Summary of microbial implications on formation/dissociation of … · 2010. 3. 12. · P (psi) Time...

Dave C. Swalm School of Chemical Engineering

Summary of microbial implications on formation/dissociation of seafloor hydrates

Principle Investigator: Rudy Rogers

Research Assistants: Guochang Zhang (Asst Research Professor)

Todd French (Microbiologist consultant)

James Radich (M.S. graduate student)

Shangmin Ziong (M.S. graduate student)

Mississippi State University

Presented to: GOM Gas Hydrate Consortium

November 5, 2009

University of South Carolina


Synthetic surfactants found to catalyze gas hydrate formation in gas-storage project

(nucleate and collect on metal surfaces)

Synthetic surfactants found to catalyze gas hydrate formation in gas-storage project

(nucleate and collect on metal surfaces)

No surfactant… Film forms No surfactant: 10 days hydrate growth

Synthetic surfactant: 1 hour Synthetic surfactant: 2 hours


Surfactant lab process scaled up: 5,300 scfgas storage demonstrated

Surfactant lab process scaled up: 5,300 scfgas storage demonstrated

1…chiller

2…feed gas

3…formation tank

4…glycol surge

5…burner/boiler


Would bio-surfactants in seafloor act similarly?Would bio-surfactants in seafloor act similarly?

Surfactin C53H93N7O13 Molecular Weight = 1036From: Rosenberg, CRC Critical Reviews in Biotechnology

L-Val

L-Asp D-Leu 7 Amino Acids, e.g., Aspartic Acid:

D-Leu L-Leu HOOCCH2CH(NH2)COOH L-Leu L-Glu

O

CH - CH2 - C = O CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2

CH3 - CH - CH3

HydrophobicTail

HydrophilicHead

Surfactin Micelle

Surfactin

from

Bacillus subtilis

Synthetic Surfactant


What kind of bio-surfactants can microbes produce?

Have any of these been found in seafloor sediments?


BiosurfactantBiosurfactant Classifications Classifications ((KosaricKosaric, 1992; , 1992; FujiiFujii, 1998), 1998)

DMPC *DPPS *POPC *

1. Thiobacillus species

2. Corynebacterium species

Phospholipids

SurfactinBacillus subtilisLipoprotein-lipopeptides

Rhamnose lipidPseudomonas aeruginosa

Glycolipids

1. Snomax2. Emulsan

1. Pseudomonas syringae

2. Acinetobacter calcoaceticus

Polysaccharide-lipid-complexes

DL-A-Hydroxystearic acid*

Corynebacterium lepus

Hydroxylated and Crosslinked Fatty Acids

BiosurfactantsEvaluatedMicrobe

BiosurfactantClassifications

Answer: Surfactin, rhamnolipidLanoil, B.D.; Sassen, R.; La Duc, M.T.; Sweet, S.T.; Nealson, K.H. Bacteria and archaea physically associated with GOM gas hydrates. Applied and Environmental Microbiology, Nov. 2001, 5143-5153.


What concentrations of surfactants necessary to catalyze hydrate formation?

0

100

200

300

400

500

600

700

0 200 400 600 800 1000 1200SDS Concentration, ppm

Hyd

rate

Indu

ctio

n Ti

me,

min

Synthetic surfactant

Bio-surfactant0

10203040506070

0 10 20 30 40 50 60

Indu

ctio

n Ti

me,

min

Rhamnolipid Concentration, ppm

Seawater Natural gas At hydrate-forming conditions


(1) Do bio-surfactants catalyze hydrates in seafloor?(2) If so, what mineral surfaces collect them?

Answers:(1) Hydrate formation rate in porous media can be increased order(s)

of magnitude with ppm of bio-surfactants.

(2) In photos below hydrates preferentially collect on smectite clays.

RhamnolipidEmulsan

Rogers, R.E., Kothapalli, C., Lee, M.S., and Woolsey, J.R., 2003. “Catalysis of gas hydrates by biosurfactants in seawater-saturated sand/clay,” Canadian J. Ch.E., 81, No. 5, 973-980.


Can microbes at seafloor conditions produce enough surfactants fast enough to influence hydrate formation?

Can the surfactants be dispersed throughout sediment?

Gas inlet

Broth

Foam

Recovered Surfactin

Shaker

pH =6.8If pH < 5.0…no foam

Zhang, G., Rogers, R.E., French, W.T., and Lao, W., 2007. “Investigation of microbial influences on seafloor gas-hydrate formations,” Marine Chemistry, 103, 359-369.


Our latest research results develop mechanisms and further involvement of the microbes…

Bio-surfactants promote hydrates. Why? How?Answers:

Why? To access the vast amount of carbon that the hydrates willsequester.

How? By providing nucleating sites for hydrate crystallization and by facilitating hydrate collection on mineral surfaces.


But microbial cells retard hydrates!!

Why?

How?

0

1

2

3

4

5

6

7

8

0 50 100 150 200 250

Tem

pera

ture

(C)

Time (min)

Cell wall polymers, peptidoglycan (PGN)And teichoic acid (TA) are kinetic andthermodynamic inhibitors


Inhibition experiments using MC-118 indigenous microbes

Treat cell mass with 1% and 5% NaCl hypertonic solutions:Geometrical irregularities created with PGN and TA polymer linkages, each of which contains dipoles that associate with water.


Biopolymer inhibition results

Cell MassInduction Time

(h)

Maximum Formation Rate

(mmol/min)MC+S+NM-0.36 0.36 18.38 2.15

MC+S+NM-0.387 0.387 174.83 1.62

MC+S+NM-0.368 0.368 151.97 1.58

NMZ+S+W Broth - 8.28 1.53

Hypertonic-1% 0.35 80.50 1.38

Hypertonic-5% 0.35 >235 N/A


Can B. subtilis be used to predict effects of indigenous microbes on hydrate formation/dissociation?

Experiment ID Cell Mass (mg/mL)Maximum Formation

Rate (mmol/min) Average Standard Deviation

B+S+NM 0.391 3.74

B+S+NM 0.405 2.69

B+S+NM 0.415 1.4

MC+S+NM 0.36 2.15

MC+S+NM 0.368 1.62

MC+S+NM 0.387 1.57

2.61 1.17

1.78 0.321

Same trend on hydrate formation rate with cell mass content

B. Subtilis

J. Radich SEM

MC-118 Microbes

J. Radich SEM


50

55

60

65

70

75

0 3 6

Surf

ace

Tens

ion

(mN

/m)

Time (d)

NWBC 1107-02 ReferenceNWBC 1101-02 EnrichmentSWBC 1110-01SWBC 1107-01

Do indigenous microbes on MC-118 seafloor surface promote hydrates uniformly?

Lag phase and proximity

J. Radich, Laboratory and theoretical investigations of direct and indirect microbial influences on seafloor gas hydrates, M.S. thesis, Mississippi State University, 2009.


Are the MC-118 indigenous microbes producing effective biosurfactant?

MC-118 seawater medium containing indigenous microbes were cultured

Biosurfactant centrifuged out of seawater medium of culture

Biosurfactant concentrated and used in hydrate formation tests

Raw saturated sediment SWBC 1110-01 used for comparison for hydrate formation


Hydrate formation tests using MC-118 biosurfactants in saturated porous media

Experiment ID

Surface Tension (mN/m) Po Pf dP Te (h) Ti (h) Td (min)

Maximum Formation Rate (mmol/min)

SW Only 71.5 402.1 350.7 51.4 72.0 15.7 112.0 1.6

SW Only 71.4 400.8 352.8 48 99.1 37.0 114.5 1.52

SW + 25% BC 69.3 401.1 350.4 50.7 47.9 15.3 64.5 1.81

SW + 50% BC 47.7 401.4 329.5 71.9 38.1 0.4 94.0 3.21

SWBC 1110-01 57.2 402.6 346.3 56.3 15.6 4.5 - 1.35

SW – seawater; BC – biosurfactant concentrate; SWBC 1110-01 – MC-118Te – elapsed time; Ti – induction time; Td – dissociation time


Comparing MC-118 and B. subtilis: Gas hydrate formation extent

0.0

20.0

40.0

60.0

80.0

100.0

120.0

MC+S+NM S+NM (MC-118) B+S+NM S+NM (B. subtilis)

Pres

sure

Dro

p (p

si)

MC-118 cultureB. subtilis culture


42.3

0.79 0.8 1.00

10

20

30

40

50

60

70

80

B+S+NM S+NM B+S+NM+Ben S+NM+Ben

Indu

ctio

n T

ime

(h)

When bentonite is present, the bad effects of microbial cell wall material on hydrate formation are negated!!!!

WITHOUT Bentonite

WITH Bentonite


In bentonite presence, more hydrates form with microbes present than without microbes—despite retarding effect of microbes. Why?

0

10

20

30

40

50

60

70

80

90

0 5 10 15 20

ΔP (p

si)

Time (hours)

B+S+NM+Ben-0.305B+S+NM+Ben-0.305S+NM+Ben-aS+NM+Ben-b

Bentonite with B. Subtilis

Bentonite without B. Subtilis

ΔP from initial 2 hours of hydrate formation

So, how do the microbes overcome this retardation of hydrate formation by the materials in the cell walls?


B. subtilis

BentoniteParticle

*Not to scaleBiosurfactant Adsorbed to Bentonite

Proposed mechanism to explain how microbes protect themselves from heat of hydrate formation, yet obtain access to the vast carbon reservoir within massive hydrates.

1st Step.

Radich, J., Rogers, R.E., French, W.T., Zhang, G., 2009. “Biochemical reaction and diffusion in seafloor gas hydrate capillaries: Implications for gas hydrate stability,” Chemical Eng. Sci., 64, Issue 20, 4278-4285.


Conceptual Model

Gas hydrate crystals cluster around B. subtilis and bentonite agglomerates

Illustrated mechanism for microbial entrance to gas hydrate macrostructures.

Step 2. Hydrates then form on biosurfactant/clay surrounding microbes—placing microbes within hydrate.

Radich, J., Rogers, R.E., French, W.T., Zhang, G., 2009. “Biochemical reaction and diffusion in seafloor gas hydrate capillaries: Implications for gas hydrate stability,” Chemical Eng. Sci., 64, Issue 20, 4278-4285.


MC-118 gas hydrate melt substantiates mechanism hypothesis?

SEM Micrographs: J. Dearman


In massive hydrate accumulations, such as seafloor mounds, capillaries provide a tortuous path by which nutrients from the surroundings, gases

generated within the mass, or even microbes could diffuse into the interior where microbes are active.

What are the diameters of these capillaries?

Hypothesis: Microbes consume hydrocarbons and metabolize other substrates from within gas hydrate interstitial and capillary spaces.

Answer: See following slide and refer to our article published in:

Dearman, J.L., Wilson, W.W., Rogers, R.E., and Zhang, G., 2009. “Gas-hydrate promotion by smectite-bioproduct interactions,” Marine Chemistry, 115, Issues 1-2, 21-30.


Determining hydrate capillary sizes (Emulsan/nontronite associate & act as hydrate nuclei)

Analyses based on Dynamic Laser Light Scattering , x-ray diffraction & Scanning Electron Microscope:

Hydrate capillary diameters found to be:

Smallest: 100 – 200 nm

Largest: 1500 nm (approx)

J. Dearman dissertation, MSU, May 2007

Nontronite-Emulsan Particle

These particles diffused through capillaries of hydrates formed in lab porous media.

X45,000 magnification

Note: Nontronite is smectite clay. Emulsan is anionic biopolymer.


THEORETICAL IMPLICATIONS OF MICROBIAL ACTIVITY WITHIN GAS HYDRATE MOUNDS ON SEAFLOOR

Microbial metabolism, diffusion rates, pH, and nutrient/hydrogen availability are directly responsible for the long-term fate of seafloor gas hydrates

Methanotrophs sequester hydrocarbon gases to solid carbonates

Methanogens enhance methane hydrate stability


Radich, J., Rogers, R.E., French, W.T., Zhang, G., 2009. “Biochemicalreaction and diffusion in seafloor gas hydrate capillaries: Implications for gas hydrate stability,” 64, Issue 20, 4278-4285.

We believe that microbial activity within gas hydrate mounds on the seafloor and within massive hydrate accumulations are major determinants of mound growths, dissociations, and longevities.

To help predict these effects, James Radich has developed the first mathematical model that considers internal microbial activities on the fate of hydrate accumulations in the seafloor. See the following publication:


Recommended future work: Extend microbial research to deep hydrate zone.

Comprehensively study microbial effects on hydrate formation/dissociation in deep gas hydrate zone– extend these findings of seafloor surface to the bottom of the gas hydrate zone.

Synthetic surfactants found to catalyze gas hydrate formation in gas-storage project�(nucleate and collect on metal surfaces)Surfactant lab process scaled up: 5,300 scf gas storage demonstratedWould bio-surfactants in seafloor act similarly?What kind of bio-surfactants can microbes produce?(1) Do bio-surfactants catalyze hydrates in seafloor?�(2) If so, what mineral surfaces collect them?�Can microbes at seafloor conditions produce enough surfactants fast enough to influence hydrate formation?�Can the surfactantsInhibition experiments using MC-118 indigenous microbesBiopolymer inhibition resultsCan B. subtilis be used to predict effects of indigenous microbes on hydrate formation/dissociation?Are the MC-118 indigenous microbes producing effective biosurfactant?Hydrate formation tests using MC-118 biosurfactants in saturated porous mediaComparing MC-118 and B. subtilis: �Gas hydrate formation extentWhen bentonite is present, the bad effects of microbial cell wall material on hydrate formation are negated!!!!In bentonite presence, more hydrates form with microbes present than without microbes—despite retarding effect of microbes. WConceptual �ModelMC-118 gas hydrate melt substantiates mechanism hypothesis?In massive hydrate accumulations, such as seafloor mounds, capillaries provide a tortuous path by which nutrients from the surDetermining hydrate capillary sizes �(Emulsan/nontronite associate & act as hydrate nuclei)

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Summary of microbial implications on formation/dissociation of … · 2010. 3. 12. · P (psi) Time...

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