Abstract and Figures

Biochar, a charcoal-like product of the incomplete combustion of organic materials, is an increasingly popular soil amendment designed to improve soil fertility. We investigated the possibility that biochar could promote direct interspecies electron transfer (DIET) in a manner similar to that previously reported for granular activated carbon (GAC). Although the biochars investigated were 1000 times less conductive than GAC, they stimulated DIET in co-cultures of Geobacter metallireducens with Geobacter sulfurreducens or Methanosarcina barkeri in which ethanol was the electron donor. Cells were attached to the biochar, yet not in close contact, suggesting that electrons were likely conducted through the biochar, rather than biological electrical connections. The finding that biochar can stimulate DIET may be an important consideration when amending soils with biochar and can help explain why biochar may enhance methane production from organic wastes under anaerobic conditions.
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Promoting Interspecies Electron Transfer
with Biochar
Shanshan Chen
1,2
, Amelia-Elena Rotaru
1
*, Pravin Malla Shrestha
1
{, Nikhil S. Malvankar
1,3
, Fanghua Liu
1,4
,
Wei Fan
5
, Kelly P. Nevin
1
& Derek R. Lovley
1
1
Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA,
2
School of Environmental Science and
Engineering, Sun Yat-sen University, Guangzhou 510275, China,
3
Department of Physics, University of Massachusetts, Amherst,
MA 01003, USA,
4
Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China,
5
Department
of Chemical Engineering, University of Massachusetts, Amherst, MA 01003, USA.
Biochar, a charcoal-like product of the incomplete combustion of organic materials, is an increasingly
popular soil amendment designed to improve soil fertility. We investigated the possibility that biochar could
promote direct interspecies electron transfer (DIET) in a manner similar to that previously reported for
granular activated carbon (GAC). Although the biochars investigated were 1000 times less conductive than
GAC, they stimulated DIET in co-cultures of
Geobacter metallireducens
with
Geobacter sulfurreducens
or
Methanosarcina barkeri
in which ethanol was the electron donor. Cells were attached to the biochar, yet not
in close contact, suggesting that electrons were likely conducted through the biochar, rather than biological
electrical connections. The finding that biochar can stimulate DIET may be an important consideration
when amending soils with biochar and can help explain why biochar may enhance methane production from
organic wastes under anaerobic conditions.
B
iochar, which is produced by heating biomass in a closed system with limited oxygen supply, has been
promoted as an amendment to improve soil quality
1,2
, but the impact of biochar on soil microbial com-
munities is poorly understood. Biochar has been used to enhance soil fertility
3
, remediate pollution,
decrease green house gas emissions
4
and sequester carbon in a worldwide quest to achieve carbon neutrality
4
.
Biochar is hypothesized to increase the microbial soil content by concentrating organic substrates and nutrients
on its surface
5
. Another feature of biochar that could potentially influence the activity of soil microbiota is that
biochar is electrically conductive
6
. Conductive minerals/materials such as magnetite or GAC facilitated DIET by
accelerating rates of anaerobic metabolism
7–10
. Under certain conditions, DIET is considered to be potentially
more effective for interspecies electron transfer than strategies such as interspecies H
2
or formate transfer that rely
on the diffusion of electron carriers between species
11,12
.
DIET has been shown possible in the absence of conductive materials via biological electrical connections such
as pili
13–17
. For example, Geobacter metallireducens and Geobacter sulfurreducens grew in co-culture via DIET in a
medium with ethanol as the electron donor and fumarate as the electron acceptor
13
. The two organisms needed to
cooperate in order to metabolize ethanol and grow, because G. metallireducens can utilize ethanol as an electron
donor, but is unable to use fumarate as an electron acceptor
18
, whereas G. sulfurreducens cannot use ethanol as an
electron donor, but can reduce the electron acceptor, fumarate
19
. In co-culture, G. metallireducens produced
acetate from ethanol and G. sulfurreducens utilized the electrons released from the ethanol oxidation as well as the
acetate produced for fumarate reduction
13
. Multiple lines of evidence
13–17
demonstrated that interspecies electron
transfer in Geobacter co-cultures proceeded via DIET rather than H
2
or formate transfer, and that the Geobacter
pili, which posses metallic-like conductivity
20,21
, were required during DIET.
However, the initial adaption of Geobacter co-cultures to interspecies electron transfer via pili-mediated DIET
took ca. 30 days
13
. GAC substantially reduced the adaptation period for growth via DIET, with ethanol meta-
bolism evident within a day
9
. Furthermore, the mechanism for DIET in the presence of GAC appeared to be
different because strains of G. sulfurreducens deficient in pili or the pili-associated cytochrome OmcS, were
incapable of DIET in the absence of GAC
13
, but functioned as well as wild-type in the presence of GAC
9
.
Instead of forming close cell-to-cell connections as observed in the absence of GAC
13
, both species attached to
the electrically conductive GAC suggesting that GAC served as a cell-to-cell conduit for electron flow between
electron-donating and electron-accepting cells. GAC also accelerated electron transfer between G. metalliredu-
cens and M. barkeri promoting methane production from ethanol, and enhanced methane production was
observed if aggregates from an anaerobic wastewater digester were amended with GAC
9
.
OPEN
SUBJECT AREAS:
ENVIRONMENTAL
MICROBIOLOGY
APPLIED MICROBIOLOGY
Received
19 November 2013
Accepted
25 April 2014
Published
21 May 2014
Correspondence and
requests for materials
should be addressed to
S.C. (447681880@
qq.com) or A.-E.R.
(arotaru@microbio.
umass.edu; arotaru@
biology.sdu.dk)
* Current address:
Nordic Center for Earth
Evolution, University of
Southern Denmark,
Campussvej 55,
Odense M, DK-5230,
Denmark.
{ Current address:
Energy Biosciences
Institute University of
California Berkeley,
CA 94704, USA.
SCIENTIFIC REPORTS | 4 : 5019 | DOI: 10.1038/srep05019 1
Biochar, is a GAC precursor
22
and chemical or physical activation
of biochar yields GAC, with a higher surface area, porosity and
increased aromaticity
23,24
. Electron conduction through a solid con-
ductive medium is substantially different than molecular diffusion of
shuttles. Conduction through solid materials involves migration of
electrons in response to a difference in voltage potential whereas
concentration gradients drive diffusion of shuttles. The term electron
shuttle typically refers to soluble molecules such as humic sub-
stances
25
, cysteine
26
, redox active metals
27
, or flavins
28
that can accept
electrons from one microorganism and donate them to another elec-
tron acceptor
29
as a result of concentration gradients.
In this study we addressed the impact of biochar on syntrophic
associations based on DIET. Syntrophic associations are at the root of
carbon cycle in the environment, and this report unravels biochar as
a potential contributor to methane emissions in the environment.
We report on studies on the impact of biochar in defined co-cultures
with G. metallireducens, an effective microorganism for evaluating
DIET because it can donate electrons via DIET, and not by inter-
species H
2
or formate transfer
13,14,16,18
.
Results
Biochar stimulates DIET in Geobacter co-cultures. The addition of
biochar to co-cultures of G. metallireducens and G. sulfurreducens in
medium with ethanol as the electron donor and fumarate as the
electron acceptor stimulated syntrophic metabolism of ethanol
(Figure 1A) with the reduction of fumarate to succinate
(Figure 1B) within two days. In contrast, a control co-culture of G.
metallireducens and G. sulfurreducens, not amended with biochar,
required ca. 30 days to adapt to ethanol metabolism
13
. Rates of
ethanol loss and succinate production with a water-soluble extract
of the biochar were only slightly faster than the biochar-free control
(Figure 1), suggesting that the particulate fraction of the biochar was
primarily responsible for stimulating co-culture metabolism.
The rates of ethanol metabolism varied somewhat with the biochar
types, but this could not be correlated with the small differences in
conductivity because the co-cultures amended with ESI biochar,
which had the lowest conductivity (Table 1), metabolized ethanol
at rates intermediate between the BEC and Kiln biochars, which both
had conductivities ca. 2-fold higher. The fast rates of ethanol meta-
bolism in the presence of biochar were comparable with those prev-
iously observed with the same quantity of GAC
9
. However, the
conductivity of GAC (3000 6 327 mS/cm) measured with the same
method
9
was substantially higher than that of biochar (Table 1). On
the other hand, a non-conductive material, glass beads, did not pro-
mote DIET under similar conditions
9
. In co-cultures amended with
biochar acetate did not accumulate, and thus the oxidation of each
mole of ethanol to carbon dioxide was coupled to the reduction of
fumarate, the electron acceptor for G. sulfurreducens, and was
expected to result in the production of six moles of succinate (reac-
tion 1).
C
2
H
6
O 1 6C
4
H
4
O
4
1 3H
2
O R 2CO
2
1 6C
4
H
6
O
4
(reaction 1)
The amounts of ethanol consumed and succinate produced in the
10 days of incubation (mean 6 standard deviation; n 5 3) in the
presence of the BEC, ESI and Kiln biochars were 8.10 6 0.72/34.98 6
0.48; 6.98 6 0.47/33.37 6 0.24; and 5.15 6 0.23/30.81 6 1.28,
respectively. Thus, the reduction of fumarate to succinate accounted
for 72–88% of the electrons, which were derived from the ethanol
removal in the biochar-amended cultures. In co-cultures amended
with biochar, most (78%) of the co-culture protein was firmly
attached to the solid particles of biochar, after 10 days of incubation
(Figure 2A). Quantitative PCR analysis of the attached cells indicated
that the majority (69%) were G. sulfurreducens (Figure 2B). Scanning
electron microscopy revealed that attached cells did not form aggre-
gates (Figure 3) like co-cultures without biochar, suggesting that
direct biological connections between the cells were unnecessary.
Cultures comprised solely of G. metallireducens slowly metabo-
lized ethanol with a corresponding increase in acetate (Figure 4A),
suggesting that biochar served as an electron acceptor (reaction 3).
C
2
H
6
O 1 H
2
O 1 (Biochar
ox.
) R C
2
H
4
O
2
1 4H
1
1 4e
2
(Biochar
red.
) (reaction 2)
There was no ethanol metabolism in the absence of biochar
(Figure 4B).
From the amount of ethanol metabolized over 10 days (2.8 mmol/
L 3 0.01 L 5 0.028 mmol), and the stoichiometry of electron release
from ethanol metabolism to acetate (4 mmol electrons/mmol eth-
anol) it can be estimated that the 0.25 g of biochar in the culture
tubes accepted 0.112 mmoles of electrons from G. metallireducens,
which is ca. 0.5 mmoles of electrons accepted per gram of biochar.
This is comparable to 0.35 mmoles of electrons that a gram of soil
humic substances is capable of accepting during microbial reduction
processes
30
. However, unlike humic substances that, once reduced,
can donate electrons to Fe(III)
25,31
, no Fe(III) was reduced when the
reduced biochar was exposed to Fe(III) citrate, suggesting that, as
previously demonstrated for GAC
9
, electron transport through bio-
char is unlikely to be attributed to quinone moieties, but rather to the
conductive properties of the materials. Our Fe(III) citrate experi-
ments with bioreduced biochar were carried out under physiological
Figure 1
|
(A) Ethanol consumption and (B) succinate production with
different types of biochar by a syntrophic co-culture of G. metallireducens
and G. sulfurreducens. As control experiments we tested all different
biochars with no cells, the co-culture without biochar and the co-culture
with soluble components released from the biochar. The error bars
represent standard deviations of the mean for triplicate cultures.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 4 : 5019 | DOI: 10.1038/srep05019 2
conditions (pH 6.5–7.0) unlike a previous study which reported that
reduced activated carbon
37
could reduce Fe(III) citrate at low pH
conditions
37
.
Biochar stimulation of DIET in methanogenic co-cultures. Some
methanogenic communities may exchange electrons via DIET
16,32
.
DIET was recently confirmed in co-culture studies with G. meta-
llireducens and Methanosaeta harundinacea
16
, a study which also
demonstrated that G. metallireducens was unable to produce H
2
or
formate to provide electrons for CO
2
reduction to methane by H
2
/
formate utilizing methanogenic partners like Methanospirillum
hungatei or Methanobacterium formicicum
16
. The impact of conduc-
tive materials on DIET and methanogenesis in defined systems was
apparent from studies with co-cultures of G. metallireducens and M.
barkeri amended with GAC
9
. Amending co-cultures of G. metallire-
ducens and M. barkeri with biochar stimulated the conversion of
ethanol to methane (Figure 5A). There was a transient accumula-
tion of acetate, but methane production was dependent on ethanol
metabolism following reaction 2.
2CH
3
CH
2
OH R 3CH
4
1 CO
2
(reaction 3)
In the presence of biochar the metabolism of 21 6 1 mM ethanol
(i.e. 210 mmol in 10 ml media) yielded 16 6 2 mmol/l methane (i.e.
272 mmol in 17 ml headspace). Thus, 86% of the electrons from
ethanol metabolism were recovered in methane. In contrast, there
was no ethanol metabolism or methane production in the G. metal-
lireducens-M. barkeri co-cultures in the absence of biochar
(Figure 5B) or with M. barkeri alone in the presence of biochar
(Figure 5C). The water-soluble biochar fraction had only a minor
stimulatory impact on ethanol metabolism in the G. metallireducens-
M. barkeri co-cultures, suggesting that the solid phase was the prim-
ary stimulatory component (Figure 5D).
Similar to Geobacter co-cultures, most of the cell protein (71%)
was firmly associated to the biochar particles in the methanogenic
co-culture (Figure 6A) and 87% of the attached cells were G. metal-
lireducens (Figure 6B). The higher abundance of G. metallireducens
in the co-culture with M. barkeri, compared with the co-culture with
G. sulfurreducens may be due to larger bio-volume of M. barkeri
versus G. sulfurreducens, as apparent from SEM micrographs
(Figure 3, 7), as well as the lower energy yield from methane produc-
tion versus fumarate reduction. Scanning electron microscopy
revealed that G. metallireducens and M. barkeri attached to the bio-
char, but did not form aggregates with each other, suggesting that the
electrical connections between the two species were through the
biochar rather than via cell-to-cell electron transfer (Figure 7).
Discussion
The results demonstrate that biochar has sufficient conductivity to
promote direct electron transfer between syntrophic partners in co-
cultures based on DIET. This provides a potential explanation for
observations that some biochar amendments can enhance methane
production in soils
33
or in small-scale digesters converting organic
waste to methane
34,35
.
The results suggest that biochar promotes interspecies electron
exchange via a conduction-based mechanism, in which electrons
migrate through the biochar from electron-donating to electron-
accepting cells. This is similar to the mechanism proposed for inter-
species electron transfer through GAC
9
, but differs significantly from
extracellular electron exchange with electron shuttles such as humic
substances
25
. In the absence of conductive materials, microorganisms
growing together, required a long adaption time and numerous
transfers
13
, to get to the same substrate consumption rates as those
observed with biochar or GAC
9
. This suggests that cells required time
to express cellular components required for extracellular electron
transfer
14
.
The ability of biochar to promote DIET with similar rates and
stoichiometries as those observed in co-cultures amended with
Table 1
|
Physical and chemical properties of biochars
Biochar
Original
feedstock Pyrolysis temperature (uC)
Particle size
(mm)
a
BET surface
area (m
2
/g)
b
Electrical conductivity per g
biochar (mS/cm)
c
BEC pine 700 for 30 sec & 500 for 15 min #0.4 15 4.41
ESI pine 500 for 2 h #1 167 2.11
Klin pine 600 for 2 h #3 209 4.33
a
Maximum particle size was estimated by inspection with transmission electron microscopy.
b
The Brunauer-Emmett-Teller (BET) surface areas were obtained from N
2
adsorption at 77 K with a Quantachrome Autosorb-1
41
.
c
The electrical conductivities were determined with a two-electrode system as previously described
20
.
Figure 2
|
(A) Protein in 10 mL of medium on day 0 and day 10 in the planktonic phase and attached to BEC biochar, and (B) quantitative PCR
analysis of the cells attached to the BEC biochar on day 10 in the G. metallireducens/G. sulfurreducens co-cultures. The error bars represent standard
deviations of the mean for triplicate cultures.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 4 : 5019 | DOI: 10.1038/srep05019 3
GAC
9
, might be surprising considering the conductivity of biochar is
1000-fold less than that of GAC. The higher conductivity of activated
carbon is likely due to increased surface area and porosity and
increased aromatization, which happens during the conversion of
biochars into activated carbon at higher temperatures
22,23
.
Aromaticity, a consequence of electron delocalization between aro-
matic rings localized on distinct neighboring planes, gives conductive
properties to graphite, charcoals or other organic polymers
24
, and as
discovered recently even to the pili of Geobacter species
21
. The con-
ductivity of the biochars evaluated here was comparable to that of G.
sulfurreducens pili preparations
20,21
,which are sufficient to effectively
promote DIET. The ability of biochar to stimulate DIET appears to
overcome the adaption period that cells require to begin expressing
high levels of the components that are required for pili-based
DIET
13,14,16
. Materials with increased aromaticity are doped by reduc-
tion or oxidation reactions
36
. If the acceptor microorganism reduces
sections of the biochar and the donor microorganism oxidizes sec-
tions of the biochar, there will be intrinsic charge differences between
sections of biochar, promoting electron flow. This has been also
noted on activated carbon, which accepted electrons from microor-
ganisms and then released the electrons to Fe(III) citrate under acidic
conditions
38
.
However, biochar is a complex material and can modify environ-
ments to which it is added with properties other than conductivity.
For example, biochar was speculated to act as a ‘‘shuttle’’ to mitigate
N
2
O emissions during denitrification in soils
38
. Whereas other stud-
ies suggested that N
2
O formation in soils is due to abiotic processes
happening on biochars surface enriched in surface charged groups,
like quinones, metal ions or radicals
39,40
.
Our observations that biochar increases methane production in
defined co-culture systems, in which partners were capable of direct
electron transfer, changes the present understanding that biochar
could mitigate methane gas emissions. Considering the potential
significant impact of methane production on global warming, and
the persistence of biochar in soil, warrants further long-term studies
on how soil methanogenic communities are affected by biochar
amendments and the impact of biochar on the global carbon cycle.
Methods
Characterization of biochars. Three pine biochars that differed somewhat in their
mode of production, particle size, and surface area were evaluated (Table 1). All
biochars were sieved (pore size 3 mm), and the size of particles was inspected with a
transmission electron microscope. The Brunauer-Emmett-Teller (BET) surface areas
of the other three kinds of biochar were calculated from the N
2
adsorption and
desorption isotherms at 77uK obtained using Quantachrome Autosorb-1 as
previously described
41
. The electrical conductivities of biochar were determined by
two-probe electrical conductance measurements using two gold electrodes separated
by 50 mm non-conductive gap, as previously described
13,20
. Biochar was placed
between the two gold electrodes to bridge the non-conductive gap. Voltage was
applied using a Keithley 2400 sourcemeter. Voltage was scanned from 0 V to
10.05 V in steps of 0.025 V. For each sample, current was measured 100 seconds
after setting the voltage to allow the exponential decay of the transient ionic current in
the gap and to measure steady state electronic current
42
. We collected data with the
Labview data acquisition system (National Instruments, TX, USA), and data analyses
were performed with Igor Pro (Wavemetrics Inc., OR USA).
Microorganisms, media and growth conditions. Geobacter metallireducens strain
GS-15 (ATCC 53774), Geobacter sulfurreducens strain PCA (ATCC 51573), and
Methanosarcina barkeri type strain, DSM 800 (ATCC 43569) were obtained from our
laboratory culture collection. Prior to initiating the co-cultures, G. metallireducens
was maintained in a medium with ethanol (10 mM) as the electron donor and Fe(III)
citrate (55 mM) as the electron acceptor as previously described
17
. G. sulfurreducens
was cultured routinely with 10 mM acetate as electron donor and 40 mM fumarate as
electron acceptor in a fresh water mineral medium as previously described
17
. M.
barkeri was maintained on DSMZ methanogenic medium 120 with 30 mM acetate as
methanogenic substrate
9
. All pure cultures and co-cultures were incubated
anaerobically, in 27 mL pressure tubes with 10 mL medium under an anoxic
atmosphere of 80520 of N
2
:CO
2
as previously described
13,17
.
Co-cultures of G. metallireducens and G. sulfurreducens were initiated as prev-
iously described
9,17
by introducing a 5% inocula of each microorganism into a med-
ium with ethanol (10 mM) as the electron donor and fumarate (40 mM) as the
electron acceptor. The incubation temperature was 30uC. Co-cultures of G. metal-
lireducens and M. barkeri were initiated with a 5% inocula of each microorganism into
DSMZ methanogenic medium 120 with 20 mM ethanol as the electron donor
9
.
Incubations of the methanogenic cocultures were done at 37 u C. All experiments were
carried on with three biological replicates.
In order to test the effect of biochar amendments, 0.25 g of biochar was added to
0.5 ml of the appropriate co-culture medium, under N
2
:CO
2
and the medium was
Figure 3
|
Scanning electron micrograph of one of the biochar tested
(BEC) with a syntrophic co-culture of
G. metallireducens
and
G.
sulfurreducens
.
Figure 4
|
Ethanol consumption, succinate and acetate production in
medium with ethanol as the electron donor with (A) G. metallireducens
and the BEC biochar or (B) G. metallireducens alone. The error bars
represent standard deviations of the mean for triplicate cultures.
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SCIENTIFIC REPORTS | 4 : 5019 | DOI: 10.1038/srep05019 4
autoclaved for 30 minutes. Additional sterile medium (9 ml) was added under
anaerobic conditions, while ethanol and cells were inoculated afterwards.
Water extracts of biochar were obtained by incubating 0.25 g of biochar in 9 ml of
culture medium on a shaker under sterile, anaerobic conditions for either 10 days (G.
metallireducens - G. sulfurreducens co-cultures) or 20 days (G. metallireducens - M.
barkeri co-cultures) to replicate the length of time that the co-cultures were exposed
to biochar. Ethanol and cells were added afterwards.
Quantitative PCR analysis. DNA was extracted from the cells attached to biochar
from triplicate 10 ml co-cultures as previously described
9
. To determine the specific
abundance of cells in co-cultures amended with biochar, quantitative PCR was
performed using G. metallireducens specific primers (Gmet_F 59-TGGCCCACAT-
CTTCATCTC-39, Gmet_R 59-TGCATGTTTTCATCCACGAT-39), versus
primers universal to both G. sulfurreducens and G. metallireducens (Geo16S_F
59-GAGGTACCGTCAAGACCAA-39, Geo16S_R 59-GCCACACTG-
GAACTGAGACA-39), or specific for Methanosarcina species (MB16S_F 59-
GGGTCTAAAGGGTCCGTAGC-39, MB16S_R 59 GTTCTGGTAAGACGCCT-
TCG-39), depending on the type of co-culture tested. Before conducting the
quantitative PCR experiment primer pairs were validated for the primer efficiency.
The quantitative PCR was carried out using Fast Syber green master mix (ABI) in Real
time PCR cycler (ABI-9500) following the manufacturer’s protocol.
Scanning electron microscopy. In order to evaluate cell attachment to biochar, the
biochar-attached fraction was studied by scanning electron microscopy at the end of
the co-culture growth (day 10 of the G. metallireducens-G.sulfurreducens cycle, and
day 20 of the G. metallireducens-M. barkeri). Samples were first fixed with 2.5%
Figure 5
|
Ethanol consumption, methane and acetate production in (A) co-cultures of G. metallireducens-M. barkeri with the BEC biochar,
(B) unamended co-cultures of G. metallireducens-M. barkeri (C) and pure culture of M. barkeri with the BEC biochar, and (D) co-cultures of G.
metallireducens-M. barkeri with the soluble components released from the BEC biochar. The error bars represent standard deviations of the mean for
triplicate cultures.
Figure 6
|
(A) Protein in 10 mL of medium on day 0 and day 20 in the planktonic phase and attached to BEC biochar, and (B) Quantitative PCR analysis
of the cells attached to the BEC biochar on day 20 in the G. metallireducens/M. barkeri co-cultures. The error bars represent standard deviations
of the mean for triplicate cultures.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 4 : 5019 | DOI: 10.1038/srep05019 5
glutaraldehyde in 0.1 M phosphate buffer for up to 12 hours at 4uC, then washed 3
times in 0.1 M phosphate buffer at 4uC for 10 min each, dehydrated further in an
ethanol/water mixture of 50%, 70%, 80%, 90%, 95% and 100% for 10 minutes each
(dehydration in 100% ethanol was done 3 times), and at last immersed twice for 30
seconds in pure hexamethyldisilazane (Sigma Aldrich, St Louis, MO, USA) followed
by 10 minutes of air-drying
43
.
Analytical techniques. Subsamples for gas chromatographic analysis of methane and
ethanol and high performance liquid chromatography analysis of acetate, fumarate,
formate and succinate were withdrawn and processed as previously described
32
.In
order to further support biomass attachment onto biochar at the end of co-culture
incubations, the liquid fraction was separated from the biochar by lysis of the biochar
in 0.5 N NaOH and bead beating for 30 seconds with sterile glass beads to ensure cell
disruption. Cell protein in the culture supernatant and biochar fractions was
determined using the bicinchoninic acid method
44
with bovine serum albumin (BSA)
as protein standard.
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Acknowledgments
We thank Trevor Woodard for facilitating HPLC and GC analysis; Louis Raboin for
facilitating SEM analysis; Kenneth H. Williams and Baoshan Xing for providing biochar.
The first author thanks the Oversea Study Program of Guangzhou Elite Project and the
Innovative Dotorial Candidates Training Project of Sun Yat-sen University. This research
Figure 7
|
Scanning electron micrograph of a syntrophic co-culture of
G.
metallireducens
(rods) and
M. barkeri
(spheres) with the BEC biochar.
The white arrow points to the representative cells.
www.nature.com/scientificreports
SCIENTIFIC REPORTS | 4 : 5019 | DOI: 10.1038/srep05019 6
was supported by the Office of Science (BER), U. S. Department of Energy, Award No.
DESC0004485.
Author contributions
D.R.L., A.E.R. and S.C. conceived the experiments. S.C. performed the experiments with
help from A.E.R., F.L. and K.P.N. P.M.S. performed qPCR experiments. N.S.M. performed
the conductivity measurements. W.F. characterized the biochars. D.R.L., S.C. and A.E.R.
analyzed the data and wrote the manuscript. All authors have seen the manuscript at all
stages, discussed the data and agreed to the content.
Additional information
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Chen, S. et al. Promoting Interspecies Electron Transfer with
Biochar. Sci. Rep. 4, 5019; DOI:10.1038/srep05019 (2014).
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SCIENTIFIC REPORTS | 4 : 5019 | DOI: 10.1038/srep05019 7
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