title: efficient metabolic exchange and electron transfer within a

1

Title: Efficient metabolic exchange and electron transfer within a syntrophic TCE degrading co-1

culture of Dehalococcoides mccartyi 195 and Syntrophomonas wolfei 2

Running Head: Syntrophic exchanges between D. mccartyi and S. wolfei 3

Authors: Xinwei Mao,1 Benoit Stenuit,

1* Alexandra Polasko,

1 Lisa Alvarez-Cohen

1,2 4

1Department of Civil and Environmental Engineering, College of Engineering, University of California, 5

Berkeley, CA, 94720; 6

2 Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 7

8

Corresponding author: 9

Lisa Alvarez-Cohen 10

Civil and Environmental Engineering 11

726 Davis Hall 12

University of California 13

Berkeley, CA 94720-1710 14

Phone: (510)643-5969 15

[email protected] 16

17

*Present address: Catholic University of Louvain, Earth & Life Institute, Applied Microbiology and 18

Bioprocess Cluster, Louvain-la-Neuve, Belgium 19

20

Key words: chlorinated solvents, reductive dechlorination, Dehalococcoides, syntrophy, cell aggregates, 21

bioremediation 22

23

24

AEM Accepted Manuscript Posted Online 9 January 2015Appl. Environ. Microbiol. doi:10.1128/AEM.03464-14Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Abstract 25

Dehalococcoides mccartyi 195 (strain 195) and Syntrophomonas wolfei (S. wolfei) were grown 26

in a sustainable syntrophic co-culture using butyrate as electron donor and carbon source and 27

TCE as electron acceptor. The maximum dechlorination rate (9.9 ± 0.1 µmol d-1

) and cell yield 28

(1.1 ± 0.3 × 108

cells µmol-1

Cl-) of strain 195 maintained in co-culture were respectively 2.6 29

times and 1.6 times higher than those measured in the pure culture. Strain 195 cells concentration 30

was about 16 times higher than S. wolfei in the co-culture. Aqueous H2 concentrations ranged 31

from 24 to 180 nM during dechlorination and increased to 350 ± 20 nM when TCE was depleted, 32

resulting in cessation of butyrate fermentation by S. wolfei with a theoretical Gibbs free energy 33

of -13.7 ± 0.2 kJ mol-1

. Carbon monoxide in the co-culture was around 0.06 µmol per bottle, 34

which was lower than that observed for strain 195 in isolation. The minimum H2 threshold value 35

for TCE dechlorination by strain 195 in the co-culture was 0.6 ± 0.1 nM. Cell aggregates during 36

syntrophic growth were observed by scanning electron microscopy. The interspecies distances to 37

achieve H2 fluxes required to support the measured dechlorination rates were predicted using the 38

Fick’s law and demonstrated the need for aggregation. Filamentous appendages and EPS-like 39

structures were present in the intercellular spaces. The transcriptome of strain 195 during 40

exponential growth in the co-culture indicated increased ABC-transporter activities compared 41

with the pure culture, while the membrane-bound energy metabolism related genes were 42

expressed at stable levels. 43

44

45

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Introduction 47

Groundwater contamination by trichloroethene (TCE), a potential human carcinogen, 48

poses a serious threat to human health and can lead to the generation of vinyl chloride (VC), 49

which is a known human carcinogen (1). Strains of Dehalococcoides mccartyi are the only 50

known bacteria that can completely degrade TCE to the benign end product ethene. 51

Biostimulation of indigenous Dehalococcoides sp. and bioaugmentation using Dehalococcoides-52

containing cultures are recognized as the most reliable in situ bioremediation technologies 53

resulting in complete dechlorination of TCE to ethene (2). However, the mechanisms that 54

regulate the activity of D. mccartyi within natural ecosystems and shape its functional robustness 55

under disturbed environments are poorly understood due to multi-scale microbial community 56

complexity and heterogeneity of biogeochemical processes involved in the sequential 57

degradation (3-4). D. mccartyi exhibits specific restrictive metabolic requirements for a variety 58

of exogenous compounds, such as hydrogen, acetate, corrinoids, biotin and thiamine, which can 59

be supplied by other microbial genera through a complex metabolic network (1,5-8). Therefore, 60

the growth of D. mccartyi is more robust within functionally-diverse microbial communities that 61

are deterministically assembled than in pure cultures (5,8-9). Previous studies have shown that D. 62

mccartyi uses hydrogen as its sole electron donor for TCE dechlorination and out-competes other 63

terminal electron-accepting processes such as methanogenesis and acetogenesis at low hydrogen 64

partial pressures (10-11). Interspecies hydrogen transfer between D. mccartyi and supportive 65

organisms is a key process of electron flow that drives reductive dechlorination in the 66

environment. Although dechlorinating microbial communities have been extensively studied 67

over the past decades (4,10,12-13), community assembly processes in the highly specialized 68

ecological niche of Dehalococcoides and the network of interactions between D. mccartyi, its 69


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syntrophic partners and other co-existing community members have yet to be deciphered. The 70

optimization of D. mccartyi-based bioremediation systems to treat TCE-contaminated 71

groundwater would be facilitated by a systems-level understanding of interspecies electron, 72

energy and metabolite transfers that shape the structural and functional robustness of TCE-73

dechlorinating microbial communities. 74

To date, only a few studies of D. mccartyi-containing constructed co-cultures and tri-75

cultures have been published (5,7,14-16). A study with D. mccartyi 195 (strain 195) revealed that 76

its growth in defined medium could be optimized by providing high concentrations of vitamin 77

B12 and that over the short term, the strain could be grown to higher densities in co-cultures and 78

tri-cultures with fermenters Desulfovibrio desulfuricans and/or Acetobacterium woodii that 79

convert lactate to generate the hydrogen and acetate required by D. mccartyi (5). Recent studies 80

demonstrated interspecies corrinoid transfer between Geobacter lovleyi and D. mccartyi strains 81

BAV1 and FL2 (16) and interspecies cobamide transfer from a corrinoid-producing methanogen 82

and acetogen to D. mccartyi (7). Another study showed that strain 195 can grow in a long-term 83

sustainable syntrophic association with Desulfovibrio vulgaris Hildenborough (DvH) as a co-84

culture as well as with hydrogenotrophic methanogen Methanobacterium congolense (MC) as a 85

tri-culture (15). The maximum dechlorination rates and cell yield of strain 195 were enhanced 86

significantly in the defined consortia. Another unexpected syntrophic association has recently 87

been discovered between carbon monoxide (CO)-producing strain 195 and CO-metabolizing 88

anaerobes which enhance the growth and dechlorination activity of strain 195 by preventing the 89

accumulation of toxic CO as an obligate by-product from acetyl-CoA cleavage (17). Although 90

these studies demonstrated the robust growth of D. mccartyi-containing co-cultures on PCE/TCE 91

with higher dechlorination activity and cell yields than isolates, a clear and mechanistic 92


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understanding of metabolic cross-feeding and electron transfer between D. mccartyi and its 93

syntrophic partner(s) is needed to establish predictive models for dechlorination activity. 94

Butyrate fermentation is an endergonic reaction under standard conditions (Table 1) that 95

can only be carried out for energy generation by syntrophic microorganisms growing with H2 96

consumers (18-20). Syntrophomonas wolfei is a model butyrate fermenter that grows 97

syntrophically with hydrogenotrophic methanogens (19-21). S. wolfei can also grow without a 98

syntrophic partner on crotonate as the sole energy source through its disproportionation to 99

acetate and butyrate (Table 1) (22). In previous studies, Syntrophomonas spp. have been detected 100

in butyrate-fed dechlorinating enrichment cultures in significant relative abundance (13,23). In 101

this study, a TCE and butyrate-fed syntrophic co-culture of strain 195 with S. wolfei was 102

established and maintained to study the physiology and transcriptome of syntrophically growing 103

D. mccartyi. Spatial architecture and the physical proximity of the cells were analyzed in the co-104

culture S. wolfei/strain 195 and another syntrophic co-culture DvH/strain 195 (15). The 105

knowledge gained from this study provides us with a more fundamental understanding of the 106

metabolic exchange and energy transfer among the key players of TCE-dechlorinating 107

communities. 108

Material and methods 109

Bacterial cultures and growth conditions 110

Bacterial co-cultures of strain 195 and S. wolfei (5% vol/vol inoculation of each bacterium) were 111

initially established in 160 mL serum bottles containing 100 mL defined medium (5) with TCE 112

supplied at a liquid concentration of 0.6 mM (corresponding to 78 µmol TCE per bottle), 10 mM 113

crotonic acid, 100 µg L-1

vitamin B12 and N2/CO2 (90:10 v/v) headspace at 34 °C with no 114


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agitation. Cultures were subsequently transferred (5% vol/vol inoculation) for growth on 4 mM 115

butyric acid with 0.6 mM TCE as the electron acceptor. The established syntrophic co-culture 116

was continuously and stably maintained on butyrate in 100 mL defined medium over 45 sub-117

culturing events before the experiments were performed. The electron donor-limited condition 118

(on a hydrogen production/consumption basis) was achieved by feeding the co-culture 0.25 mM 119

butyrate (25 µmol per bottle that could theoretically generate 50 µmol H2 based on stoichiometry) 120

and 0.6 mM TCE (78 µmol per bottle that requires 234 µmol H2 to reductively dechlorinate TCE 121

to ethene). The strain 195 isolate was grown in defined medium with H2/CO2 (90:10 v/v) 122

headspace, 0.6 mM TCE as electron acceptor and 2 mM acetate as carbon source. Pure S. wolfei 123

was grown on crotonate in 160 mL serum bottles as described previously (Beaty et al. 1987). Co-124

culture DvH and strain 195 were grown in the same medium with the substitutions of 5 mM 125

lactate. Pure culture and co-culture purities were routinely checked by phase-contrast microscopy 126

and DGGE (Denaturing Gradient Gel Electrophoresis). 127

Chemical analysis 128

Chloroethenes and ethene were measured by FID-gas chromatograph using 100 µL headspace 129

samples, and hydrogen and carbon monoxide was measured by RGD-gas chromatography using 130

300 µL headspace sample as described previously (23-24). Organic acids, including butyrate and 131

acetate, were analyzed with a high-performance liquid chromatograph as described previously

132

(23). Mass of each compound was calculated based on gas-liquid equilibrium by using Henry’s 133

law constants at 34 °C according to: mass (μmol/bottle) = Cl×Vl + Cg×Vg, 𝑘Hcc = 𝐶l/𝐶g. Cl: liquid 134

phase concentration (μM); Vl : volume of liquid phase (L); Cg: gas phase concentration (μM); Vg: 135

volume of gas phase (L); 𝑘Hcc : Henry’s law constant (unitless). 136


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Scanning electron microscopy (SEM) 137

The co-cultures (S.wolfei and strain 195 grown on butyrate, and DvH and strain 195 on lactate) 138

provided with 0.6 mM TCE were collected when cell growth had ceased and treated by the 139

following standard protocols for SEM observation (http://em-140

lab.berkeley.edu/EML/protocols/psem.php). Briefly, cultures were collected by slowly filtering 1 141

mL fresh liquid sample through a 0.2µm GTTP filter (IsoporeTM

membrane, polycarbonate, 142

hydrophilic, 25 mm in diameter from Millipore), followed by chemical fixation, dehydration, 143

critical point drying and conductive coating (25). SEM images were obtained according to 144

standard operation protocols (26). The maximum interspecies distances between strain 195 and 145

S.wolfei that would enable the observed dechlorination rates for butyrate fermentation were 146

estimated using Fick’s diffusion law according to the procedure described by Ishii (27): 147

148

JH2 = H2 flux across the total surface areas (As,tot) of S. wolfei (pmol m-2

cell d-1

); 149

As = 2.1× 10-12

m2 cell

-1, S. wolfei surface area calculated based on the assumption of 0.25 by 150

2.5 µm cells (based on SEM observation). As,tot =As× S.wolfei cell number 151

DH2 = 6.31 10-5

cm2 s

-1, molecular diffusion coefficient in water for hydrogen at 35 °C (28); 152

CH2-sw = maximum H2 concentration enabling exergonic fermentation (μM) (the highest H2 153

level at which S.wolfei can ferment butyrate.); 154

CH2-195 = theoretical minimum H2 concentration useable by strain 195 for energy generation 155

(μM) (the lowest H2 level at which strain 195 can dechlorinate TCE); 156

JH2= 𝐷H2

×𝐶H2−𝑠𝑤 − 𝐶𝐻2−195

d𝑠𝑤−195


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dsw-195 = allowed interspecies distance for accomplishing syntrophic oxidation at an observed 157

substrate utilization rate (μm). 158

DNA extraction and cell number quantification 159

1.5 mL liquid samples were collected for cell density measurements and cells were harvested by 160

centrifugation (21,000 × g, 10 min at 4°C). Genomic DNA was extracted from cell pellets using 161

Qiagen DNeasy Blood and Tissue Kit according to the manufacturer’s instructions for Gram-162

positive bacteria. qPCR using SYBR Green-based detection reagents was applied to quantify 163

gene copy numbers of each bacterium with S.wolfei 16S rRNA gene primers (forward primer 5’-164

GTATCGACCCCTTCTGTGCC-3’, and reverse primer 5’-CCCCAGGCGGGATACTTATT-3’) 165

(19), and D. mccartyi tceA gene primers (forward primer 5’-166

ATCCAGATTATGACCCTGGTGAA-3’ and reverse primer 5’-167

GCGGCATATATTAGGGCATCTT-3’), as previously described (29). The electron equivalents 168

(µmol) diverted to biomass were calculated based on empirical biomass formula C5H7O2N (113 169

g mol-1

cell, 20 e- eq mol

-1 cells). 170

RNA preparation 171

Cultures were sampled for RNA on day 6 during exponential growth when around 75% of 78 172

µmol TCE was dechlorinated (~20 µmol TCE remained). In order to collect sufficient material 173

for transcriptomic microarray analysis, 18 bottles of pure strain 195 and 18 bottles of the co-174

culture were inoculated and grown from triplicate bottles of the isolate and co-culture, 175

respectively. For each biological triplicate, cells from six bottles were collected by vacuum 176

filtration on day 6 during active dechlorination for the co-culture and day 12 for the strain 195 177

pure culture (200 mL culture per filter, 0.2-µm autoclaved GVWP filter (Durapore membrane, 178


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Millipore, Billerica, MA). Each filter was placed in a 2 mL orange-cap micro-centrifuge tube, 179

frozen with liquid nitrogen and stored at -80 °C until further processing. 180

RNA was extracted using the phenol-chloroform method described previously (31) with the 181

following minor modifications. The ratio of phenol (pH 4.0)-chloroform-isoamylalcohol used for 182

extraction was 25:24:1 (vol:vol), and the RNA pellet was re-suspended in 100 µL of nuclease-183

free water. RNA samples were purified following manufacturer’s instructions with the AllPrep 184

DNA/RNA Mini Kit (Qiagen). Additional DNA contamination was removed with Turbo DNA 185

free kit (Ambion, Austin, TX) according to the manufacturer’s instructions. The quality of RNA 186

samples was checked by electrophoresis (1.0 µL RNA sample), and the concentrations of RNA 187

samples were quantified using a nano-photometer (IMPLEN, Westlake Village, CA, USA). The 188

ratio of A260/A280 for all samples was between ~1.80-2.0. Purified RNA was stored at -80 °C 189

prior to further use. 190

Transcriptomic microarray analysis 191

A complete description of the Affymetrix GeneChip microarray used in this study has been 192

reported elsewhere (4). Briefly, the chip contains 4,744 probe sets that represent more than 98% 193

of the ORFs from four published Dehalococcoides genomes (strain 195, VS, BAV1, and 194

CBDB1). cDNA was synthesized from 9 µg RNA, then each cDNA sample was fragmented, 195

labeled and hybridized to each array. All procedures were performed with minimal modifications 196

to the protocols in section 3 of the Affymetrix GeneChip Expression Analysis Technical Manual 197

(Affymetrix, Santa Clara, CA http://www.affymetrix.com). Microarray data analysis methods 198

were described previously (15,32). 199


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Results 200

Syntrophic co-culture degradation characteristics 201

TCE did not inhibit growth of the S. wolfei isolate at concentrations up to 0.6 mM 202

(Supplemental material, Part 1, Fig. S1); therefore, co-cultures were maintained with 0.6 mM 203

TCE and 4 mM butyrate. After subculturing this co-culture 45 times, the maximum 204

dechlorination rate of strain 195 was approximately 2.6 fold (9.9 ± 0.1 µmol d-1

) (Fig. 1a) greater 205

than the strain 195 isolate (3.8 ± 0.1 µmol d-1

) (15). The calculation of strain 195 cell yield was 206

based on the metabolic reductive TCE dechlorination to VC, and the cell yield of strain 195 was 207

1.6 times greater in the co-culture (1.1 ± 0.3 × 108 cells µmol

-1 Cl

-) (Fig. 1b) compared to the 208

pure culture (6.8 ± 0.9 × 107 cells µmol

-1 Cl

-) (15), similar to results from a co-culture containing 209

a sulfate-reducing bacterium (9.0 ± 0.5 × 107

cells µmol-1

Cl-) (15) (Table 2). The initial 210

concentration of butyrate (from 1 mM to 20 mM) did not affect the dechlorination rate (data not 211

shown), showing that high butyrate concentrations are not inhibitory to dechlorination. Cell 212

numbers of strain 195 (1.3 ± 0.2 × 108 cells mL

-1) were consistently about 16 times higher than S. 213

wolfei (7.7 ± 0.1 × 106

cells mL-1

) in the co-cultures growing on butyrate. In contrast, when 214

maintaining the co-culture on crotonate, the cell number ratio was about 1.3:1 (Supplemental 215

material, Part 1, Fig. S2). In the syntrophic co-culture with DvH growing on lactate, the cell 216

number ratio of strain 195 to DvH was reported to be about 5:1 (15). 217

When the ratio of butyrate to TCE in the co-culture was maintained under electron 218

acceptor limitation with 79 ± 6.7 µmol TCE and 440 ± 29 µmol butyrate (measured by HPLC) as 219

the electron donor, H2 levels remained steady at ~24-180 nM during active dechlorination (from 220

day 0 to day 7) and increased to 350 ± 20 nM right after TCE and cis-DCE were consumed on 221


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day 8 (Fig. 1c and Supplemental material, Part 2, Table S2). This H2 concentration was similar to 222

the level (480 nM) that resulted in cessation of S. wolfei growth on butyrate in isolation (33). At 223

the end of the experiment (day 18), 88.4% of the amended TCE could be accounted for in 224

dechlorination products VC (7.4± 0.3 % molar equivalents) and ethene (81.0± 0.1% molar 225

equivalents). The missing 11.6% of initial TCE was likely due to the numerous sampling events 226

during the experiment, as confirmed by losses in the abiotic controls (8.7% loss of initial TCE 227

added). In addition, 115 ± 3.0 µmol butyrate was consumed while 234 ± 9 µmol acetate and 3.9 228

±1.1 µmol H2 remained in the bottles (in the abiotic control, the amount of butyrate decreased 26 229

µmol with no production of acetate or H2). This indicates that around 88.6% of electrons (0.886, 230

this corresponds to 𝑓𝑒° close to 0.84 from Fig. 1d) (in the form of H2) generated from butyrate 231

fermentation (ca. 230 µmol H2) supported dechlorination. A calculation based on biomass cell 232

numbers (Assumed 0.5 gram dry weight per gram cell, i.e. water accounts for 50% of the cell 233

weight. The cell formula was C5H7O2N, with 20 electron equivalent per mol biomass) indicates 234

that 7.4 % (17 µmol H2) of the electrons from butyrate fermentation (in the form of H2) were 235

diverted to biomass production, giving a total electron recovery of about 98 %. This result 236

demonstrates that butyrate was efficiently used as the electron donor in the co-culture with high 237

electron transfer efficiency between the two bacteria. 238

The calculated Gibbs free energy of reaction (∆𝐺𝑟) (butyrate fermentation and associated 239

hydrogen generation by S.wolfei) was -21.7 ± 0.3 kJ mol-1

on day 6 and decreased to -13.7 ± 0.2 240

kJ mol-1

on day 8 when TCE and cis-DCE was depleted. This number was smaller than the 241

hypothetical minimum energy (-20 kJ mol-1

) required by a bacterium to exploit the free energy 242

change in a reaction and support growth (34) (detailed calculations in Supplemental material, 243

Part 2). After TCE and cis-DCE depletion, we observed a slight decrease in butyrate (36.0 ± 6.6 244


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µmol) during co-metabolic VC dechlorination (Fig. 1c) with H2 concentrations increasing to a 245

stable level of 1.2 ± 0.3 µM after day 10 while the cell numbers of strain 195 and S. wolfei were 246

observed to decrease (Fig. 1b and 1c). The calculated ∆𝐺𝑟 of butyrate fermentation during the 247

cometabolic process was less negative and reached -5.7 ± 1.4 kJ mol-1

by the end of the 248

experiment (Supplemental material, Part 2, Fig. S3). 249

In order to determine the hydrogen threshold of strain 195 in the co-culture, it was grown 250

under electron donor-limited conditions (TCE fed in excess). H2 concentrations dropped to 251

thresholds of 0.6 ± 0.1 nM at which point TCE was not further degraded by strain 195 and 252

butyrate depletion ceased (Supplemental material, Part 3, Figure S4). In order to validate our 253

method for quantifying the minimum H2-threshold concentration, we measured the hydrogen 254

threshold of the sulfate reducing microorganism Desulfovibrio vulgaris Hildenborough (DvH), 255

and obtained 15.2 ± 1.4 nM, which is in agreement with literature results (14.8 nM) for this 256

bacterium (35). 257

We tested the inhibitory effect of CO on S. wolfei by exposing cells to 0.6 to 8 µmol CO 258

per bottle and observed no significant effect on cell growth (Fig. 2a). CO accumulation for the S. 259

wolfei isolate growing on crotonate was 0.02 µmol per bottle throughout the experiment (Fig. 2b), 260

which was at the same level as the abiotic control (data not shown). In the co-culture growing on 261

butyrate, CO was measured at around 0.06 µmol per bottle. This was lower than that detected for 262

the strain 195 isolate (0.5 ± 0.1 µmol per bottle after one dose of TCE (77 µmol) was depleted) 263

(17). Furthermore, we found that pure S. wolfei could consume CO from 14.5 ± 1.5 µmol to 0.4 264

± 0.1 µmol in 42 days (Fig. 2c) when growing on crotonate. 265


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Formation of cell aggregates 266

Strain 195 and S. wolfei isolates as well as the co-cultures of S. wolfei and strain 195 267

maintained on butyrate were grown to late exponential to early stationary phase before samples 268

were analyzed by scanning electron microscopy (SEM). Figures 3a and 3b show specific cell 269

morphology of the isolates with strain 195 growing in individual coccus-shapes with diameters 270

of around 0.5-1 µm while most S. wolfei cells grew individually or in pairs as 0.25 - 0.5 by 2.5 - 271

5 µm rods as observed in previous studies (1,21). When butyrate was amended to the co-culture 272

as electron donor with TCE as electron acceptor, the cells form cell aggregates with size ranges 273

from 2 µm to 10 µm (Fig. 3c-e). In relatively small aggregates, cells were found connected with 274

flagellum-like filaments (Fig. 3c,d). In large aggregates, EPS-like structures were observed (Fig. 275

3d,e). Cell aggregates were also observed during exponential growth phase (Supplemental 276

material, Part 4, Figure S5). However, when strain 195 was grown as a co-culture with DvH on 277

lactate, the two strains grew together but did not form obvious cell aggregates (Fig. 3f). 278

In order to determine whether the co-cultures may be capable of sharing electrons via 279

direct interspecies electron transfer (DIET), the crotonate and butyrate co-cultures were sent to 280

Professor Lovley’s laboratory for conductivity testing (36). The co-cultures exhibited three 281

orders of magnitude lower conductivity than Geobacter, indicating that H2 rather than DIET is 282

the transfer mechanism. In addition, since S. wolfei could potentially use both hydrogen and 283

formate as electron carriers for interspecies electron transfer (37), we tested the expression levels 284

of several formate dehydrogenase genes (fdhA: Swol_0786, Swol_0800, Swol_1825) in co-285

cultures grown on crotonate or butyrate and S. wolfei grown in isolation. The relative expression 286

of fdhA subunits to gyrB gene was generally in the range of 0.6-1.2 units for all three conditions 287


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(38), confirming that hydrogen rather than formate was the exclusive electron shuttle between 288

these two bacteria. 289

The theoretical maximum interspecies distance for an observed rate of molecular transfer 290

between two microorganisms can be calculated using Fick’s diffusion law (20). Here, this 291

distance was calculated for co-culture growth on butyrate (Table 3, with detailed calculations in 292

Supplemental material, Part 4) by defining JH2 as the hydrogen flux between S. wolfei and strain 293

195. JH2 was calculated from the total surface areas of S. wolfei and substrate oxidation rate 294

(hydrogen production rate = 2 × butyrate oxidation rate) measured in the co-culture experiment 295

using equation 2 in Table 1. CH2-sw (0.35 ± 0.02 µM) is the maximum H2 concentration 296

immediately outside of an S. wolfei cell when fermentation by S. wolfei stopped on butyrate and 297

CH2- 195 is the theoretical minimum H2 concentration above which strain 195 can gain energy, 298

estimated in this study as 0.6 ± 0.1 nM. dsyn-195 is then the calculated maximum interspecies 299

distance for accomplishing syntrophic oxidation at the observed substrate oxidation rate. 300

The theoretical mean cell-cell distance of randomly dispersed cells is calculated based on 301

the total cell numbers (quantified using qPCR) suspended in the unstirred liquid culture (Table 3). 302

In this study, cell settling was not observed for the strain 195 isolate or co-cultures growing on 303

butyrate (strain 195 and S. wolfei) or lactate (strain 195 and DvH). Considering that the cell 304

number ratio of strain 195 to S. wolfei was about 20:1 on day 4 (Fig. 1c), strain 195 cells account 305

for the majority of all cells in the bottle, therefore the average cell-cell distance between 306

suspended strain 195 and S. wolfei cells (i.e., >> 27.1 µm, Table 3) would be larger than the 307

calculated theoretical maximum cell-cell distance for achieving butyrate fermentation (Table 3). 308

In order to accomplish syntrophic butyrate oxidation at the rate observed, the average 309

interspecies distance must be much less than the distance between randomly dispersed cells 310


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(Table 3), necessitating the formation of aggregates. In addition, according to the 311

thermodynamically consistent rate law, the calculated free energy available for S. wolfei growth 312

on day 4 was -27.4 ± 1.1 kJ mol-1

which is small (Supplemental material, Part 2, Fig. S3), 313

indicating that close physical contact between the two species is particularly important for 314

efficient syntrophic butyrate fermentation. In contrast, when strain 195 was grown with DvH as a 315

co-culture, the maximum calculated interspecies distance was large enough (755 μm) to enable 316

interspecies hydrogen transfer between the two bacteria (Supplemental material, Part 4, Table 317

S4), and aggregates were not formed. 318

Strain 195 Transcriptome during syntrophic growth 319

Transcriptomic microarray analysis comparing the co-culture growing on butyrate and 320

strain 195 growing in isolation identified 214 genes that were differentially transcribed (Fig. 4, 321

Table S7). Among these differentially expressed genes, 18 were up-regulated and 196 were 322

down-regulated in the syntrophic co-culture compared to the strain 195 isolate. Among the up-323

regulated genes, most significantly expressed genes (signal level 5000~20,000) belonged to 324

transport and metabolism functions including several types of ABC (ATP binding cassette) 325

transporters. Genes located within an operon of a Fec-type ABC transporter (from DET1173 to 326

DET1176) which are involved in periplasmic iron-binding were up-regulated 2.7 to 4.2 times, 327

respectively; genes (DET1491, DET1493) from a cluster encoding a peptide ABC transporter 328

responsible for ATP binding were up-regulated 2.1 and 2.3 times, respectively; genes 329

(DET0140-0141) encoding a phosphate ABC transporter and ATP-binding protein were up-330

regulated 2.0 to 2.6 times, respectively. There were also a few genes with unknown functions up-331

regulated at high signal levels compared to the isolate. DET1008, with highest homology to a 332


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gene encoding a cell-division initiation protein (39) was up-regulated 3.2 times and acetyl-CoA 333

synthase (DET1209) was up-regulated 2.4 times at a high signal level (~20,000). 334

Genes associated with membrane-bound oxidoreductase complexes, which are related to 335

energy metabolism, such as RDases, hydrogenases, molybdopterin oxidoreductases, putative 336

formate dehydrogenases, as well as NADH-oxidoreductases, showed no significant differential 337

expression patterns between the co-culture and pure culture throughout the experiment (Table 338

S5). Among the 196 down-regulated genes in the co-culture compared to the isolate, 75 were 339

genes with signal levels lower than 1000 in both treatments (suggesting low expression level), 340

and most of them encode for hypothetical proteins (Table S5). Many of the down-regulated 341

genes were not related to energy metabolism, such as those encoding a phage domain protein 342

(DET0354); mercuric reductase (DET0732); Mg chelatase-like protein (DET0986) and 343

virulence-associated proteinE (DET1098). 344

Discussion 345

In the strain 195 and S. wolfei syntrophic co-culture studied here, strain 195 grew 346

exponentially at the rate of 0.69 d-1

with a doubling time of 1.0 day, calculated from cell numbers 347

(tceA copies) from day 2 to day 6. This doubling time is shorter than previously reported for the 348

isolates of D. mccartyi sp. (31,40-42). The dechlorination rate was 9.9 ± 0.1 µmol d-1

which is 349

similar to the value observed with strain 195 and DvH co-cultures (11.0 ± 0.01 µmol d-1

, (15)) 350

and higher than the dechlorination rates observed with other D. mccartyi-containing co-cultures 351

(4.2-6.8 µmol d-1

) (5,7,14-16). The cell yield of strain 195 in this co-culture (1.1 ± 0.3 × 108 cells 352

µmol-1

Cl- released) was similar to that observed in other D. mccartyi co-culture studies, e.g., 9.0 353


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± 0.5 ×107 cells µmol

-1 Cl

-1 (15) and 9.0 ± 1.4 ×10

7 cells µmol

-1 Cl

- (7). Given that the minimum 354

free energy change required for ATP synthesis is in the range of 15-25 kJ mol-1

for most 355

syntrophic fermentations (43), only a small amount of energy saved as ATP is expected to be 356

available during S. wolfei syntrophic butyrate fermentation (19). Indeed, S. wolfei in the co-357

culture was growing very near the thermodynamic threshold (delta G from -41.9 to -5.7 kJ mol-1

), 358

and consequently the cell numbers remained at low levels (1:16 ratio with strain 195 cells) when 359

growing on butyrate compared to growth on crotonate. The ratio of calculated free energy 360

available for strain 195 to reduce TCE to VC (-272.0 kJ mol-1

) and S. wolfei (-43.6 kJ mol-1

, 361

Table S2) growing on butyrate was about 6.2:1 at the beginning of the experiment. Considering 362

that the cell size of S. wolfei is about 2.5 times larger than strain 195, the theoretical cell 363

production ratio (based on free energy) of strain 195 to S. wolfei (15.6:1) is similar to our 364

observation (16:1). While growing on crotonate, the ratio of available energy for strain 195 (-365

295.3kJ mol-1

) and S. wolfei (-465.2 kJ mol-1

) was 0.6, and the theoretical cell production ratio of 366

1.2:1 is similar to our observation of 1.3:1 (Supplemental material, Part 1). Roden and Jin (44) 367

found a linear correlation between microbial growth yields (YXS, g cell mol-1

substrate) and 368

estimated catabolic ∆𝐺𝑟 for metabolism of short-chain fatty acids and H2 coupled metabolic 369

pathways. Table 4 compares the growth yields calculated using the Roden and Jin (44) method 370

(YXS,cal) with observed growth yields (YXS,obs) of syntrophs growing in a variety of co-cultures. 371

Our results confirmed that it is possible to estimate syntrophic cell yields by percentage error of 372

less than 100% by applying an empirical equation for the listed syntrophic co-culture studies (44), 373

suggesting that the growth yield of syntrophic bacteria and the ratio maintained in the co-cultures 374

were mainly controlled by thermodynamics. Furthermore, we found H2 concentrations to be the 375


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key factor affecting the ∆𝐺𝑟 in the co-cultures, which in turn affects predictions of microbial 376

growth yields (calculation in Tables S2 and S6). 377

When TCE was supplied in excess to the co-culture (on a H2 production/consumption 378

basis), the H2 level dropped to the threshold concentration of 0.6 ± 0.1 nM and dechlorination 379

ceased. Although the calculated ∆𝐺𝑟available for dechlorination by strain 195 was still negative 380

(-145.5 kJ mol-1

) at this concentration (Supplemental material, Part 5, Table S6), it did not 381

support growth. Thermodynamic calculations indicate that hydrogen concentrations would have 382

to reach liquid concentrations of 10-27

nM to drive the ∆𝐺𝑟 for strain 195 positive, a value that is 383

unrealistic in the environment (45). Therefore, the minimum H2 threshold for strain 195 cells is 384

likely not based on thermodynamics, but is rather based upon other factors such as enzyme 385

binding affinity and specificity (46). This finding is consistent with previously published H2 386

thresholds for dechlorination by Dehalcoccoides-containing communities (10,42), and falls in the 387

same range as thresholds for sulfate reduction (47). Other dechlorinating isolates have been 388

reported to have minimum H2 thresholds around ~0.04-0.3 nM (11,47,48). In previous studies of 389

S. wolfei growing with H2-oxidizing hydrogenotrophic methanogens, sulfate reducers, and nitrate 390

reducers, the estimated energy available for S. wolfei ranged from 0.9 to 15.0 kJ mol-1

(18) when 391

butyrate fermentation ceased and the ratio of acetate to butyrate was around 900. While in this 392

study we observed estimated energy available for S. wolfei was -13.7 ± 0.2 kJ mol-1

with the 393

acetate to butyrate ratio of 0.4:1. Previous studies have shown calculated ∆𝐺𝑟 value fell in the 394

range (-10 to -15 kJ mol-1

) observed in H2-dependent terminal electron accepting processes under 395

starvation conditions in sulfate reducing bacteria and methanogenic archaea (49,50,63). 396

Furthermore, the value obtained in this study was close to the average threshold value of butyrate 397


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metabolism (-13.8 ± 1.2 kJ mol-1

) reported in co-culture S. aciditrophicus and Desulfovibrio 398

strain G11 at different acetate to butyrate ratios by Jackson and McInerney (51). 399

It is interesting that the amount of butyrate in the bottles continued to decrease slightly 400

(36 µmol) after TCE was depleted and ethene was being produced from VC (from day 8 to day 401

18). According to thermodynamics, microbial metabolism ceases when the ∆𝐺𝑟 available from a 402

reaction becomes positive. At the beginning of the experiment, the ∆𝐺𝑟 available for S. wolfei 403

butyrate fermentation was negative (-41.9 ± 1.5 KJ mol-1

), and on day 6, this number increased 404

to -21.7 ± 0.3 kJ mol-1

, then on day 8, it increased further to -13.7 ± 0.2 kJ mol-1

(Supplemental 405

material, Fig. S3), which is less than the minimum energy (-20 kJ mol-1

) required by a bacterium 406

to synthesize ATP (34). During this time, the cell numbers decreased indicating cell death and 407

lysis. It has been reported that butyrate fermentation by S. wolfei stops when aqueous H2 408

concentrations reach 480 nM (33). In this work, the H2 concentration was 350 ± 20 nM in the co-409

culture on day 7 when all TCE was reduced to VC. However, the concentration of H2 steadily 410

increased to 1,200 ± 300 nM by the end of the experiment on day 18, indicating that additional 411

butyrate fermentation occurred although cell growth had ceased, suggesting that regulation of the 412

fermentation enzymes may not be stringent. During active dechlorination, H2 concentrations 413

remained at levels below 200 nM (Fig. 1b) indicating that the H2 generation rate was at about the 414

same level as the consumption rate (Fig. 1d) and that the growth rate of the two species was 415

strictly coupled by hydrogen transfer. 416

In strain 195, acetyl-CoA is cleaved in an incomplete Wood-Ljungdahl pathway to 417

provide the methyl group for methionine biosynthesis, whereby carbon monoxide (CO) is 418

produced as a byproduct that accumulates and eventually inhibits D. mccartyi growth and 419

dechlorination (17,52). However, in microbial communities, CO can serve as an energy source 420


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for many anaerobic microorganisms (53). A previous study showed a high CO concentration 421

(15%) to have an inhibitory effect on S. wolfei (Sieber et al. 2014). Here we found that not only 422

did low levels of CO not exhibit inhibitory effects on S. wolfei cell growth, but S. wolfei could 423

consume CO (µmol per bottle) while growing on crotonate. CO levels in the co-culture growing 424

on butyrate were maintained at low levels during the feeding cycle rather than accumulating as it 425

does with the 195 isolate. Therefore it is possible that CO serves as a supplemental energy source 426

for S. wolfei during syntrophic fermentation with strain 195. This finding is interesting since 427

there were no anaerobic carbon monoxide dehydrogenase genes annotated in the S. wolfei 428

genome (19). However Swol_1136 and Swol_1818, which were originally annotated as iron-429

sulfur cluster binding domain-containing protein and 2Fe-2S binding protein in S. wolfei, were 430

more recently annotated in the KEGG database (54) as carbon-monoxide dehydrogenase small 431

sub-units for CO conversion to CO2, suggesting the genomic potential for CO metabolic ability 432

in S. wolfei. 433

Formation of cell aggregates has been shown to be a distinctive feature of obligate 434

syntrophic communities containing acetogenic bacteria and methanogenic archaea (20). 435

Clustering of cells with decreasing inter-microbial distances leads to increased fluxes and 436

increased specific growth rates (55). Previous studies reported that the syntrophic co-culture of a 437

propionate degrader and a hydrogenotrophic methanogen could form cell aggregates while 438

growing on propionate for optimal hydrogen transfer (27,56). A similar phenomenon was also 439

observed in mixed cultures containing propionate degraders and methanogens enriched from an 440

anaerobic bio-waste digester where flocs formed showing that reducing the interspecies distances 441

by aggregation was advantageous in complex ecosystems (57). In engineered dechlorinating 442

systems, D. mccartyi species have been observed in biofilms within a membrane bioreactor (58) 443


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and in bioflocs maintained in a continuous flow bio-reactor fed with butyrate (13). Based on 444

morphological observation in this study, strain 195 cells grown in isolation are more likely to 445

grow in planktonic form and when grown with DvH as a syntrophic co-culture on lactate, no 446

significant aggregates were observed. However when grown with S. wolfei on butyrate, the co-447

cultures formed aggregates during syntrophic growth. Thermodynamic calculations show that 448

syntrophic butyrate oxidation is endergonic unless the circumstantial H2 partial pressure is 449

maintained very low (Table 1 and Supplemental material, Table S2). Furthermore fdh 450

dehydrogenase expression in S. wolfei under different growth conditions confirmed that these 451

genes were not up-regulated in in the co-culture grown on butyrate compared to growth on 452

crotonate, or in isolation (38). This is the first study to report D. mccartyi forming cell aggregates 453

with a syntrophic partner. In addition, because the average inter-microbial distances are smaller 454

for syntrophic aggregates, the H2 and metabolite flux between cells would be expected to 455

increase, leading to higher material transfer efficiencies, which could partly explain the observed 456

increased cell yields of strain 195. Another reason for the observed increased cell yields of strain 457

195 is likely due to the continuous removal of CO, which was shown to exert an inhibitory effect 458

on D.mccartyi growth (17). 459

In a previous study of Methanococcus maripaludis growing in syntrophic association 460

with Desulfovibrio vulgaris under hydrogen limitation in chemostats (59), M. maripaludis was 461

growing close to the thermodynamic threshold for growth, and the acetyl-CoA synthase 462

transcripts levels of M. maripaludis in the co-culture decreased compared to the isolate. While in 463

our study, the acetyl-CoA synthase transcripts levels of strain 195 in the co-culture increased 464

compared to the isolate. A recent study (60) showed a division of metabolic labor and mutualistic 465


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interactions were the reason for increased rates of growth in a vast majority of cross-feeding 466

strains. The benefit of cross-feeding was larger than the cost (60, 64). 467

Acknowledgements 468

We Acknowledge Professor Michael McInerney at University of Oklahoma kindly provided us 469

the pure strain Syntrophomonas wolfei for this study. We also thank Dr. Nikhil S. Malvankar and 470

Professor Derek Lovley at University of Massachusetts carried out the experiments of 471

conductivity test for the co-cultures. This work was funded through research grants from NIEHS 472

(P42-ES04705-14) and NSF (CBET-1336709) 473

474

475

476

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680


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0 2 4 6 8 10 12 14 16 180

20

40

60

80

100

Time (day)

Eth

enes

(µ

mol/bottle

)

a

0 2 4 6 8 10 12 14106

107

108

109

1010

1011

Time (day)

cell n

um

ber

/bottle

b

0 100 200 3000

50

100

150

200

250

butyrate consumed in H2 equivalent (mmol)Chlo

rinat

ed e

then

es r

educe

d in H

2 e

quiv

alen

t (m

mol)

d fe=0.84, R2=0.9772

0 2 4 6 8 10 12 14 16 180

100

200

300

400

500

600

0

1

2

3

4

5

Time (day)

Fat

ty a

cids

(µm

ol/bottle

)

c

Liq

uid

H2 co

ncen

tration (µ

M)


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Table 1. Stoichiometric energy generating reactions in the syntrophic co-culture.

Process Redox reaction ΔGo’

kJ/mol Number of equation

Crotonate oxidation and reduction 2𝐶4𝐻5𝑂2− + 2𝐻2𝑂 → 2𝐶2𝐻3𝑂2

− + 𝐶4𝐻7𝑂2− + 𝐻+ -350 1

Butyrate fermentation reaction 𝐶4𝐻7𝑂2− + 2𝐻2𝑂 → 2𝐶2𝐻3𝑂2

− + 𝐻+ + 2𝐻2 (g) 46.8a 2

TCE reduction to cis-DCE 𝐶2𝐻𝐶𝑙3 + 𝐻2 → 𝐶2𝐻2𝐶𝑙2 + 𝐻+ + 𝐶𝑙− -133 3

cis-DCE reduction to VC 𝐶2𝐻2𝐶𝑙2 + 𝐻2 → 𝐶2𝐻3𝐶𝑙 + 𝐻+ + 𝐶𝑙− -144 4

VC reduction to ETH 𝐶2𝐻3𝐶𝑙 + 𝐻2 → 𝐶2𝐻4 + 𝐻+ + 𝐶𝑙− -154 5

a. ΔGo’ (pH 7.0) was corrected to 307.15K


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Table 2. Dechlorination rate and cell yield of D. mccartyi strains in various co-culture studies

Co-culturesa Dechlorination

rate

(µmol d-1)

Specific dechlorination

rate of strain 195

(µmol cell-1 d-1)

D. maccartyi cell yield

(cells µmol-1 Cl- released)

Reference

Strain 195 and Desulfovibrio desulfuricans b 0.4 2.3 × 10-10 2.4 × 108 5

Strain 195 and Sedimentibacter sp. c 6.8 N.A. 4.2 × 108 14

Strain 195 and Desulfovibrio vulgaris Hildenborough 11 ± 0.01 N.A. 9.0 ± 0.5 × 107 15

Strain BAV1 and Geobacter lovleyi d

Strain FL2 and Geobacter lovleyi

6.1

4.2

7.9 × 10-10

1.5 × 10-9

6.7 × 107

3.3 × 107

16

Strain BAV1 and Sporomusa sp. strain KB1 e

Strain GT and Sporomusa sp. strain KB1

Strain FL2 and Sporomusa sp. strain KB1

3.3

2.1

2.5

2.7 × 10-10

1.4 × 10-10

7.2 × 10-10

2.1 ± 0.2 × 108

2.1 ± 0.3 × 108

9.0 ± 1.4 × 107

7

Strain 195 and S. wolfei 9.9 ± 0.1 7.6 ×10-10 1.1 ± 0.3 ×108 This study


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aDehalococcoides mccartyi strain 195 (strain 195), Dehalococcoides mccartyi strain BAV1 (strain BAV1), Dehalococcoides mccartyi strain GT

(strain GT), Dehalococcoides mccartyi strain FL2 (strain FL2). b Calculated from Fig. 4B and Table 2 therein.

c Calculated from Fig. 1C and Fig. 3 therein.

d Calculated from Fig. 2 and Table 1 therein.

e Calculated from Fig. 6 and Table 1 therein.


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Table 3. Calculation of cell-cell distancea.

Total cell number

(strain 195 + S. wolfei)

Mean cell-cell

distanceb(µm)

Maximum average interspecies distance for

H2 transfer (S. wolfei to strain 195) at the

observed substrate oxidation ratec (µm)

day2 6.2×106 cells mL-1 55.3 14

day4 5.0×107 cells mL-1 27.1d 6.3d

day6 9.2×107 cells mL-1 22.2 2.7

day8 13.7×107 cells mL-1 19.6 1.9

aThe cell-cell distance calculated in the table was based on one sample.

bCalculated for suspended cells using the cell number quantified from quantitative PCR analysis.

cCalculated by using Fick’s Diffusion Law.

dDetailed calculation in Supplemental material, Part 4.


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Table 4 Estimation of syntrophic bacterial growth yields based on Gibbs free energy (∆𝐺𝑟) calculations

e-donor Organism ∆𝑮𝒓a

(kJ/mol donor)

YXS,cal b

(g cells/mol)

YXS,obsc

(g cells/mol)

(Percent error)

Reference

Lactate DVH/ M. bakeri co-culture -61.4 3.4 6.7 (97%)d 61

Lactate DVH/ strain 195 co-culture -61.4 3.4 3.3 (3%) 15

Lactate DVH/strain 195/Methanobacterium conglense tri-culture -61.4 3.4 5.3 (56%) 15

Butyrate S. wolfei/ M. hungatei co-culture -9.1 2.3 1.36 (41%) 62

Butyrate S. wolfei/ Desulfovibrio G11 co-culture -9.1 2.3 0.55 (76%) 62

Butyrate S. wolfei/M. hungatei/M.barkeri tri-culture -9.1 2.3 1.08 (53%) 62

Butyrate S. wolfei/strain 195 co-culture -9.1 2.3 2.7 (17%) This study

a. ∆𝐺𝑟 (Gibbs free energy change) was calculated for H2 in the gaseous state at 1.3 Pa (~10 nM in the aqueous phase). All other compounds are

calculated at 10 mM.

b. Calculated cell yields (Ycal ) were based on the equation of Y= 2.08+ 0.0211× (-ΔG’) (44).


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c. Observed cell yields (Yobs) were based on directly measured cell masses (61) or masses converted from protein concentrations by assuming 2 g

cells/ g protein (62), or converted from cell numbers based on the assumption of 6×10-13 g per syntrophic cell (15) (44) (62).

d. Absolute error E = 3.3 g cells mol-1; Relative error = 0.97 ; Percent error = 97%


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title: efficient metabolic exchange and electron transfer within a

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