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.
Content may be subject to copyright.
Promoting Interspecies Electron Transfer
with Biochar
Shanshan Chen
, Amelia-Elena Rotaru
*, Pravin Malla Shrestha
{, Nikhil S. Malvankar
, Fanghua Liu
Wei Fan
, Kelly P. Nevin
& Derek R. Lovley
Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA,
School of Environmental Science and
Engineering, Sun Yat-sen University, Guangzhou 510275, China,
Department of Physics, University of Massachusetts, Amherst,
MA 01003, USA,
Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China,
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
Geobacter sulfurreducens
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.
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
, but the impact of biochar on soil microbial com-
munities is poorly understood. Biochar has been used to enhance soil fertility
, remediate pollution,
decrease green house gas emissions
and sequester carbon in a worldwide quest to achieve carbon neutrality
Biochar is hypothesized to increase the microbial soil content by concentrating organic substrates and nutrients
on its surface
. Another feature of biochar that could potentially influence the activity of soil microbiota is that
biochar is electrically conductive
. Conductive minerals/materials such as magnetite or GAC facilitated DIET by
accelerating rates of anaerobic metabolism
. Under certain conditions, DIET is considered to be potentially
more effective for interspecies electron transfer than strategies such as interspecies H
or formate transfer that rely
on the diffusion of electron carriers between species
DIET has been shown possible in the absence of conductive materials via biological electrical connections such
as pili
. 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
. 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
, whereas G. sulfurreducens cannot use ethanol as an
electron donor, but can reduce the electron acceptor, fumarate
. 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
. Multiple lines of evidence
demonstrated that interspecies electron
transfer in Geobacter co-cultures proceeded via DIET rather than H
or formate transfer, and that the Geobacter
pili, which posses metallic-like conductivity
, were required during DIET.
However, the initial adaption of Geobacter co-cultures to interspecies electron transfer via pili-mediated DIET
took ca. 30 days
. GAC substantially reduced the adaptation period for growth via DIET, with ethanol meta-
bolism evident within a day
. 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
, but functioned as well as wild-type in the presence of GAC
Instead of forming close cell-to-cell connections as observed in the absence of GAC
, 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
19 November 2013
25 April 2014
21 May 2014
Correspondence and
requests for materials
should be addressed to
S.C. (447681880@
qq.com) or A.-E.R.
umass.edu; arotaru@
* Current address:
Nordic Center for Earth
Evolution, University of
Southern Denmark,
Campussvej 55,
Odense M, DK-5230,
{ 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
and chemical or physical activation
of biochar yields GAC, with a higher surface area, porosity and
increased aromaticity
. 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-
, cysteine
, redox active metals
, or flavins
that can accept
electrons from one microorganism and donate them to another elec-
tron acceptor
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
or formate transfer
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
. 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
. However, the
conductivity of GAC (3000 6 327 mS/cm) measured with the same
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
. 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).
O 1 6C
1 3H
1 6C
(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).
O 1 H
O 1 (Biochar
) R C
1 4H
1 4e
) (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
. However, unlike humic substances that, once reduced,
can donate electrons to Fe(III)
, no Fe(III) was reduced when the
reduced biochar was exposed to Fe(III) citrate, suggesting that, as
previously demonstrated for GAC
, 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.
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
could reduce Fe(III) citrate at low pH
Biochar stimulation of DIET in methanogenic co-cultures. Some
methanogenic communities may exchange electrons via DIET
DIET was recently confirmed in co-culture studies with G. meta-
llireducens and Methanosaeta harundinacea
, a study which also
demonstrated that G. metallireducens was unable to produce H
formate to provide electrons for CO
reduction to methane by H
formate utilizing methanogenic partners like Methanospirillum
hungatei or Methanobacterium formicicum
. 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
. 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.
1 CO
(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).
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
or in small-scale digesters converting organic
waste to methane
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
, but differs significantly from
extracellular electron exchange with electron shuttles such as humic
. In the absence of conductive materials, microorganisms
growing together, required a long adaption time and numerous
, to get to the same substrate consumption rates as those
observed with biochar or GAC
. This suggests that cells required time
to express cellular components required for extracellular electron
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
feedstock Pyrolysis temperature (uC)
Particle size
BET surface
area (m
Electrical conductivity per g
biochar (mS/cm)
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
Maximum particle size was estimated by inspection with transmission electron microscopy.
The Brunauer-Emmett-Teller (BET) surface areas were obtained from N
adsorption at 77 K with a Quantachrome Autosorb-1
The electrical conductivities were determined with a two-electrode system as previously described
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.
SCIENTIFIC REPORTS | 4 : 5019 | DOI: 10.1038/srep05019 3
, 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
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
, and as
discovered recently even to the pili of Geobacter species
. The con-
ductivity of the biochars evaluated here was comparable to that of G.
sulfurreducens pili preparations
,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
. Materials with increased aromaticity are doped by reduc-
tion or oxidation reactions
. 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
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
O emissions during denitrification in soils
. Whereas other stud-
ies suggested that N
O formation in soils is due to abiotic processes
happening on biochars surface enriched in surface charged groups,
like quinones, metal ions or radicals
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.
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
adsorption and
desorption isotherms at 77uK obtained using Quantachrome Autosorb-1 as
previously described
. 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
. 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
. 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
. 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
. M.
barkeri was maintained on DSMZ methanogenic medium 120 with 30 mM acetate as
methanogenic substrate
. 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
as previously described
Co-cultures of G. metallireducens and G. sulfurreducens were initiated as prev-
iously described
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
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
and the medium was
Figure 3
Scanning electron micrograph of one of the biochar tested
(BEC) with a syntrophic co-culture of
G. metallireducens
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.
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
. 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-
primers universal to both G. sulfurreducens and G. metallireducens (Geo16S_F
GAACTGAGACA-39), or specific for Methanosarcina species (MB16S_F 59-
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.
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
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
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
with bovine serum albumin (BSA)
as protein standard.
1. Lehmann, J. A handful of carbon. Nature 447, 143–144 (2007).
2. Zheng, H. et al. Characteristics and nutrient values of biochars produced from
giant reed at different temperatures. Bioresour. Technol. 130, 463–471 (2013).
3. Chan, K., Van Zwieten, L., Meszaros, I., Downie, A. & Joseph, S. Using poultry
litter biochars as soil ammendments. Soil Res. 46, 437–444 (2008).
4. Woolf, D., Amonette, J. E., Street-Perrott, F. A., Lehmann, J. & Joseph, S.
Sustainable biochar to mitigate global climate change. Nat. Commun. 1, 56 (2010).
5. Lehmann, J. et al. Biochar effects on soil biota: A review. Soil Biol. Biochem. 43,
1812–1836 (2011).
6. Xu, W., Pignatello, J. & Mitch, W. Role of black carbon electrical conductivity in
mediating hexahydro-1,3,5-triazine (RDX) transformation on carbon surfaces by
sulfides. Environ. Sci. Technol. 34, 2472–2478 (2013).
7. Kato, S., Hashimoto, K. & Watanabe, K. Methanogenesis facilitated by electric
syntrophy via (semi)conductive iron-oxide minerals. Environ. Microbiol. 14,
1646–1654 (2012).
8. Kato, S., Hashimoto, K. & Watanabe, K. Microbial interspecies electron transfer
via electric currents through conductive minerals. Proc. Natl. Acad. Sci. USA 109,
10042–10046 (2012).
9. Liu, F. et al. Promoting direct interspecies electron transfer with activated carbon.
Energy Environ. Sci. 5, 8982–8989 (2012).
10. Liu, F. et al. Magnetite compensates for the lack of a pilin-associated c-type
cytochrome in extracellular electron transfer. Environ. Microbiol. DOI: 10.1111/
1462-2920.12485. (2014).
11. Lovley, D. R. Live wires: direct extracellular electron exchange for bioenergy and
the bioremediation of energy-related contamination. Energy Environ. Sci. 4,
4896–4906 (2011).
12. Lovley, D. R. Electromicrobiology. Annu. Rev. Microbiol. 66, 391–409 (2012).
13. Summers, Z. M. et al. Direct exchange of electrons within aggregates of an evolved
syntrophic coculture of anaerobic bacteria. Science 330, 1413–1415 (2010).
14. Shrestha, P. M. et al. Transcriptomic and genetic analysis of direct interspecies
electron transfer. Appl. Environ. Microbiol. 79, 2397–2404 (2013).
15. Shrestha, P. M. et al. Syntrophic growth with direct electron transfer as the sole
mechanism for for energy exchange. Environ. Microbiol. Rep. 5, 904–910 (2013).
16. Rotaru, A.-E. et al. A new model for electron flow during anaerobic digestion:
direct interspecies electron transfer to Methanosaeta for the reduction of carbon
dioxide to methane. Energy Environ. Sci. 7
, 408–415 (2014).
17. Rotaru, A.-E. et al. Interspecies electron transfer via hydrogen and formate rather
than direct electrical connections in co-cultures of Pelobacter carbinolicus and
Geobacter sulfurreducens. Appl. Environ. Microbiol. 78, 7645–7651 (2012).
18. Lovley, D. R. et al. Geobacter metallireducens gen. nov. sp. nov., a microorganism
capable of coupling the complete oxidation of organic compounds to the
reduction of iron and other metals. Arch. Microbiol. 159, 336–344 (1993).
19. Caccavo, F., Jr. et al. Geobacter metallireducens sp. nov., a hydrogen- and acetate-
oxidizing dissimilatory metal-reducing microorganism. Appl. Environ. Microbiol.
60, 3752–3759 (1994).
20. Malvankar, N. S. et al. Tunable metallic-like conductivity in microbial nanowire
networks. Nat. Nanotechnol. 6, 573–579 (2011).
21. Vargas, M. et al. Aromatic amino acids required for pili conductivity and long-
range extracellular electron transport in Geobacter sulfurreducens. mBio 4,
e00105–13 (2013).
22. Azargohar, R. & Dalai, A. in 27th Symposium on Biotechnology for Fuels and
Chemicals, Vol. 129–132. (eds . McMillan, J., Adney, W., Mielenz, J. & Klasson, K.)
762–773 (Humana Press, Denver, CO, USA; 2006).
23. Lehmann, J. & Joseph, S. (eds.) Biochar for environmental management: science
and technology. (Earthscan, London, Sterling, VA; 2009).
24. Bourke, J. et al. Do all carbonized charcoals have the same chemical structure? 2. A
model of the chemical structure of carbonized charcoal. Ind. Eng. Chem. Res. 46,
5954–5967 (2007).
25. Lovley, D. R., Coates, J. D., Blunt-Harris, E. L., Phillips, E. J. & Woodward, J. C.
Humic substances as electron acceptors for microbial respiration. Nature 382,
445–448 (1996).
26. Kaden, J., Galushko, A. & Schink, B. Cysteine-mediated electron transfer in
syntrophic acetate oxidation by cocultures of Geobacter sulfurreducens and
Wolinella succinogenes. Arch. Microbiol. 178, 53–58 (2002).
27. Nevin, K. P. & Lovley, D. R. Potential for nonenzymatic reduction of Fe (III) via
electron shuttling in subsurface sediments. Environ. Sci. Technol. 34, 2472–2478
28. Hernandez, M. & Newman, D. Extracellular electron transfer. Cell. Mol. Life Sci.
58, 1562–1571 (2001).
29. Lovley, D. R. Bug juice: harvesting electricity with microorganisms. Nat. Rev.
Microbiol. 4, 497–508 (2006).
30. Lovley, D. et al. Humic substances as a mediator for microbially catalyzed metal
reduction. Acta Hydrochim. Hydrobiol. 26, 152–157 (1998).
31. Scott, D. T., McKnight, D. M., Blunt-Harris, E. L., Kolesar, S. E. & Lovley, D. R.
Quinone moieties act as electron acceptors in the reduction of humic substances
by humics-reducing microorganisms. Environ. Sci. Technol. 32, 2984–2989
32. Morita, M. et al. Potential for direct interspecies electron transfer in methanogenic
wastewater digester aggregates. mBio 2, e00159–00111 (2011).
33. Yu, L., Tang, J., Zhang, R., Wu, Q. & Gong, M. Effects of biochar applicati on on
soil methane emission at different soil moisture levels. Biol. Fert. Soils 49, 119–128
34. Inthapanya, S., Preston, T. & Leng, R. Biochar increases biogas production in a
batch digester charged with cattle manure. Livest. Res. Rural Dev. 24, Article #212
35. Das, K., Balagursamy, N., Chinnasamy, S., Martinez Castro, G. J. & Espino Lopez,
C. in WO/2011/019871. (ed. WO Patent 2, 019,871) (WO Patent 2, 011,019,871,
WO Patent 2,011,019,871; 2011).
36. Snook, G. A., Kao, P. & Best, A. S. Conducting-polymer-based supercapacitor
devices and electrodes. J. Power Sources 196, 1–12 (2011).
37. van der Zee, F. P., Bisschops, I. A. E., Lettinga, G. & Field, J. A. Activated Carbon as
an Electron Acceptor and Redox Mediator during the Anaerobic
Biotransformation of Azo Dyes. Environ. Sci. Technol. 37, 402–408 (2002).
38. Cayuela, M. L. et al. Biochar and denitrification in soils: when, how much and why
does biochar reduce N2O emissions? Sci. Rep. 3 (2013).
39. Joseph, S. D. et al. An investigation into the reactions of biochar in soil. Soil Res. 48,
501–515 (2010).
40. Heymann, K., Lehmann, J., Solomon, D., Schmidt, M. W. I. & Regier, T. C 1 s K-
edge near edge X-ray absorption fine structure (NEXAFS) spectroscopy for
characterizing functional group chemistry of black carbon. Org. Geochem. 42,
1055–1064 (2011).
41. Fan, W. et al. Hierarchical nanofabrication of microporous crystals with ordered
mesoporosity. Nature Mater. 7, 984–991 (2008).
42. Du, K. et al. Self-Assembled Electrical Contact to Nanoparticles Using Metallic
Droplets. Small 5, 1974–1977 (2009).
43. Araujo, J. C. et al. Comparison of hexamethyldisilazane and critical point drying
treatments for SEM analysis of anaerobic biofilms and granular sludge. J. Electron.
Microsc. 52, 429–433 (2003).
44. Smith, P. et al. Measurement of protein using bicinchoninic acid. Anal. Biochem.
150, 76–85 (1985).
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
(rods) and
M. barkeri
(spheres) with the BEC biochar.
The white arrow points to the representative cells.
SCIENTIFIC REPORTS | 4 : 5019 | DOI: 10.1038/srep05019 6
was supported by the Office of Science (BER), U. S. Department of Energy, Award No.
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).
This work is licensed under a Creative Commons Attribution-NonCommercial-
ShareAlike 3.0 Unported License. The images in this article are included in the
article’s Creative Commons license, unless indicated otherwise in the image credit;
if the image is not included under the Creative Commons license, users will need to
obtain permission fromthe license holder in order to reproduce the image. To view a
copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
SCIENTIFIC REPORTS | 4 : 5019 | DOI: 10.1038/srep05019 7
... a) Magnetite particles, indicated in the micrograph by white arrows, lodge in the e-pili, replacing the role of type C cytochromes [44]. b) The microorganisms cells, indicated in the micrograph by white arrows, are adhered to the surface of the biochar [46]. the process via CMs [12]. ...
In recent years, many researches have reported that anaerobic digestion and methane production can be significantly improved with the addition of conductive materials to the process. Despite advances, a number of questions about this strategy remain unsolved, including the mechanism and impact of material properties on methanogenic pathways. In order to provide an update on the current state of knowledge and future application perspectives, this work analyzed some of the most recent studies using conductive materials in the anaerobic digestion of effluents through a systematic review of the literature and statistical analysis, as well as the enriched microorganisms, the influence of the dosage, size and conductivity of the materials used. It was found that the change in the microbial community is associated with the use of conductive material (p < 0.05). Additionally, a slight trend was suggested that millimeter-scale materials appear to be more effective in increasing methane production ; in contrast to expectations, it was not possible to find a correlation between electrical conductivity and an increase in methane production, supporting the idea that other properties may also perform important roles in the process and require further research. In general, the use of conductive materials is a promising approach to improve anaerobic digestion and methane production, however, the need for future research was indicated to expand the scale of application of this technology.
... Since the DIET among methanogenesis was proposed, conductive materials have been extensively reported to enhance methane production in AD system. Up to now, much work has shown that adding conductive materials like granular active carbon [67,68], biochar [69], carbon fiber [70], magnetite [71], hematite [72], activated carbon, and nano-zero-valent iron [31] could promote the methanogenesis through improving the effectiveness of syntrophic interaction [73], interspecies electron transfer [74,75]. The addition of conductive materials in AD system could promote the utilization of electrons for methanogenesis [64]. ...
The low efficiency and stability of anaerobic digestion (AD) has always hindered its wide application. The accumulation of volatile fatty acids (VFAs) cause acidification and inhibition of anaerobic digester. The conversion of some VFAs (propionate and butyrate) to acetate, CO2, and H2 is endergonic under standard conditions, which make it difficult to occur spontaneously in AD system. Thus, the degradation of VFAs is regarded as the rate-limiting step in AD. Studies proved that VFAs could be degraded through the syntrophic cooperation between bacteria and methanogens. Therefore, clear insight into the mechanism of syntrophic methanogenesis as well as the process optimization in syntrophic metabolism of VFAs are essential to improve the efficiency of AD. The present review summarized the roles of syntrophic bacteria and methanogens in AD process, containing metabolism pathways, biochemical reactions, microorganisms characteristics, substrate oxidation, and electron transfer in syntrophic methanogenesis. The main forms and mechanisms of interspecies electron transfer were detailly and deeply described. In addition, the process control and optimization of the syntrophic metabolism of VFAs were also summarized in the review for improving the efficiency of syntrophic methanogenesis. This review provides a deeper understanding of efficiency enhancement of syntrophic metabolism in AD system.
... The biochar has electron shuffling properties where it can act as an electron donor; e.g., the electron can be donated to the target element in the soil or the biochar ability to exchange ions such as Ca 2+ and K + , and this ability is due to the o-functional groups and can act as an electron acceptor where electrons can be transferred from microbial cell to the functional group capable to accept the electron such as quinone or the ability of the biochar to retain anions such as NO 3− and PO 4 3− [55]. Most biochar created at > 600 are electrical conductors [56]. ...
Full-text available
The accumulation of heavy metals in water bodies degrades the water quality and availability. Heavy metals are toxic and can be fatal if consumed. Various techniques such as ion-exchange, precipitation, and adsorption have been used to extract heavy metals in wastewater. The process of adsorption will be reviewed in this study since it uses various adsorbents from industrial waste to agricultural waste and is inexpensive. The production of adsorbents from industrial waste produces large amounts of toxins such as greenhouse gases and it is also costly to produce; thus, it was suggested that adsorbents are produce using biomass, which supports both circular economy and sustainability. The most effective biomass adsorbent is activated carbon; however, it has high production costs than biochar. This study will review on synthesis of biochar, its contribution to circular economy, biochar adsorption mechanisms, heavy metals extraction techniques, and peanut shells as an effective adsorbent to extract heavy metals, namely, chromium, cadmium, lead, zinc, and copper and as a low-cost adsorbent. Furthermore, limitations to using peanut shell-derived biochar are identified. Studies were conducted using peanut shells and it was found that even using peanut shell without pyrolysis is effective to remove heavy metals. In one study, raw peanut shells (non-pyrolyzed) were used to extract lead and the peanut shells’ dosages were at 0.5 g, 1.0 g, and 1.5 g and it was found that 74.36%, 74.57%, and 74.05% of lead was extracted, respectively. In other study, the peanut shells were pyrolyzed to produce biochar and used to extract Cr(I II), Cu(II), and Pb(II) and it was found that it extracted 80%, 85%, and 90% of the metal ions, respectively. This shows that biochar adsorbs more heavy metal ions; thus, it is necessary to thermally degrade the biomass before usage. More literature on the usage of peanut shells to extract heavy metals in wastewater are reviewed in this article to further show that peanut shells have potential to be used as an adsorbent.
... Inhibitory compounds in the digester are adsorbed by the biochar, promoting Direct Interspecies Electronic Transfer (DIET) and acting as microbial immobilization. The porosity of the biochar creates a surface area on which methanogenic archaea develops and adheres, reducing the inhibition of absorbent inhibitors [43,44]. Biochar also acts as an adsorbent to support the development of a stable bacterial community [45]. ...
This study aims to perform an LCA of the co-digestion of pig manure with two co-substrates (corn silage and elephant grass) and an additive (biochar) to produce biogas for electricity generation. Four scenarios were defined: considering only the digestion of pig manure (Base scenario); co-digestion of pig manure with elephant grass-silage (scenario II); co-digestion of pig manure with corn silage (scenario III), and digestion of pig manure with biochar as an additive (scenario IV). The biogas produced was used for electricity generation in a cogeneration system. The thermal energy obtained from the cogeneration system was used to heat the substrate, maintain a mesophilic digestion temperature (38 °C), and heat the pig stalls. Digestate was considered an organic fertilizer in this analysis. Scenario III presented the worst results for most of the impact categories considered in the LCA, with only a positive impact on ozone depletion (ODP). Scenario II presented a lower impact than Scenario III. Using co-substrates and additives in the digestion of pig manure has allowed a considerable increase in electricity production. However, the environmental impacts of using co-substrates are significant in most impact categories, mainly due to the use of fossil fuels in the chain of silage production.
The effects of combining magnetized cellulase (MC) with Ni-graphite (NG) coated materials on the anaerobic digestion of corn stover were investigated. In pretreatment of the corn stover, cellulase showed higher glycosylation efficiency in a 30 mT magnetic field environment than in other magnetic fields, and the reducing sugar content reached 0.182 mg/L. Treatment with the combination of magnetized cellulase and Ni-graphite coated material resulted in a 74.35% increase in cumulative CH4 yield. Analysis of the composition of the microbial community showed that the addition of Ni-graphite coating significantly influenced the composition of the microbial community. The electrically active microorganisms Bacteroidota (46.98%) and Methanomicrobiales (6.08%) became the dominant bacteria and archaea when the corn stover was treated with the NG+MC combination. Carbon balance calculations showed that the combination treatment resulted in carbon flow distributions that differed from other treatments, and the energy conversion efficiency (ECE) reached 57.1%.
Prevailing global increases in population, urbanization, and agricultural production are causing increased pressures on water resources, especially as the use of chemicals in agriculture, industry, and medicine provide new challenges for water treatment and reuse. Organohalogen compounds are persistent contaminants that often evade current wastewater treatment technologies, resulting in their accumulation in the environment and posing a serious threat to ecosystem health. Recent advances in understanding pyrogenic carbons as electron shuttling and storing materials have exposed their potential for enhancing the dehalogenation and overall degradation of organohalide contaminants in soil, sediment, surface water, and wastewater systems. Biochar is a porous carbonaceous material produced during the thermochemical decomposition of biomass feedstock in the presence of little or no oxygen (pyrolysis). Interest in biochar for application towards environmental remediation is largely based on its three distinct benefits: (1) carbon sequestration to offset greenhouse gas emissions, (2) adsorption of (in-) organic contaminants and nutrients, and (3) a strong electron exchange capacity. Due to the innate complexity of biochar materials, several electron transfer mechanisms exist by which biochar may mediate contaminant degradation. These electron transfer pathways include electron-accepting and donating cycles through redox-active functional groups and direct electron transfer via conductive carbon matrices. These mechanisms are responsible for biochar's participation in multiple redox-driven biogeochemical transformations with proven consequences for effective organohalogen remediation. This literature review summarizes the current knowledge on the mechanisms and processes through which biochar can directly or indirectly mediate the transformation of organohalogen compounds under various environmental conditions. Perspectives and research directions for future application of biochars for targeted remediation strategies are also discussed.
Permafrost thaw in northern peatlands is likely to create a positive feedback to climate change, as microbes transform soil carbon into carbon dioxide (CO2) or methane (CH4). While the microbiome's encoded C-processing potential changes with thaw, the impact on substrate utilization and gas emissions is less well characterized. We therefore examined microbial C cycling dynamics from a partially thawed Sphagnum-dominated bog to a fully thawed sedge-dominated fen in Stordalen Mire (68.35°N, 19.05°E), Sweden. We profiled C substrate utilization diversity and extent by Biolog Ecoplates™, then tested substrate-specific hypotheses by targeted additions (of glucose, the short chain fatty acids (SCFAs) acetate and butyrate, and the organic acids galacturonic acid and p-hydroxybenzoic acid, all at field-relevant concentrations) under anaerobic conditions at 15 °C. In parallel we characterized microbiomes (via 16S rRNA amplicon sequencing and quantitative polymerase chain reaction) and C gas emissions. The fen exhibited a higher substrate use diversity and faster rate of overall substrate utilization than in the bog, based on Biolog Ecoplate™ incubations. Simple glucose additions (akin to a positive control) to peat microcosms fueled fermentation as expected (reflected in enriched fermenter lineages, their inferred metabolisms, and CO2 production), but also showed potential priming of anaerobic phenol degradation in the bog. Addition of SCFAs to bog and fen produced the least change in lineages and in CO2, and modest suppression of CH4 primarily in the fen attributed to inhibition. Addition of both organic acids greatly increased the CO2:CH4 ratio in the deep peats but had distinct individual gas dynamics and impacts on microbiota. Both organic acids appeared to act as both C source and as a microbial inhibitor, with galacturonic acid also likely playing a role in electron transfer or acceptance. Collectively, these results support the importance of aboveground-belowground linkages - and in particular the role of Sphagnum spp.- in supplying substrates and inhibitors that drive microbiome C processing in these dynamically changing systems. In addition, they highlight an important temporal dynamic: responses on the short time scale of incubations (which would reflect transition conditions in the field) differ from those evident at the longer scales of habitat transition, in ways consequential to C gas emissions. In the short term, substrate addition response reflected microbiome legacy (e.g., bog communities were slower to process C and better tolerated inhibitors than fen communities) but led to little overall increase in C gas production (and a high skew to CO2). At the longer time scale of bog and fen thaw stages (which are used to represent these systems in models) the concomitant shifts in plants, hydrology and microbiota attenuate microbiome legacy impacts on substrate processing and C gas emissions over time. As habitat transition areas expand under accelerating change, we hypothesize an increased role of microbiome legacy in the landscape overall, leading to a lag in the increase of CH4 emissions expected from fen expansion.
The simultaneous removal of nitrate and bisphenol A (BPA), two highly concerning contaminants in fluvial systems, is limited due to the low activity of the denitrifying community and poor degradability of BPA. Iron-loaded biochars (FeBCs) are efficient in promoting electron transfer efficiency and accelerating redox active processes, e.g., nitrate reduction and pollutant degradation. Nevertheless, the physicochemical properties of FeBCs and their potential strengthening mechanism during simultaneous removal of nitrate and BPA from aquatic systems are largely unknown. This study explored the potential of FeBCs to accelerate the simultaneous removal of nitrate and BPA. The FeBCs were prepared at 300–700 °C, with a BET surface area that increased from 10.78 to 207.97 m² g⁻¹ and oxygen content that decreased from 12.16% to 3.89%. Maximum nitrate and BPA removal of 99.0% and 74.1%, respectively, was achieved when the biochar was pyrolyzed at 500 °C (FeBC5). FeBC5 increased the levels of nicotinamide adenine dinucleotide (NADH), 5′-adenylate triphosphate (ATP), and electron transport system activity (ETSA) of the microbes, which were 485.50%, 371.88%, and 68.85% higher than the control values. After adding 0.05 g L⁻¹ FeBC, the denitrifying enzyme activity and the level of nitrate-reducing genes increased by 48.22% and 45.6% respectively, and the genera responsible for denitrification and BPA degradation were increased by 5.56% and 33.33%, respectively. The BPA degradation pathway analysis suggested that the enhanced biotransformation of BPA resulted from co-metabolic degradation by denitrifying bacteria. The ferric-containing functional groups and their types were significantly correlated with nitrate-reducing enzymes and metabolic activities, facilitating the simultaneous removal of nitrate and BPA.
Full-text available
Anaerobic conversion of organic wastes and biomass to methane is an important bioenergy strategy, which depends on poorly understood mechanisms of interspecies electron transfer to methanogenic microorganisms. Metatranscriptomic analysis of methanogenic aggregates from a brewery wastewater digester, coupled with fluorescence in situ hybridization with specific 16S rRNA probes, revealed that Methanosaeta species were the most abundant and metabolically active methanogens. Methanogens known to reduce carbon dioxide with H2 or formate as the electron donor were rare. Although Methanosaeta have previously been thought to be restricted to acetate as a substrate for methane production, Methanosaeta in the aggregates had a complete complement of genes for the enzymes necessary for the reduction of carbon to methane, and transcript abundance for these genes was high. Furthermore, Geobacter species, the most abundant bacteria in the aggregates, highly expressed genes for ethanol metabolism and for extracellular electron transfer via electrically conductive pili, suggesting that Geobacter and Methanosaeta species were exchanging electrons via direct interspecies electron transfer (DIET). This possibility was further investigated in defined co-cultures of Geobacter metallireducens and Methanosaeta harundinacea which stochiometrically converted ethanol to methane. Transcriptomic, radiotracer, and genetic analysis demonstrated that M. harundinacea was accepting electrons via DIET for the reduction of carbon dioxide to methane. The discovery that Methanosaeta species, which are abundant in a wide diversity of methanogenic environments, are capable of DIET has important implications not only for the functioning of anaerobic digesters, but also for global methane production.
Full-text available
The aim of this study was to investigate the effects of biochar application on soil methane (CH4) emission. Experiments were conducted over an 84-day incubation period with the following treatments: each of two soils (a paddy soil and a forest soil) was treated with or without biochar at three soil moisture levels (35, 60, and 100 % water-filled pore space (WFPS) for the paddy soil; 35, 60, and 85 % WFPS for the forest soil). Biochar application (P < 0.05) significantly increased soil pH and stimulated C mineralization at the early incubation stage. The effects of biochar application on CH4 emission were related to the soil moistures, with reduction of CH4 emission at 35 and 60 % WFPS and stimulation at the highest soil moisture. While both soils changed from CH4 sinks to sources by increasing soil moisture regardless of biochar addition, the effect was enhanced with biochar application. At lower soil moistures, the CH4 oxidation activity in soils was higher with biochar than without biochar, while the trend became opposite at higher soil moistures. Therefore, the CH4 production and consumption processes were influenced by different soil moisture levels and microbial communities of different soils.
Two in vitro incubation experiments were conducted to test the hypothesis that biochar would serve as support media for biofilm development in a biodigester and would as a result increase the yield of biogas whether added separately or enclosed in a nylon bag The treatments in experiment 1 were: control (no biochar), biochar added at 1% of the substrate DM in the biodigester, biochar added at 3% of the substrate DM in the biodigester. The substrate was fresh manure from cattle fed dried cassava root, fresh cassava foliage and urea. Proportions of water and manure were arranged so that the manure provided 5% of the solids in the biodigester. Gas production was measured daily over the fermentation period of 30 days; methane in the gas was measured after 21 and 28 days. In experiment 2, a 2*2 factorial arrangement with 4 replications was used to compare level of biochar: 1% of solids in the digester or none; and presence or absence of a cloth bag in the biodigester. The fermentation was followed over 21 days with daily measurement of gas production and content of methane in the gas at the end of the fermentation. In experiment 1, incorporation of 1% (DM basis) of biochar in the biodigester increased gas production by 31% after 30 days of continuous fermentation; there were no benefits from increasing the biochar to 3% of the substrate DM. The methane content of the gas increased with the duration of the fermentation (24% higher at 28 compared with 21 days) but was not affected by the presence of biochar in the incubation medium. In experiment 2, adding 1% of biochar (DM basis) to the substrate increased gas production by 35%, reduced methane content of the gas by 8%, increased the DM solubilized (by 2%) and increased methane production per unit substrate solubilized by 25%. Presence of the cloth bag increased gas production when it also contained biochar but decreased it when added to the biodigester without biochar. There was a similar interaction for methane produced per unit substrate solubilized.
We present a fast procedure for scanning electron microscopy (SEM) analysis in which hexamethyldisilazane (HMDS) solvent, instead of the critical point drying, is used to remove liquids from a microbiological specimen. The results indicate that the HMDS solvent is suitable for drying samples of anaerobic cells for examination by SEM and does not cause cell structure disruption.
Nano-scale magnetite can facilitate microbial extracellular electron transfer that plays an important role in biogeochemical cycles, bioremediation, and several bioenergy strategies, but the mechanisms for the stimulation of extracellular electron transfer are poorly understood. Further investigation revealed that magnetite attached to the electrically conductive pili of Geobacter species in a manner reminiscent of the association of the multi-heme c-type cytochrome OmcS with the pili of Geobacter sulfurreducens. Magnetite conferred extracellular electron capabilities on an OmcS-deficient strain unable to participate in interspecies electron transfer or Fe(III) oxide reduction. In the presence of magnetite wild-type cells repressed expression of the OmcS gene, suggesting that cells might need to produce less OmcS when magnetite was available. The finding that magnetite can compensate for the lack of the electron transfer functions of a multi-heme c-type cytochrome has implications not only for the function of modern microbes, but also for the early evolution of microbial electron transport mechanisms.
Direct interspecies electron transfer (DIET) through biological electrical connections is an alternative to interspecies H2 transfer as a mechanism for electron exchange in syntrophic cultures. However, it has not previously been determined whether electrons received via DIET yield energy to support cell growth. In order to investigate this, co-cultures of Geobacter metallireducens, which can transfer electrons to wild-type G. sulfurreducens via DIET, were established with a citrate synthase-deficient G. sulfurreducens strain that can receive electrons for respiration through DIET only. In a medium with ethanol as the electron donor and fumarate as the electron acceptor, co-cultures with the citrate synthase-deficient G. sulfurreducens strain metabolized ethanol as fast as co-cultures with wild-type, but the acetate that G. metallireducens generated from ethanol oxidation accumulated. The lack of acetate metabolism resulted in less fumarate reduction and lower cell abundance of G. sulfurreducens. RNAseq analysis of transcript abundance was consistent with a lack of acetate metabolism in G. sulfurreducens and revealed gene expression levels for the uptake hydrogenase, formate dehydrogenase, the pilus-associated c-type cytochrome OmcS and pili consistent with electron transfer via DIET. These results suggest that electrons transferred via DIET can serve as the sole energy source to support anaerobic respiration.
Microorganisms that can form direct electrical connections with insoluble minerals, electrodes, or other microorganisms can play an important role in some traditional as well as novel bioenergy strategies and can be helpful in the remediation of environmental contamination resulting from the use of more traditional energy sources. The surprising discovery that microorganisms in the genus Geobacter are capable of forming highly conductive networks of filaments that transfer electrons along their length with organic metallic-like conductivity, rather than traditional molecule to molecule electron exchange, provides an explanation for the ability of Geobacter species to grow in subsurface environments with insoluble Fe(III)oxides as the electron acceptor, and effectively remediate groundwater contaminated with hydrocarbonfuels or uranium and similar contaminants associated with the mining and processing of nuclear fuel. A similar organic metallic-like conductivity may be an important mechanism for microorganisms to exchange electrons in syntrophic associations, such as those responsible for the conversion of organic wastes to methane in anaerobic digesters, a proven bioenergy technology. Biofilms with conductivities rivaling those of synthetic polymers help Geobacter species generate the high current densities in microbial fuelcells producing electric current from organic compounds. Electron transfer in the reverse direction, i.e. from electrodes to microbes, is the basis for microbial electrosynthesis, in which microorganisms reduce carbon dioxide to fuels and other useful organic compounds with solar energy in a form of artificial photosynthesis that is more efficient and avoids many of the environmental sustainability concerns associated with biomass-based bioenergy strategies. The ability of Geobacter species to produce highly conductive electronic networks that function in water opens new possibilities in the emerging field of bioelectronics.