Geology 591 Fall 2005

Final Proposal

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Problem Statement

The Geology 591 class experiment was executed during Fall 2005 in an attempt to

answer the main research question; how do increases in silicate-Ni concentration impact

microbially-mediated silicate weathering by native, anaerobic microbial consortia? This was

accomplished by collecting the native microbial consortia and its inhabitant groundwater

samples from the US Geological Survey Bemidji Research Site, Minnesota, adding the

groundwater in treatment bottles with Ni-doped glass with varying Ni-content, and analyzing

both the solution, biomass, and headspace gases over 12 weeks.

Unfortunately, the scope of this experiment did not adequately answer the research

question. Apparently oxygen contamination to both treatment and sterile-control bottles resulted

in increasing activity of iron-reducing microbes and hampering of anaerobic methane-producing

microbes (Figures 1, 2 & 3), as well as an overall decrease in biomass (Figure 4). Also, silicate

weathering was not adequately demonstrated, nor was there a definite observable correlation

between weathering rate and Ni-content (Figure 5).

This proposal seeks to address these deficiencies by addressing them and proposing

alternative methods for the experiment. Also, it will project and compare proposed long-term

results of the University of Kansas anaerobic experiment with those of the Alleghany College

aerobic experiment, both originating from the Bemidji, Mn.

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Proposal for Future Experimental Design

This proposal is based upon the original proposal used for the Geology 591 class Fall

2005 experiment. It will build upon the original with minor changes to more adequately answer

the original research question. As such, this proposal will be presented with the component in

question followed by a justification section that explains how this deviates (if any) from the

original and why a particular approach was taken.

Research Question:

How do increases in silicate-Ni concentration impact microbially-mediated silicate

weathering by native, anaerobic microbial consortia?

Justification: The research question does not vary form the original. It is still a valid

question in regards to the Bemidji site, and will remain the focus of this proposal.

Hypotheses:

1) Accelerated dissolution of Si will occur at low Ni concentrations due to Ni micronutrient

requirements by methanogens;

2) High Ni concentrations are toxic to the native microbial consortia and will cause a decrease in

biomass.

Justification: The hypotheses follow directly from the original. With no change on the

research question, the hypotheses remain valid as well.

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Expected Results:

Under anaerobic conditions there should be accelerated dissolution at low Ni

concentrations due to high Ni requirements from methanogens (Bennett et al. 2001). At high

concentrations (5%) there should be a decrease in microbial biomass due to Ni toxicity at high

levels. The decrease in biomass should correlate to a lowered rate of dissolution in the reactors.

Justification: The expected results are essentially the same as the original. The one

major change, is that the expected toxic level of Ni has increased from 1-2% to 5%. This is due

the 1-2% Ni concentration having no apparent differing results than those of lower

concentrations (Figures 4 & 5).

Variables:

• Solid-phase Ni- and Cu-doped glass (Table 1)

• Anaerobic native microbial consortia (Bemidji well site), and a treatment with

Arthrobacter sp. (Brantley, Liermann and Bau 2001)

Justification: The doped glass used in each reactor is the same as the original with the

exception of an added Ni-6 treatment at 5 mole %. This is due to the apparent non-

toxicity of 1-2% (Ni-4 and Ni-5) treatments in biomass and carbon dioxide production

(Figures 2 & 4). The native consortia is also used as was the original due to the nature of

the question asked, with the exception of a reactor with sterilized, filtered Bimidji well

water and another with known methanogen, archeobacter, for each Ni concentration.

Jennifer Roberts� 12/21/05 7:32 AMComment [1]: Not a reasonable ref for this statement.

Jennifer Roberts� 12/21/05 7:42 AMComment [2]: What is justification for Cu

Jennifer Roberts� 12/21/05 7:40 AMComment [3]: Good idea, but this is not a methanogen. Archeobacter is a pathogen, and Arthrobacter is a soil bacterium which happens to be an obligate aerobe. Neither are good choices here.

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Controls:

• Solid-phase no Ni or Cu glass (Table 1)

• Sterility

Justification: As per the original experiment, controls will include a sterile reactor.

Unlike the original, the groundwater will be filtered after autoclaving to insure no biotic

contamination. Also, there will be an additional reactor microcosm consisting of a glass

with no Ni or Cu, as was the original experiment.

Replicates:

• Triplicate reactors

Justification: Triplicate experiments are the most cost effective way of eliminating

inconsistent results and identifying outliers in data.

Material used:

• Cell type and abundance: Native microbes from Bemidji well 9014, and cultured

Arthrobacter sp., both at 105 cells/ml-1.

• Composition of initial solution: Formation groundwater collected at Bemidji well 9014

diluted 1:1 and addition of P in the form of Na2HPO4 (Disodium Hydrogen Phosphate)

available from FisherScientific(2005)

• Composition of initial solids: Manufactured glass (borosilicate) with Ni (0-5%) and

Ni,Cu (.01-.1 %)

• Composition of initial headspace: Initial headspace will be anaerobic atmosphere from

the anaerobic chamber.

Jennifer Roberts� 12/21/05 8:03 AMComment [4]: Why is it filtered after? Autoclaving will render it sterile and filtration afterward risks contamination. The loss of sterility was likely a sampling issue not an initial problem with autoclaving.

Jennifer Roberts� 12/21/05 8:03 AMComment [5]: This is minimum for statistical relevancy.

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• Batch reactors sampled throughout the experiment (See Figure 6 & Table 2)

Justification: The original materials will be utilized with some additions and adjustments.

A known methanogen will be used in a reactor batch to compare with the native consortia

at the each Ni concentration (see Table 2). Also P will be added to the groundwater in

minor amount (1 mole %) to ensure nutrients are available for microbial growth and to

eliminate “cannibalism” for micronutrients. A reactor with 0% Ni concentration will be

added to the experiment to as an additional control with the sterile controls. Finally the

headspace will be anaerobic atmosphere of the anaerobic chamber to eliminate any initial

oxygen contamination as may have happened in the original experiment (Figures 1 & 2).

Methodology

There will be 50 reactor bottles total (Table 2, Figure 6). Each reactor will use the

following methodology:

• Deionized water (DIW) will have Na2HPO4 added until it reaches 1 mole %. This will

ensure P does not become a limiting micronutrient in the reactors, as is typical in most

systems (Madigan and Martinko 2005).

• Next, the Bemidji well 9014 groundwater will be diluted with the DIW-P enriched solution at

1:1 ratio.

• The nine different glass compositions will be added to the reactor bottles( ~ 1 mg)

• 100 ml of the solution will be added to the reactor bottles

• Innoculation with ~2 ml of native microbial consortia derived from sediments from Bemidji

well 9014 into all the “live’ reactors, except the control Arthrobacter sp. reactor, which will

be inoculated with ~ 2 ml of cultured Arhtrobacter sp.

Jennifer Roberts� 12/21/05 8:06 AMComment [6]: This is what we did in the initial experiment. Oxygen was introduced in the field during sampling. This whole scenario needs to be explained in greater detail.

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• Inoculation of the reactors with 10mg/L toluene; a food source for the methanogens (Lovely

and Longergan 1990)

• Reactors will be incubated at room temperature in the dark without agitation for 6 weeks.

• Sampling will be conducted of each reactor bottle within the anaerobic chamber to ensure

that oxygen contamination does not occur. Also separate syringes, needles and filters will be

used for each reactor bottle to ensure cross contamination does not occur.

• Biomass, pH and alkalinity will be measured biweekly starting at week 6 (Table 3). 1 ml of

solution will be withdrawn from the reactors and filtered under vacuum. The solution will be

diluted with 2 ml DIW and tested for pH and alkalinity. The filter will be stained with DAPI,

and biomass counted under epiflourescent microscope.

• If methanogens are thriving in the reactor bottles then it will be expected that methane will be

produced and reach equilibrium with the headspace (Madigan and Martinko 2005). Since the

headspace will be provided in the anaerobic chamber, there should be no initial methane.

Methane and carbon dioxide of the headspace will be tested biweekly. 250 µl of headspace

gas will be withdrawn form the reactors and analyzed using gas chromatography with

detection by TCD on a Haysept Q column. 250 µl of anaerobic air will be injected into the

reactors to maintain headspace gas volumes and solution equilibrium.

• Filtered solution will also be analyzed for ferrous Fe and Ni content using the

spectrophotometer. The trace metals will react with 2,2’-bipyridine (Fe) and 1-2 Pyridylazo-

2-Naphthol PAN (Ni) (Hach 2005) and the spectrophometer will analyze for the

concentration of the metals. 1 ml of solution will be drawn at the same time as that for

biomass, pH, and alkalinity.

Jennifer Roberts� 12/21/05 8:10 AMComment [7]: Can you do mass balance on toluene, CO2, CH4, and biomass? Is CO2 primary substrate for methanogens?

Jennifer Roberts� 12/21/05 8:07 AMComment [8]: This is fine—however, you need to describe how you are going to outgas the solutions---adding headspace gas isn’t enough.

Jennifer Roberts� 12/21/05 8:09 AMComment [9]: Are detection limits adequate for our solutions?

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• Silica will be analyzed tri-weekly starting at week 6. 1 ml of removed solution from the

reactors will be analyzed using the molybdate method, with absorbance measured on UV-

Vis. Finally at the end of the experiment the solids will be filtered from the solution and

weighed and analyzed on a XRD (for elemental composition) and the ICPMS for elemental

abundance.

Justification: The methodology follows the original experiment with a few minor

adjustments. Ni in solution will be analyzed to see if Ni is removed from solid phase as well as

if it is taken up by the microbial community. Also, the solids will be analyzed for changes in

elemental composition (due to uptake of Ni or Cu preferentially, or precipitation of new

minerals) and mass. This in combination with solution analysis for Fe, Ni, and SI will allow a

better understanding of weathering of the solid phase and help to answer the research question.

Sampling Frequency and Length of Experiment:

Sampling will be conducted at approximately the same rate as the original experiment;

however, it will take place over a longer time frame (Table 2) to allow for the reactors to

develop. As was seen from methane production (Figure 3), it is possible that the methanogens

were not given enough time to “thrive” in each reactor during the original experiment. This will

also allow several more data points within each database and allow trends of change and

anomalies to be discerned.

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Prediction of longer-term biogeochemical changes to the experiment

Allegheny College

If the Allegheny College experiment were to be “shelved”, it would become a closed

system since oxygen would no longer be introduced to the reactors on a weekly basis. Since

oxygen would become a limiting electron acceptor, then the reactors with native microbial

consortia would be expected to undergo community temporal succession similar to soil spatial

succession (Baedecker and Back 1979; Stumm and Morgan 1981).

First aerobic respiration would take place, consuming oxygen, with an increase in carbon

dioxide, pH and alkalinity. Fe would decrease in solution and precipitation of Fe-oxides would

occur. Unless the microbes had a Ni requirement, silica dissolution should not occur. As the

oxygen became depleted, the microbial community would shift in abundance from cyanobacteria

to nitrifiers, and denitrification would occur. This would be followed by Mn reduction (if Mn is

present in the groundwater) and the sulfate reduction (again, if sulfate was present). Finally,

when all these processes had used up the electron acceptors available in the closed system, the

methanogens would become the dominant active microbes, methanogenesis would take place,

and have similar results as the KU proposed future experiment. Overall, I would not expect

much change n biomass, as one microbe took over the metabolic activities from the last which

would die out.

University of Kansas

If the experiment was allowed to become a “drawer” experiment I would not expect

much change. Unlike Allegheny College experiment, ideally this proposed “rerun” of the

Jennifer Roberts� 12/21/05 8:21 AMComment [10]: pH would not increase. Jennifer Roberts� 12/21/05 8:19 AMComment [11]: There is no Fe in solution. This is nonsensical. Jennifer Roberts� 12/21/05 8:19 AMComment [12]: No cyanos—no sunlight underground.

Jennifer Roberts� 12/21/05 8:20 AMComment [13]: Is it present? You should be able to give a definitive concentration and reference it.

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experiment would be anaerobic from the start. Due to this, I would not expect a temporal

“succession” of microbial communities and their products (Baedecker and Beck 1979; Stumm

and Morgan 1981). If allowed to continue on its own in the closed system reactors, I would

expect to see increase in methanogens and thusly increases in methane in solution and

headspace. This would probably continue until the “food-source” of toluene was consumed, at

which point the methanogens would probably “cannibalize” each other for nutrients. At some

point the microbes would deplete their nutrients to such a point that the community would

collapse, resulting in mass death.

Conversely, during maximum methane production, a clumsy graduate student might

inadvertently bump a bottle and cause a chain-reaction of methane “bombs” exploding. Needless

to say this would terminate the experiment, and the graduate student’s career. But, if this

unfortunate event were not too occur, then I would expect the community to be dead after a 6

month shelving, and thus to see no change after 12 months either.

If the community was capable of being self-sustaining during a six month period (and

barring an explosive end), then I would expect to see marked increases in methane, decrease in

carbon dioxide, increase in Si and Ni in solution, and no change in solution ferrous Fe. Biomass

would also be too high to count without a dilution of 1000:1 or greater. Again, I would be

greatly surprised if the community hadn’t burned itself out by the twelfth month, within the

closed system and limited macro- and micro-nutrients.

Jennifer Roberts� 12/21/05 8:22 AMComment [14]: Methanogens use CO2 as well.

Jennifer Roberts� 12/21/05 8:23 AMComment [15]: I disagree---metabolic rate is quite low, turnover about once a year. I think it would take several years for them to die out.

Jennifer Roberts� 12/21/05 8:25 AMComment [16]: Good Jennifer Roberts� 12/21/05 8:26 AMComment [17]: Probably not.

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Figures and Tables

0

0.02

0.04

0.06

0.08

0.1

0.12

Fe I

I (m

mol

/L)

Ni-0

Ni-0 Sterile

Ni-1

Ni-1 Sterile

Ni-2

Ni-2 Sterile

Ni-3

Ni-3 Sterile

Ni-4

Ni-4 Sterile

Ni-5

Ni-5 Sterile

Cu/Ni-1

Cu/Ni-1 Sterile

Cu/Ni-3

Cu/Ni-3 Sterile

Figure 1. Fe II concentration comparison between averaged replic ates and sterile controls (day 35 vs. day 50). Fe in solution decreased between the two sample po ints, in conjucntion with an observable increase in Fe -oxide precipitation, indicates an increase in Fe -reducing microbe acitvity in an aerobic environment. This is most likely a product of co ntamination during sampling.

Figure 2. Solution carbon dioxide: Ni -doped glass (dayn - day10). Co2 increases through time for all the Ni concentrations. This in conjunction with the decreas e in soluble Fe indicates an increase in microbial respiration are active and thus oxygen con tamination to the bottles during sampling.

-3-2-1012345678

0 5 10 15 20 25 30 35 40 45 50

Time (days)

Δ [C

O2]

(mm

ol/L

)

Ni-0Ni-1Ni-2Ni-3Ni-4Ni-5

12

-80

-60

-40

-20

0

20

40

60

80

100

0 5 10 15 20 25 30 35 40 45 50

Time (days)

Ni-0Ni-1Ni-2Ni-3Ni-4Ni-5Δ

[CH4

] (µ

mol

/L)

Figure 3. Change in solution methane: Ni -doped glass (dayn - day10). Methane increases through time similar to CO 2,however, the increase is an order of magnitude smaller than that of CO 2 (micromoles versus millimoles ). Thusly, the methanogens are a minor constituent to the microbial consortia, and mehtane change is negligible.

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

Cel

l Con

cent

ratio

n (c

ells

/ml) Ni 0

Ni 0 sterile

Ni 1

Ni 1 sterile

Ni 2

Ni 2 sterile

Ni 3

Ni 3 sterile

Ni 4

Ni 4 sterile

Ni 5

Ni 5 sterile

Cu, Ni 1

Cu, Ni 1 sterile

Cu, Ni 3

Cu, Ni 3 sterile

Figure 4. Biomass comparison of microcosms (day 2 vs. day 50) . With the exception of Ni -1 (which was too numerous to accurately count on day 50), biomasse s of the microcosms appear to deminish through time by ~ one order of magnitude, although generally wi thin the error given. This indicates that the microcosms are not conductive to microbi al growth. One possibilty is a switch in microbial consortia composition (as indicated by incre ase CO2 and decrease soluble Fe -Figs. 1 &2) from anerobic to aerobic. Another is that the microbes are “cannabilizing ” and maintaining a steady -state of biomass due to unforseen nutrient limitations

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-600

-400

-200

0

200

400

600

800

1000

day 20 day 24 day 31

Time (days)

Si (

mm

ol/L

)

Ni-0Ni-1Ni-2Ni-3Ni-4Ni-5Cu/Ni-1Cu/Ni-3

Figure 5. Silica solubility through time. With only three data p oint per microcosm, it appears that there is a slight increase in Si in solution with the exception on Ni -5 and Ni -1, both with an initial decrease large decrease. Unfortunately, three sampling times is not enough to discern any appreciable trend in silica dissolution.

Silicate glass (w/ trace elements)

Nativegroundwater

Gas

Native consortium

Organic carbon

Liquid

Solid

Figure 6. Typical laboratory reactor bottles: the microcosms! E ach bottle will contain the appropriate glass, groundwater/DIW/P solution, toluene, native m icrobes, and anaerobic chamber atmosphere in the headspace. From Roberts 2005.

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Table 1. Compositions of manufactured Cu and Ni-doped silicate glasses. Glass SiO2 B2O3 Na2O Al2O3 CuO NiO Ni-0 80.8 12.0 4.3 2.2 ___ ___ Ni-1 80.8 12.0 4.3 2.2 ___ 0.01 Ni-2 80.8 12.0 4.3 2.2 ___ 0.05 Ni-3 80.8 12.0 4.3 2.2 ___ 0.1 Ni-4 80.8 12.0 4.3 2.2 ___ 1.0 Ni-5 80.8 12.0 4.3 2.2 ___ 2.0 Ni-6 80.8 12.0 4.3 2.2 ___ 5.0 Cu/Ni-1 80.8 12.0 4.3 2.2 0.01 0.01 Cu/Ni-2 80.8 12.0 4.3 2.2 0.1 0.01 Cu/Ni-3 80.8 12.0 4.3 2.2 0.01 0.1 All values expressed as mole percent

Table 2. Experiment design Microcosm Arthrobater Native Microbes Sterile Solution Ni-0 Bottle 1 Bottles 2-4 Bottle 5 Ni-1 Bottle 6 Bottles 7-9 Bottle 10 Ni-2 Bottle 11 Bottles 12-14 Bottle 15 Ni-3 Bottle 16 Bottles 17-19 Bottle 20 Ni-4 Bottle 21 Bottles 22-24 Bottle 25 Ni-5 Bottle 26 Bottles 27-29 Bottle 30 Ni-6 Bottle 31 Bottles 32-34 Bottle 35 Cu/Ni-1 Bottle 36 Bottles 37-39 Bottle 40 Cu/Ni-2 Bottle 41 Bottles 42-44 Bottle 45 Cu/Ni-3 Bottle 46 Bottles 47-49 Bottle 50 Table 3. Experiment timeline Week 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Si (1) X X X X X X X CO2 (.250 g) X X X X X X X X X X CH4 (.250 g) X X X X X X X X X X Fe+Ni (1) X X X X X X X X X X pH+alkal.(1) X X X X X X X X X X Biomass (0) X X X X X X X X X X Solids EA X Total Fluid (ml)

3 3 2 1 2 3 2 1 2 3 2 3 (27 total)

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References and Bibliography

Baedecker, M., & Back, W., 1979. Modern marine sediments as a natural analog to the chemically stressed environment of a landfill: Journal of Hydrology, v. 43, p. 393-414. Bennet, P., Rogers, J., Choi, W., 2001. Silicates, silicate weathering and microbial ecology: Geomicrobiology Journal, v. 18, p. 3-19. Brantley, S., Liermann, L., and Bau, M., 2001. Uptake of trace metals and rare earth elements from hornblende by a soil bacterium. Geomicrobiology Journal, v. 18, p. 37-61. Brock Biology of Microorganisms (2005) Madigan and Martinko, Prentice Hall, Upper Saddle River, NJ. Fischer Scientific (2005), website https://www1.fishersci.com USGS (2005), Bemidji Crude-Oil Research Project, website http://mn.water.usgs.gov/bemidji/ Geomicrobiology (2002) Ehrlich, Marcel Dekker, Inc., New York. Ground-Water Microbiology and Geochemistry (2001) Chapelle, John Wiley & Sons, Inc., New York. Geomicrobiology: Interactions Between Microbes and Minerals (1997) Banfield and Nealson, Eds., Reviews in Mineralogy Vol. 35, Mineralogical Society of America, Washington D.C. Hach (2005), website http://www.hach.com/hc/browse.parameter.list/PAR061/NewLinkLabel=Nickel Lovley, D., & Lonergan, D., 1990. Anaerobic oxidation of toluene, phenol, an p-cresol by the dissimilatory Iron-reducing organism, GS-15: Applied and Environmental Microbiology, June, p. 1858-1864. Microbial Ecology: Fundamentals and Applications, (1998) Atlas and Bartha, Benjamin/Cummings, Menlo Park. Roberts, J., 2005. Applied Techniques in Microbiology, Geology 591, Lecture 1 PowerPoint. Subsurface Microbiology and Biogeochemistry (2001) Fredrickson and Fletcher, Eds., Wiley-Liss, New York. Stumm, W., and Morgan, J., 1981. Aquatic Chemistry. 2nd edition, John Wiley & Sons, new York, 780 pp.