-up with a (co-) culture known

to exchange/accept electrons. Without proof of concept of the set-up itself, it is difficult to make

conclusions regarding the biology.

5 Acknowledgments Given the short amount of time for conceptualizing the idea for these experiments, building and

troubleshooting the set-ups employed, performing the actual experiments and the subsequent data

analysis, to finally writing and preparing a presentation and this report (three weeks), it is obvious that this

accomplishment involved the aid of many people. I want to thank Lina and Sebastian for their help with

anything anaerobic, which basically means for help with every step of this miniproject (e.g.,

MFC/potentiostat set-ups and GC/HPLC analysis). Thank you Kurt H., for your help with thinking about the

thermodynamics of my cultures. I owe Scott and Srijak gratitude for aiding with both attempts at creating

a clone library (and for their general kind-heartedness and ability to bring a smile to my face), and Emil for

his concise instructions regarding preparing and analyzing CARD and mono FISH samples. Thank you

Cristian for dealing with all the unhappy people and for allowing all lab-operations to go smoothly, as well

as Kristina and Rachel for their continuous help in making media/plates and finding random stuff. On a

different note, I am very thankful for Becky’s and Dave’s presence at the hospital after my unfortunate

collision with an impulsive 5 year old who definitely should be kept on a leash at all times. I want to thank

the construction engineers and architects of the LOEB building for providing us with such a great

conference room by day and epic movie theater by night (the FishBowl will never be forgotten).

A special call out to Dianne and Jared for creating such an enthusiastic and inspiring environment that

enabled us all to continuously push ourselves and to search for excellence in every aspect of our lives at

Woods Hole (although it seems fair to say that excellence was not reached, our quality of life in pursuit of

this goal was phenomenal). In conclusion, I want to thank all students, staff, and visitors for creating the

great atmosphere throughout the last 7 weeks that allowed us all to work hard while playing hard at the

same time.


11

6 References Chen, S., Rotaru, A.-E., Liu, F., Philips, J., Woodard, T.L., Nevin, K.P., and Lovley, D.R. (2014). Carbon cloth

stimulates direct interspecies electron transfer in syntrophic co-cultures. Bioresource Technology, 173(0), 82-86. doi: http://dx.doi.org/10.1016/j.biortech.2014.09.009

Conklin, A., Stensel, H.D., and Ferguson, J. (2006). Growth Kinetics and Competition between Methanosarcina and Methanosaeta in Mesophilic Anaerobic Digestion. Water Environment Research, 78(5), 486-496. doi: 10.2307/25053536

Domingues, M.R., Araujo, J.C., Varesche, M.B.A., and Vazoller, R.F. (2002). Evaluation of thermophilic anaerobic microbial consortia using fluorescence in situ hybridization (FISH). Water Science and Technology, 45(10), 27-33.

Dong, X., Cheng, G., and Stams, A.J.M. (1994). Butyrate oxidation by Syntrophospora bryantii in co-culture with different methanogens and in pure culture with pentenoate as electron acceptor. Applied Microbiology and Biotechnology, 42(4), 647-652. doi: 10.1007/BF00173934

Fu, Q., Kuramochi, Y., Fukushima, N., Maeda, H., Sato, K., and Kobayashi, H. (2015). Bioelectrochemical Analyses of the Development of a Thermophilic Biocathode Catalyzing Electromethanogenesis. Environmental Science & Technology, 49(2), 1225-1232. doi: 10.1021/es5052233

Liu, F., Rotaru, A.-E., Shrestha, P.M., Malvankar, N.S., Nevin, K.P., and Lovley, D.R. (2012). Promoting direct interspecies electron transfer with activated carbon. Energy and Environmental Science, 5, 8982-8989.

Nagarajan, H., Embree, M., Rotaru, A.-E., Shrestha, P.M., Feist, A.M., Palsson, B.Ø., Lovley, D.R., and Zengler, K. (2013). Characterization and modelling of interspecies electron transfer mechanisms and microbial community dynamics of a syntrophic association. Nat Commun, 4. doi: 10.1038/ncomms3809

Rotaru, A.-E., Shrestha, P.M., Liu, F., Shrestha, M., Shrestha, D., Embree, M., Zengler, K., Wardman, C., Nevin, K.P., and Lovley, D.R. (2014). A new model for electron flow during anaerobic digestion - DIET to Methanosaeta for the reduction of carbon dioxide to methane. Energy and Environmental Science, 7, 408-415.

Shrestha, P.M., Malvankar, N.S., Werner, J.J., Franks, A.E., Elena-Rotaru, A., Shrestha, M., Liu, F., Nevin, K.P., Angenent, L.T., and Lovley, D.R. (2014). Correlation between microbial community and granule conductivity in anaerobic bioreactors for brewery wastewater treatment. Bioresource Technology, 174, 306-310. doi: http://dx.doi.org/10.1016/j.biortech.2014.10.004

Shrestha, P.M., Rotaru, A.E., Summers, Z.M., Shrestha, M., Liu, F., and Lovley, D.R. (2013). Transcriptomic and genetic analysis of direct interspecies electron transfer. Applied and environmental microbiology, 79(7), 2397-2404.

Solera, R., Romero, L.I., and Sales, D. (2001). Determination of the microbial population in thermophilic anaerobic reactor: Comparative analysis by different counting methods. Anaerobe, 7(2), 79-86. doi: 10.1006/anae.2001.0379

Summers, Z.M., Fogarty, H.E., Leang, C., Franks, A.E., Malvankar, N.S., and Lovley, D.R. (2010). Direct Exchange of Electrons Within Aggregates of an Evolved Syntrophic Coculture of Anaerobic Bacteria. Science, 330(6009), 1413-1415. doi: 10.1126/science.1196526

Zhao, Z., Zhang, Y., Woodard, T.L., Nevin, K.P., and Lovley, D.R. (2015). Enhancing syntrophic metabolism in up-flow anaerobic sludge blanket reactors with conductive carbon materials. Bioresource Technology, 191, 140-145. doi: http://dx.doi.org/10.1016/j.biortech.2015.05.007

http://dx.doi.org/10.1016/j.biortech.2014.09.009



Appendix A – Media preparation (2015 Microbial Diversity Course

Manual) Use fresh-water mineral media, with, for 1 liter:

100X FW-Base 10 ml

1 M NH4Cl solution 5 ml

100 mM K phos, pH 7.2 10 ml

1 M MOPS buffer, pH 7.2 5 ml

Trace elements 1 ml

1% Resazurin 100 µl

Boil this mixture for 10 minutes in 3 L flasks under N2/CO2 (80/20 v%). Cool under stream of N2/CO2 gas

Multivitamin solution 1 ml

Sodium bicarbonate, 1 M 69 ml

Medium should become pink. Stopper round the bottom flask and bring into the anaerobic chamber. Add 1 mL per liter of 1 M sodium sulfide and stopper the flask. Wait for the color to become clear. Dispense 25 ml each each to all bottles and stopper. Crimp the bottles outside the anaerobic chamber and autoclave such that the bottles have an autoclave bin covering the top to minimize harm caused in case of an explosion. Once out of the autoclave, wait to remove the top auctoclave bin till they are cool to touch. Slight precipitation might occur post-autoclaving. This will disappear once the medium is cool. Store in dark.

Make 100X FW-base (for 100 X stock)

Component Amount MW (g/mol) 100X conc (stock) Final conc (1X)

NaCl 100 g 58.44 1711 mM 17.1 mM

MgCl2.6H2O 40 g 203.30 197 mM 1.97 mM

CaCl2.2H2O 10 g 147.02 68 mM 0.68 mM

KCl 50 g 74.56 671 mM 6.71 mM

Make 100X/100 mM K phoshate, pH 7.2 (for 100 X stock)


KH2PO4/K2HPO4 4.0/12.7 g 136.1/714.2 100 mM 1 mM

Make MOPS buffer (per liter)


MOPS free acid 209.26 g 209.26 1000 mM 5-20 mM

5M NaOH 100 ml 500 mM

Dissolve MOPS in 750 ml H2O, add 100 ml NaOH. The pKa of MOPS is 7.2, so 0.5 M NaOH should put it spot on pH 7.2, but do confirm and adjust the pH to 7.2 with dilute HCl or dilute NaOH as needed, and then adjust volume to 1000 ml.

Note that some aerobic organisms can use MOPS as C and/or S source, this is rare, but in rate cases ‘negative controls’ that are otherwise C and N free will yield growth


13

Filter sterilize concentrated stock and keep out of light

Add to base media before autoclaving unless otherwise stated

Make 1000X HCl-Dissolved trace elements stock solution (per liter)


20 mM HCl 1.7 ml conc HCl Na 20 mM 20 µM

FeSO4.7H2O 2100 mg 278.01 197 mM 1.97 mM

H3BO3 30 mg 61.83 0.48 mM 0.48 µM

MnCl2.6H2O 100 mg 197.91 0.5 mM 0.5 µM

CoCl2.6H2O 190 mg 237.93 6.8 mM 6.8 µM

NiCl2.6H2O 24 mg 237.69 1.0 mM 1.0 µM

CuCl2.2H2O 2 mg 170.48 12 µM 12 nM

ZnSO4.7H2O 144 mg 287.56 0.5 mM 0.5 µM

Na2MoO4.2H2O 36 mg 241.95 0.15 mM 0.15 µM

NaVO3 3 mg 121.93 25 µM 25 nM

Na2WO4.2H2O 3 mg 329.85 9 µM 9 nM

Na2SeO3.5H2O 6 mg 263.01 23 µM 23 nM

Make multivitamin solution

Component Amount 1000X conc. Final conc.

10 mM MOPS, pH 7.2 1000 ml 10 mM 10 µM

Riboflavin 100 mg 0.1 mg/ml 0.1 µg/ml

Biotin 30 mg 0.03 mg/ml 0.03 µg/ml

Thiamine HCl 100 mg 0.1 mg/ml 0.1 µg/ml

L-Ascorbic acid 100 mg 0.1 mg/ml 0.1 µg/ml

d-Ca-pantothenate 100 mg 0.1 mg/ml 0.1 µg/ml

Folic acid 100 mg 0.1 mg/ml 0.1 µg/ml

Nicotinic acid 100 mg 0.1 mg/ml 0.1 µg/ml

4-aminobenzoic acid 100 mg 0.1 mg/ml 0.1 µg/ml

Pyridoxine HCl 100 mg 0.1 mg/ml 0.1 µg/ml

Lipoic acid 100 mg 0.1 mg/ml 0.1 µg/ml

NAD 100 mg 0.1 mg/ml 0.1 µg/ml

Thiamine pyrophosphate

100 mg 0.1 mg/ml 0.1 µg/ml

Cyanocobalamin 10 mg 0.01 mg/ml 0.01 µg/ml

Titrate with 5 M NaOH (5-10 drops) until dissolved.

Filter sterilize (0.2 µm filter) and regriferate in the dark in 10 ml aliquote


14

Appendix B: Hyphomicrobium medium (2015 Microbial Diversity Course

Manual)

Appendix C: CARD-FISH protocols (2015 Microbial Diversity Course

Manual) Fixation of planktonic samples

Embedding

Permeabilization


15

Inactivation of endogenous peroxidases

Hybridization

Washing

CARD


16

Appendix D: Mono-FISH protocols (2015 Microbial Diversity Course

Manual) Hybridization


17

looking for syntrophic · 4). glass slides were also added to bottles #5 and 6 to promote biofilm...

Documents