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Geology 591 Fall 2005
Final Proposal
2
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.
3
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.
4
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.
5
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.
6
• 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.
7
• 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?
8
• 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.
9
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.
10
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.
11
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
13
-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.
14
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)
15
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.