Humic Substances and Electron Transport Reactions in Aquatic Ecosystems: the Role

of Quinone Moieties

Diane McKnightCollaborators: Robin Fulton, Derek Lovley, Pat

Hatcher, Mark Williams

Students: Lisa Klapper, Durelle Scott, Eran Hood

Humic substances: Ubiquitous, chemically functional, biomolecular junk

• Humic substances modulate ecosystem function by controlling light regime, soil fertility, electron transfer, etc.

• Biogeochemical reactions involving humic substances control elemental cycles for C, N, S, P, Fe, Mn and other trace metals

• Heterogeneous class with similar properties (color, hydrophobicity, acidic groups, etc)

Humic substances as electron acceptors

• All humics have some electron accepting capacity

• Anoxic microbial respiration

• Reduced humics can readily transfer electrons to ferric iron

• ESR studies indicates that quinones are involved.

Biological reduction of humics

• Microbial reduction by GS15 (Geobacter metallireducens) with acetate

Humics-reducingmicroorganism

Acetate

CO2

OxidizedHS

ReducedHS

The electron mediator role of humics

• Electrons are microbially transferred from acetate to HS in an anoxic environment.

• Electrons are transferred from HS to Fe(III), reducing the metal to Fe(II).

Lovley, D. R.; Coates, J. D.; Blunt-Harris, E. L.; Phillips, E. J. P.; Woodward, J. C. Nature, 1996, 382, 445-447

Humics-reducingmicroorganism

Acetate

CO2

OxidizedHS

ReducedHS

Fe(II)

Fe(III)

Quinones

O

O

OH

O

OH

OH

e-, H+ e-, H+

quinone semiquinone dihydroquinone

- fluorescent properties dependent on attached

substituents

- fluorescent - highly fluorescent

Redox state of humic substances in natural environments?

• Necessary to detect redox state in order to compare to other microbially mediated redox processes

• Measurement by reduction of iron not possible in natural systems containing iron

• ESR is not practical, requiring high concentrations

Typical excitation emission matrices (EEMs) of fulvic acids

Suwannee River FAterrestrial source

Lake Fryxell FAmicrobial source

Senesi, N.; Miano, T. M.; Provenzano, M. R.; Brunetti, G, Soil Science, 1991, 152, 259-271

Fluorescence

Heat(radiationless

decay)

Fluorescence

(10-6 - 10-9 s)

Ground State

Excited Statesincluding

vibrational

S1

S0

S2

Absorption

(10-15 s)

Vibrational relaxation

(10-12 s)

Internal conversion

Emission spectra (excit = 370 nm) varies with source of fulvic acid

Fluorescence index: intensity ratio between 450 and 500 nmMcKnight, D. M.; Boyer, E. W.; Westerhoff, P K.; Doran, P T.; Kulbe, T.; Andersen, D. T.

Limnology and Oceanography, 2001, 46, 38-48

0.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

400 450 500 550

Wavelength (nm)

Inte

nsi

ty

Lake Fryxell FA

Suwannee River FA

Quinones mediate electron transfer

• Play an important role in electron transfer several biologically important enzymes

• Redox potential is tunable by attaching various substrates to the quinone

O

O

OH

O

OH

OH

e-, H+ e-, H+

quinone semiquinone dihydroquinone

EEM’s of AQDS and AHDS

O

O

NaO3S

SO3Na

OH

OH

NaO3S

SO3Na

AQDS AHDS

red.

oxid.

San Diego Bay

Coronado Cayes

Paleta Creek

Shelter Island

Humics as electron acceptors in San Diego Bay sediments

• Paletta Creek– PAH contaminated– High rates of anaerobic degradation of PAH’s have been observed– Terrestrial source of humics

• Shelter Island– Commercial/recreational boat basin– Contaminants: copper and tin (from anti-fouling hull paints)– Contains humic substances from both terrestrial and microbial

sources

• Coronado Cayes– “Pristine” site– Microbial source of humics

Fluorescence index and % aromaticity of SDB sediment FA’s

* Fluorescence index of sediment FA’s are lower than aquatic FA’s; however, the same source trend is followed.

Source Fluorescence Index % Aromaticity

Paleta Creek 1.04 28

Shelter Island 1.07 29

Coronado Cayes 1.13 24

Green Lakes 4 (upper sediment) 1.13 NA

Humic substances isolated from San Diego Bay sediments

• Microbial reduction by GS15 (Geobacter metallireducens) with acetate as substrate

• Measured amount of ferrous iron produced after removal of cells (electron accepting capacity)

• Recorded EEM’s before and after reduction

Electron accepting capacity of isolated humic substances

0

100

200

300

400

500

600

700

800

900

CoronadoCayes

SuwanneeRiver

LakeFryxell

ShelterIsland

PaletaCreek

NymphLake

IHSS SoilHumics

mo

l Fe

/g h

um

ics

Change in EEM of Paletta Creek FA with microbial reduction

Isolated Paletta Creek FA Reduced Paletta Creek FA

Change in EEM of Shelter Island FA with microbial reduction

Isolated Shelter Island FA Reduced Shelter Island FA

Change in EEM of Coronado Cayes FA with microbial reduction

Isolated Coronado Cayes FA Reduced Coronado Cayes FA

Continuing experiments…

• Isolated humic substances from sediments stored at 4 ºC for 13 months

• Discovered that EEM’s of these humic samples were same as those of microbially reduced humics!!!

Aged sediment FA’s have similar EEM’s to that of reduced FA’s

• FA’s isolated after 13 months incubation at 4°C

Aged Paletta Creek FA Reduced Paletta Creek FA

Aged sediment FA’s have similar EEM’s to that of reduced FA’s

• Microbial processes did not shut down at 4oC• Limited supply of Fe(III)???

Aged Coronado Cayes FA Reduced Coronado Cayes FA

Aged FA’s can be oxidized by Fe(III):

Original Coronado Cayes FAReoxidized Coronado Cayes FA

Change in EEM’s for reduced humics from sediments

• Possible to detect in natural environment

• Consistent with quinone moieties as main electron accepting moiety

• Greater alteration of EEM’s for microbially-derived fulvic acids with lower eelctron accepting capacity, suggesting more of quinones act as electron acceptors

Lake Fryxell fluorescence index depth profile

-18

-16

-14

-12

-10

-8

-6

-4

-2

0

1.5 1.6 1.7 1.8 1.9 2 2.1

Flouresence IndexD

ep

th (

m )

Lake Fryxell: Whole water samples

• Samples filtered through 4 micron filters• Samples sealed directly after acquisition

5 m 16 m

Similar changes in EEM’s seen in laboratory experiments

• Photo-oxidation of FA’s lead to change in EEM

16 m wholewater8 hours under solar simulator 10 m

Hypothesis for relationship of humic source to electron accepting capacity

• Quinone moieties incorporated into humics from quinone-containing “enzymes” involved in electron transfer reactions

• Quinone moieties incorporated into humics from degradation products of lignin

• Quinones in humics from “enzymes” are more effective as electron acceptors than lignin-derived quinones

Implications of relationship of humic source to electron accepting capacity

• From geologic perspective, humics from microbial biomass came “first”, potentially preceding O2 atmosphere.

• Evolutionary advantage to the preservation of the electron transfer function in detrital organic material (humics) to release nutrients to sustain growth

Implications of relationship of humic source to electron accepting capacity

• Because of O2 atmosphere, terrestrial ecosystems not limited by availability of electron acceptors for degradation of OM.

• The land surface is “green”-higher plants have a higher standing biomass, more structural material, compete for light, water and nutrients

Implications of relationship of humic source to electron accepting capacity

• In terrestrial ecosystems, organic material cycling is slower than in aquatic environments

• Degradation of lignin is slow and is not “optimized” to produce humic substances containing “highly functional” quinone moieties for electron transfer.

San Diego Bay

Coronado Cayes

Paleta Creek

Shelter Island

Conclusions

• Humic substances are probably involved as an electron shuttles or terminal electron acceptors in many aquatic environments

• Significant for designing C sequestration!!

• Significant for fate of organic contaminants in context of land use change!!

Humics as electron acceptors in San Diego Bay sediments

• Microbial reduction by GS15 (Geobacter metallireducens) with acetate

• Fluorescence of humics potentially caused by quinone moieties

• Humics with microbial source have less electron accepting capacity

• Electron accepting capacity may be related the lower excitation/emission maximum

• Potential for EEM’s to be used to map distribution of reactivity with concentration of extractable fulvic acid