Metabolism of Multiple Aromatic Compounds in ... - ACS Publications

Subscriber access provided by NEW YORK UNIV

Article

Metabolism of multiple aromatic compounds in corn stover hydrolysate by Rhodopseudomonas palustris Samantha Austin, Wayne S Kontur, Arne Ulbrich, Julian Zachary Oshlag, Weiping Zhang, Alan Higbee, Yaoping Zhang, Joshua J. Coon, David B Hodge, Timothy J. Donohue, and Daniel R. Noguera Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02062 • Publication Date (Web): 29 Jun 2015 Downloaded from http://pubs.acs.org on July 2, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

Environmental Science & Technology

TOC Art 47x26mm (600 x 600 DPI)

ACS Paragon Plus Environment


1

Metabolism of multiple aromatic compounds in corn stover hydrolysate

2

by Rhodopseudomonas palustris

3

By

4

Samantha Austin,1,3 Wayne S. Kontur,2,3 Arne Ulbrich,4 J. Zachary Oshlag,1,3 Weiping Zhang,1,3

5

Alan Higbee,3,4 Yaoping Zhang,3 Joshua J. Coon,3,4,5 David B. Hodge,6,7 Timothy J. Donohue,2,3

6

Daniel R. Noguera,1,3*

7 8

1

Department of Civil and Environmental Engineering, 2Department of Bacteriology, 3DOE Great

9

Lakes Bioenergy Research Center, 4Department of Chemistry, 5Department of Biomolecular

10

Chemistry, University of Wisconsin-Madison, Madison, WI

11 12

6

Department of Chemical Engineering & Materials Science, 7Department of Biosystems & Agricultural Engineering, Michigan State University, East Lansing, MI

13 14

* Corresponding author

15

Daniel R. Noguera

16

Department of Civil and Environmental Engineering

17

University of Wisconsin-Madison

18

1415 Engineering Drive

19

Madison, WI 53706

20

Phone: (608) 263-7783

21

Fax: (608) 262-5199

22

Email: [email protected]


Page 2 of 31

Page 3 of 31

23


ABSTRACT

24

Lignocellulosic biomass hydrolysates hold great potential as a feedstock for microbial

25

biofuel production, due to their high concentration of fermentable sugars. Present at lower

26

concentrations are a suite of aromatic compounds that can inhibit fermentation by biofuel-

27

producing microbes. We have developed a microbial-mediated strategy for removing these

28

aromatic compounds, using the purple non-sulfur bacterium Rhodopseudomonas palustris.

29

When grown photoheterotrophically in an anaerobic environment, R. palustris removes most of

30

the aromatics from ammonia fiber expansion (AFEX) treated corn stover hydrolysate (ACSH),

31

while leaving the sugars mostly intact. We show that R palustris can metabolize a host of

32

aromatic substrates in ACSH that have either been previously described as unable to support

33

growth, such as methoxylated aromatics, and those that have not yet been tested, such as

34

aromatic amides. Removing the aromatics from ACSH with R. palustris, allowed growth of a

35

second microbe that could not grow in the untreated ACSH. By using defined mutants, we show

36

that most of these aromatic compounds are metabolized by the benzoyl-CoA pathway. We also

37

show that loss of enzymes in the benzoyl-CoA pathway prevents total degradation of the

38

aromatics in the hydrolysate, and instead allows for biological transformation of this suite of

39

aromatics into selected aromatic compounds potentially recoverable as an additional bioproduct.

2 ACS Paragon Plus Environment


40

Page 4 of 31

INTRODUCTION

41

The increasing world-wide demand for energy is accelerating fossil fuel consumption,

42

depleting natural resources, and contributing to climate change.1 With roughly 80% of the

43

world’s primary energy supply derived from fossil fuels, there is significant interest in increasing

44

the contribution of renewable fuels to the overall energy production portfolio. Liquid fuels

45

generated from lignocellulosic biomass are of particular interest as transportation fuels for long-

46

term environmental and economic sustainability.

47

The Energy Independence and Security Act created a roadmap for increased industrial

48

production of biofuels from lignocellulosic biomass in the United States.2 According to the

49

roadmap, the production of renewable fuels from lignocellulosic biomass was expected to reach

50

1.75 billion gallons by 2014.2 The actual production was only 683,643 gallons,3 and the first

51

generation of commercial-scale biorefineries in the U.S., to be in full operation in 2015, will not

52

exceed an annual capacity of 50 million gallons.4

53

Clearly, major bottlenecks still exist for the cost-effective production of biofuels from

54

lignocellulosic biomass. Some of the challenges are economic and brought about by the large

55

amounts of fossil fuels that can now be tapped with horizontal drilling and hydraulic fracturing,

56

which contribute to instability in the price of fossil fuels. Other challenges are technical,

57

requiring new scientific and engineering innovation to bring transformational changes and cost

58

reductions to the lignocellulosic biofuels industry.

59

One of the persistent challenges to implement cost-effective fermentation processes is the

60

presence of plant-derived aromatic compounds and other small bioactive molecules in

61

hydrolysates derived from lignocellulosic biomass.5,6 Some of these molecules have been shown

62

to diminish biofuel production by inhibiting growth and metabolism of sugars in fermenting


Page 5 of 31


63

organisms. For instance, acetic acid is known to affect cellular processes, reduce ethanol yields,

64

and decrease sugar consumption in wild type and engineered strains of Saccharomyces

65

cerevisiae,7,8 whereas the negative effects of a variety of furans or aromatic compounds on

66

ethanologens such as S. cerevisiae, Zymomonas mobilis, and Escherichia coli are well

67

documented.9-14

68

The suite of inhibitory molecules in lignocellulosic hydrolysates is diverse and highly

69

dependent on the biomass pretreatment used to produce the hydrolysates. However, aromatic

70

compounds are reported to be found in hydrolysates independent of the plant species or

71

hydrolysis method.6 Strategies to overcome the effect of these inhibitory bioactive molecules

72

range from physical or chemical removal,15 to microbial and enzymatic degradation.16,17

73

Although removal can be achieved by different approaches, in most cases the removal of the

74

inhibitory compounds is accompanied by an undesirable decrease in the amount of sugars (e.g., 5

75

to 35%).17

76

In this study, we showed that Rhodopseudomonas palustris, a bacterium known to

77

anaerobically degrade aromatic compounds and utilize short chain organic acids,18 can remove

78

inhibitory aromatics from corn stover hydrolysate, without consuming the sugars needed for

79

biofuel production. In addition, genetic removal of selected enzymes in the benzoyl-CoA

80

pathway, used for anaerobic aromatic metabolism in R. palustris, resulted in the

81

biotransformation of the large variety of plant-derived aromatics into selected phenolic

82

compounds that could be potentially recovered and used for other applications. To our

83

knowledge, this is the first demonstration that the diversity of aromatics in hydrolysates can be

84

biotransformed to a single aromatic, a strategy that could add value to lignocellulosic biomass

85

biorefineries, where production of multiple products can aid the cost-effective and sustainable



Page 6 of 31

86

production of biofuels and chemicals from lignocellulosic biomass.4 Thus, the data in this

87

manuscript (1) provides the first demonstration of using a microorganism for metabolism and

88

removal of mixed aromatics present in hydrolysates produced by the ammonia fiber expansion

89

(AFEX)19 process; (2) demonstrates a process to remove these compounds that does not reduce

90

the sugar content of the hydrolysates; and (3) demonstrates the possibility of not only removing

91

inhibitory compounds from the hydrolysates, but also converting a chemically diverse suite of

92

aromatics into single compounds (either benzoic acid or p-hydroxybenzoic acid), which can

93

potentially be recovered as an additional valuable chemical co-product in the biorefinery.

94 95

EXPERIMENTAL SECTION

96

Corn Stover Hydrolysate. AFEX-pretreated corn stover hydrolysate (ACSH) was

97

prepared as described by Schwalbach et al.20 and diluted ~1:3 with sterile deionized water to

98

reach ~ 2% glucose content. The hydrolysates were filtered in series through 0.5 µm and 0.22

99

µm filters (Nalgene Disposable Bottle Top Filter, Thermo Fisher Scientific, Waltham, MA) prior

100

to storage at 4°C. Before inoculation, the pH of each hydrolysate batch, originally between 4.6

101

and 4.8, was adjusted to 7.0 using potassium hydroxide (KOH) pellets. After pH adjustment, the

102

hydrolysates were filter-sterilized by passing through 0.22 µm filters.

103

Microbial strains. R. palustris CGA009,21 R. palustris CGA606 (CGA009-derived

104

mutant lacking benzoyl-CoA reductase activity),22 and R. palustris CGA506 (CGA009-derived

105

mutant lacking 4-hydroxybenzoyl-CoA reductase activity)23 were used in this study. In addition,

106

R. sphaeroides 241EDD, an R. sphaeroides 2.4.124,25-derived mutant with a modification to

107

restore function of the edd gene (See Supporting Information) was also used.


Page 7 of 31


108

Minimal Media. Photosynthetic medium (PM), prepared as described in Kim et al.26 and

109

containing succinate as the organic substrate, was used for R. palustris growth before inoculation

110

in hydrolysate. Sistrom’s minimal medium (SIS) containing succinate as the organic substrate,

111

prepared as previously described,27 was used for growing cultures of R. sphaeroides before

112

inoculation into hydrolysate.

113

Experimental Conditions. Most experiments were conducted in an Applikon

114

biofermenter (3L Autoclavable Microbial BioBundle, Applikon Biotechnology, Foster City, CA

115

94404) using 1,000 mL of ACSH. In these experiments the pH was controlled between 6.95 and

116

7.10 with 1M H2SO4 and 10M KOH, and the cultures were placed in front of continuous light

117

generated by 10W tungsten lamps. The temperature was kept at 28˚C, oxygen was removed by

118

flushing with N2 gas, and cell densities were measured using the Klett-Summerson colorimeter

119

with a no. 66 filter (Klett MFG Co., NY). R. sphaeroides 241EDD and R. palustris CGA009

120

were pre-grown in minimal media, and for each inoculation, 20 mL of culture were added to

121

1,000 mL of ACSH.

122

Experiments with R. palustris CGA506 and CGA606 were conducted with ACSH (~ 1%

123

glucose content) in the presence of 100 µg kanamycin mL-1. The strain was initially grown in

124

PM, and for each incubation, 150 µL of culture were added to 15 mL of hydrolysate. Glass

125

reactor tubes were closed with rubber stoppers to ensure anaerobic conditions and placed in

126

continuous illumination at 30˚ C.

127

Analytical procedures. Organic acids and sugars were analyzed by high performance

128

liquid chromatography (HPLC) and quantified with a refractive index detector (RID-10A,

129

Shimadzu) using a Bio-Rad Aminex HPX-87H column at 60˚C and mobile phase of 5 mM

130

H2SO4 at 0.6 mL/min as described by Schwalbach et al.20 Samples were prepared by filtering



Page 8 of 31

131

(0.22 µm) aliquots of the culture and diluting the filtrate ten-fold before injection into the HPLC.

132

The majority of phenolic compounds were quantified by reverse phase HPLC – high

133

resolution/accurate mass mass spectrometry, as described in Keating et al.28 Benzoic acid and 4-

134

hydroxybenzoic acid were measured by high performance anion exchange chromatography –

135

tandem mass spectrometry, using procedures also described in Keating et al.28

136 137

RESULTS AND DISCUSSION

138

Metabolism of plant-derived aromatics in corn stover hydrolysates by R. palustris

139

CGA009. An effective microbial strategy to remove aromatic inhibitors from hydrolysates will

140

require an organism that specifically degrades these compounds without compromising the

141

conversion of glucose and xylose to biofuel. In addition to being deficient in sugar utilization,21

142

R. palustris CGA009 efficiently grows using short chain organic acids29 and is also known for its

143

ability to utilize aromatic compounds as sole carbon sources under anaerobic conditions, using

144

the benzoyl-CoA pathway.18,30,31 Entrance into this pathway (Figure 1) occurs through activation

145

of either benzoate or p-hydroxybenzoate by ligation to coenzyme A (CoA). A subsequent

146

stepwise reduction of the aromatic ring yields 1-ene-cyclohexanoyl-CoA, leading to ring

147

cleavage and further transformations to metabolites that enter central metabolism.22,32

148

Although R. palustris CGA009 has been shown to degrade several aromatic

149

hydrocarbons (Table 1), little is known about its ability to utilize more complex plant-derived

150

aromatics present in hydrolysates (Figure 2), which contain up to two methoxy functional groups

151

and an alkyl side-chain with characteristics that depend on the methods used for biomass

152

deconstruction.6 Table 1 summarizes the aromatics that were known to be degraded by R.

153

palustris and compares them to aromatics found in ACSH. In general, R. palustris CGA009 has 7 ACS Paragon Plus Environment

Page 9 of 31


154

been shown to completely degrade phenolic acids without ring substitutions or with only one

155

hydroxyl group in the meta or para position (Table 1).18 In addition, of the phenolic acids with a

156

propanoid side-chain, p-coumaric acid can be completely degraded by R. palustris GGA009

157

(Figure 1),33 while only partial degradation, without ring fission, was shown to occur with

158

aromatic acids having more than one ring substitution, such as ferulic or caffeic acid (Table 1).

159

R. palustris CGA009 has also been reported not to grow on vanillin, vanillic acid or syringic acid

160

when present as a sole organic carbon source.18 Growth of R. palustris CGA009 on the aromatic

161

amides that are both found in hydrolysates prepared with the AFEX pretreatment19 and known to

162

have inhibitory effects on ethanologenic microbes6 has not been investigated so far.

163

Given the knowledge gap on the ability of R. palustris CGA009 to degrade aromatic

164

amides and phenolics with more than one ring substitution, we evaluated the extent of aromatic

165

transformation by this organism when grown in ACSH (Figure 3). Coumaroyl amide and

166

feruloyl amide, the two aromatic amides present at the highest concentrations in ACSH, were

167

removed from the medium (Figure 3A). Concomitant with the loss of these aromatics, 4-

168

hydroxybenzoic acid, vanillic acid, and 3,4-dihydroxybenzoic acid accumulated (Figures 3C,

169

3D), suggesting that these aromatics are intermediates in the degradation of the propanoyl

170

amides. Ferulic acid transiently accumulated, but was almost completely absent in the medium

171

toward the end of the experiment (Figure 3D). Consistent with prior knowledge on its ability to

172

serve as a carbon source,34 p-coumaric acid was removed from the medium (Figure 3C). In

173

addition, aromatic benzaldehydes (Figure 3E) were also transformed, regardless of the number of

174

methoxy groups that they contained, while the extracellular level of aromatic phenones did not

175

change, and a small accumulation of 4-hydroxyacetophenone was observed (Figure 3F).



Page 10 of 31

176

This experiment demonstrated for the first time that R. palustris CGA009 has the ability

177

to degrade coumaroyl amide and feruloyl amide, although three other aromatic amides present in

178

ACSH, vanillamide, syringamide, and 4-hydroxybenzamide were not transformed (Figure 3B).

179

Based on the predicted pathway for p-coumaric acid degradation (Figure 1),34,35 a possible route

180

for coumaroyl amide degradation may include an initial removal of the amine group and

181

activation to coumaroyl-CoA, followed by removal of the alkyl chain leading to p-

182

hydroxybenzaldehyde and oxidation to p-hydroxybenzoic acid, which then enters the benzoyl-

183

CoA pathway after CoA-ligation. The accumulation of p-hydroxybenzoic acid in the medium

184

(Figure 3C) suggests that CoA ligation of p-hydroxybenzoic acid is a limiting step in the use of

185

coumaroyl amide and other aromatics in ACSH.

186

transformations, with the removal of the alkyl chain after CoA ligation resulting in the formation

187

of vanillin, and then accumulation of vanillic acid. Although it has been shown that vanillic acid

188

is not degraded by R. palustris CGA001,18 the parent strain of R. palustris CGA009 (CGA009 is

189

a chloramphenicol resistant derivative of CGA001), there is evidence that other R. palustris

190

strains can use vanillic acid as a sole carbon source.18 In the experiment with ACSH, the molar

191

concentration of vanillic acid in the medium was about one half of the initial concentration of

192

feruloyl amide (Figure 3), suggesting that some degradation of vanillic acid occurred. In separate

193

experiments with more dilute ACSH and longer incubation times (see supporting information)

194

we observed complete removal of vanillic acid, and therefore, we propose that the accumulation

195

of vanillic acid in Figure 3C reflects a transient extracellular build up of this putative pathway

196

metabolite. In support of this hypothesis, we note that the extracellular accumulation of p-

197

hydroxybenzoic acid is also transient (see supporting information); this is not surprising since

198

this compound is known to be metabolized via the benzoyl-CoA pathway (Figure 1).23 Likewise,

Feruloyl amide may undergo similar


Page 11 of 31


199

the seemingly stable accumulation of benzoic acid in the medium over the course of this

200

experiment is likely due to it being actively produced and consumed during the experiment, as it

201

is also a readily degradable aromatic by R. palustris (see supporting information). The

202

extracellular accumulation of 3,4-dihydroxybenzoic acid (protocatechuic acid) is also of note,

203

since it is difficult to explain the source of this compound. One possibility is the removal of a

204

methoxy group from the mono methoxylated aromatics during the degradation of vanillic acid

205

and ferulic acid, although there is no prior knowledge that such a transformation is catalyzed by

206

R. palustris. Another possibility is that protocatechuic acid is produced from the degradation of

207

unidentified plant-derived aromatics present in ACSH. Regardless, we propose that

208

protocatechuic acid is slowly degraded via the benzoyl-CoA pathway based on previous studies

209

which showed that R. palustris CGA009 will degrade protocatechuic acid only when benzoic

210

acid or p-hydroxybenzoic acid are also present, suggesting that these later compounds induce the

211

benzoyl-CoA pathway, which does not get induced in the presence of protocatechuic acid

212

alone.36 The same synergy that allows R. palustris to degrade protocatechuic acid in the presence

213

of other aromatics may be an explanation for the degradation of vanillic acid in ACSH.

214

While these experiments extend the knowledge on the range of plant-derived aromatics

215

that R. palustris CGA009 can degrade, some compounds remained unutilized in ACSH.

216

Specifically, there is no evidence that R. palustris CGA009 can degrade acetophenones, or

217

dimethoxylated aromatics other than syringaldehyde present in ACSH (Figure 3).

218

R. palustris CGA009 removes the short chain organic acids but does not consume

219

the sugars found in ACSH. The removal of inhibitory compounds from hydrolysates is needed

220

to alleviate metabolic stress in microorganisms used for fermentative production of ethanol or

221

other compounds from lignocellulosic biomass hydrolysates.12,28,37 However, an effective



Page 12 of 31

222

hydrolysate pretreatment needs to selectively remove the inhibitors, while leaving the sugars and

223

other essential nutrients available for subsequent fermentation. As shown in Figure 4, R.

224

palustris CGA009 does not consume glucose and xylose, the main sugars present in corn stover

225

hydrolysate. In addition to metabolizing the aromatics in ACSH, acetate was the other main

226

organic substrate that was removed from the medium in these cultures.

227

Biotransformation of aromatics with accumulation of benzoic acid derivatives using

228

R. palustris mutants. If the most abundant aromatics present in ACSH are biotransformed via

229

the benzoyl-CoA pathway, we hypothesized that blocking this pathway could lead to partial

230

transformations of some aromatics, but without ring cleavage. We tested this hypothesis with R.

231

palustris CGA606, a mutant with an insertion in the badE gene that inactivates benzoyl-CoA

232

reductase (Figure 1) and prevents de-aromatization of benzoyl-CoA.22 Experiments with the

233

BadE mutant showed transformation of aromatic compounds, with a significant accumulation of

234

benzoic acid in the medium (Figure 5). Since benzoic acid is not present at high levels in ACSH

235

and remained at low concentrations during growth using R. palustris CGA009 (Figure 3), we

236

conclude that its accumulation when using R. palustris CGA606 is a consequence of losing BadE

237

activity (which normally uses benzoyl-CoA as a substrate).

238

We also tested growth of R. palustris CGA506, which lacks 4-hydroxybenzoyl-CoA

239

reductase (HbaBCD) activity,23 in ACSH. Based on what is known about the benzoyl-CoA

240

pathway, the loss of HbaBCD should block degradation of para-hydroxylated aromatics but not

241

benzoic acid. In experiments using CGA506, we found loss of aromatic compounds and

242

accumulation of 4-hydroxybenzoic acid in the medium (Figure 5). HbaBCD uses 4-

243

hydroxybenzoyl-CoA as a substrate (Figure 1), so the accumulation of 4-hydroxybenzoic acid in

244

the medium predicts that metabolism of the aromatics in ACSH also uses this enzyme. More


Page 13 of 31


245

importantly, these experiment show that most of the aromatics are metabolized through the

246

common benzoyl-CoA pathway where the aromatic ring is reduced (Figure 1). They also

247

demonstrate the ability of engineered R. palustris strains to convert a diverse pool of aromatics

248

into a single compound (either benzoic acid, 4-hydroxybenzoic acid, or possibly others based on

249

the genetic block in the benzoyl-CoA pathway).

250

Biological removal of aromatics from ACSH improves growth of a second

251

bacterium. The negative effect of aromatic compounds on ethanologenic fermentations has

252

been well documented, with p-coumaric acid,38 benzoic acid,39 p-hydroxybenzaldehyde,11,12

253

vanillin,11,40 4-hydroxyacetophenone,11 acetovanillone,11, and aromatic amides28 reported to be

254

inhibitory to bacterial or yeast ethanologens. Our experiments show that most of these

255

compounds can be removed from ACSH using R. palustris CGA009 (Figure 3 and Table 1).

256

Therefore, we considered the possibility that R. palustris can be used as a bio-based method to

257

reduce metabolic stress and improve ethanologenic or other biofuel fermentations. To test this

258

hypothesis we used Rhodobacter sphaeroides, an organism that we are investigating for

259

production of advanced biofuels, such as long-chain fatty acids and furans.41,42

260

R. sphaeroides does not grow at the ACSH concentrations used in this study. However,

261

when we inoculated R. sphaeroides 241EDD, a strain with an improved rate of glucose

262

utilization (See Supplementary Materials) in filter-sterilized ACSH that had been used to grow R.

263

palustris CGA009, we found that R. sphaeroides could grow and metabolize the sugars. This

264

demonstrates that biological conditioning of ACSH with R. palustris CGA009 allows growth and

265

improved sugar metabolism by a second bacterium (Figure 6). From control experiments where

266

R. sphaeroides is grown in the presence of the aromatic compounds found in ACSH, we know

267

that p-coumaric acid, p-hydroxybenzaldehyde, and p-hydroxyacetophenone inhibited glucose



Page 14 of 31

268

utilization, while the other aromatics were not inhibitory.43 ACSH conditioning with R. palustris

269

effectively

270

hydroxyacetophenone, which remained at low concentrations in the hydrolysate (Figure 3). Thus,

271

the removal of the aromatics that were present at highest concentrations in ACSH by R. palustris

272

was sufficient to allow R. sphaeroides growth and sugar utilization.

removed

p-coumaric

acid

and

p-hydroxybenzaldehyde,

but

not

p-

273

Implications of the biological removal of aromatics from lignocellulosic biomass

274

hydrolysates. Our results demonstrate the possibility to exploit R. palustris metabolism for

275

removal of aromatic compounds from lignocellulosic biomass hydrolysates. A key observation

276

is that R. palustris CGA009 grown in ACSH leaves the sugars unaltered and available for biofuel

277

production by a second microbe because it preferentially uses acetate and aromatics as organic

278

electron donors. While demonstrated in this study using ACSH, removal of inhibitory aromatics

279

with R. palustris could be applied to other biomass pretreatments where aromatics in the

280

hydrolysates are also a concern.6 In addition the removal of acetate from ACSH by R. palustris

281

could also provide added benefit to the use yeast and other microbes in which fermentation

282

performance is inhibited by the presence of this organic acid in the biomass hydrolysate.5,44 To

283

achieve the full potential of this process it is necessary to engineer strains capable of growing in

284

more concentrated hydrolysates, and extend the range of plant-derived aromatics that can be

285

metabolized. Moreover, by engineering strains capable of channeling the aromatics into specific

286

phenolic compounds, as demonstrated here with several R. palustris mutants, it should be

287

possible to both remove inhibitors and convert them to valuable bioproducts recoverable from

288

the hydrolysates. The accumulation of well-defined phenolic compounds by engineered strains of

289

R. palustris adds to the diversity of biochemicals that could be produced in a biorefinery,45 and


Page 15 of 31


290

contributes to increasing the fraction of the carbon present in the hydrolysates that is recovered

291

as a valuable product instead of being released as organic waste.

292 293

ACKNOWLEDGEMENTS

294

We thank Jackie Bastyr-Cooper for assistance performing analytical tests, and facilities at

295

MSU and UW for the production of corn stover hydrolysates. This work was funded by the US

296

Department of Energy Great Lakes Bioenergy Research Center (DOE Office of Science BER

297

DE-FC02-07ER64494). J. Zachary Oshlag was supported by a traineeship from the NIGMS

298

Biotechnology Training grant (Grant T32 GM08349). We extend our thanks to Carrie S.

299

Harwood for providing the R. palustris strains used in this study.

300 301

SUPPORTING INFORMATION AVAILABLE

302

Text describing the construction of R. sphaeroides 241EDD strain, additional figures

303

supporting the reproducibility of aromatic degradation in ACSH by R. palustris CGA009 (Figure

304

S1) and the lack of growth of R. sphaeroides 241EDD in ACSH that has not been biologically

305

conditioned by R. palustris CGA009 (Figure S2), and a table showing aromatic degradation in

306

diluted ACSH (Table S1). This information is available free of charge via the Internet at

307

http://pubs.acs.org.

308 309

REFERENCES

310 311

1.

312

Washington, D.C., 2011, www.eia.gov/forecasts/archive/ieo11/pdf/0484(2011).pdf.

International Energy Outlook 2011; U.S. Energy Information Administration:



Page 16 of 31

313

2.

Energy Independence and Security Act of 2007. Public Law 110-140; 2007,

314

www.gpo.gov/fdsys/pkg/BILLS-110hr6enr/pdf/BILLS-110hr6enr.pdf.

315

3.

316

http://epa.gov/otaq/fuels/rfsdata/2014emts.htm

317

4.

318

DOE/SC-0170; U. S. Department of Energy Office of Science: Washington D.C., 2015,

319

http://genomicscience.energy.gov/biofuels/lignocellulose/.

320

5.

321

inhibition and detoxification. Bioresour. Technol. 2000, 74, (1), 17-24; DOI 10.1016/S0960-

322

8524(99)00160-1.

323

6.

324

Landick, R., Death by a thousand cuts: the challenges and diverse landscape of lignocellulosic

325

hydrolysate inhibitors. Frontiers in Microbiology 2014, 5, 1-8; DOI 10.3389/fmicb.2014.00090.

326

7.

327

on the kinetics of xylose fermentation by an engineered, xylose-isomerase-based Saccharomyces

328

cerevisiae strain. FEMS Yeast Res. 2009, 9, (3), 358-364; DOI 10.1111/j.1567-

329

1364.2009.00487.x.

330

8.

331

The fraction of cells that resume growth after acetic acid addition is a strain-dependent parameter

332

of acetic acid tolerance in Saccharomyces cerevisiae. FEMS Yeast Res. 2014, 14, (4), 642-653;

333

DOI 10.1111/1567-1364.12151.

U. S. Environmental Protection Agency 2014 RFS2 Data,

Lignocellulosic biomass for advanced biofuels and bioproducts: Workshop Report,

Palmqvist, E.; Hahn-Hägerdal, B., Fermentation of lignocellulosic hydrolysates. I:

Piotrowski, J. S.; Zhang, Y. P.; Bates, D. M.; Keating, D. H.; Sato, T. K.; Ong, I. M.;

Bellissimi, E.; van Dijken, J. P.; Pronk, J. T.; van Maris, A. J. A., Effects of acetic acid

Swinnen, S.; Fernandez-Nino, M.; Gonzalez-Ramos, D.; van Maris, A. J. A.; Nevoigt, E.,


Page 17 of 31


334

9.

Chambel, A.; Viegas, C. A.; Sa-Correia, I., Effect of cinnamic acid on the growth and on

335

plasma membrane H+-ATPase activity of Saccharomyces cerevisiae. Int. J. Food Microbiol.

336

1999, 50, (3), 173-179; DOI 10.1016/s0168-1605(99)00100-2.

337

10.

338

Induces Messenger Ribonucleoprotein (mRNP) Granule Formation in Saccharomyces cerevisiae:

339

Application and Validation of High-Content, Image-Based Profiling. PLoS One 2013, 8, (4), 1-

340

10; DOI 10.1371/journal.pone.0061748.

341

11.

342

oxidation of wheat straw and their effect on ethanol production of Saccharomyces cerevisiae:

343

Wet oxidation and fermentation by yeast. Biotechnol. Bioeng. 2003, 81, (6), 738-747; DOI

344

10.1002/Bit.10523.

345

12.

346

products on ethanol fermentations of glucose and xylose by Saccharomyces cerevisiae,

347

Zymomonas mobilis, Pichia stipitis, and Candida shehatae. Enzyme Microb. Technol. 1996, 19,

348

(3), 220-225; DOI 10.1016/0141-0229(95)00237-5.

349

13.

350

fermentation of ethanologenic Escherichia coli. Biotechnol Bioeng 1999, 65, (1), 24-33; DOI

351

10.1002/(SICI)1097-0290(19991005)65:13.0.CO;2-2.

352

14.

353

Qin, L.; Busalacchi, D.; Deshpande, S.; Gasch, A.; Hodge, D., Harnessing Genetic Diversity in

354

Saccharomyces cerevisiae for Improved Fermentation of Xylose in Hydrolysates of Alkaline

355

Hydrogen Peroxide Pretreated Biomass Appl Environ Microbiol 2014, 80, (2), 540-554; DOI

356

10.1128/AEM.01885-13.

Iwaki, A.; Ohnuki, S.; Suga, Y.; Izawa, S.; Ohya, Y., Vanillin Inhibits Translation and

Klinke, H. B.; Olsson, L.; Thomsen, A. B.; Ahring, B. K., Potential inhibitors from wet

Delgenes, J. P.; Moletta, R.; Navarro, J. M., Effects of lignocellulose degradation

Zaldivar, J.; Martinez, A.; Ingram, L. O., Effect of selected aldehydes on the growth and

Sato, T.; Liu, T.; Parreiras, L.; Williams, D.; Wohlbach, D.; Bice, B.; Ong, I.; Breuer, R.;



Page 18 of 31

357

15.

Larsson, S.; Reimann, A.; Nilvebrant, N. O.; Jonsson, L. J., Comparison of different

358

methods for the detoxification of lignocellulose hydrolyzates of spruce. Appl. Biochem.

359

Biotechnol. 1999, 77-9, 91-103; DOI 10.1385/abab:77:1-3:91.

360

16.

361

wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor.

362

Appl. Microbiol. Biotechnol. 1998, 49, (6), 691-697; DOI 10.1007/s002530051233.

363

17.

364

lignocellulose hydrolysates for ethanol production: review. Crit. Rev. Biotechnol. 2011, 31, (1),

365

20-31; DOI 10.3109/07388551003757816.

366

18.

367

compounds by the photosynthetic bacterium Rhodopseudomonas palustris. Appl. Environ.

368

Microbiol. 1988, 54, (3), 712-717.

369

19.

370

using Saccharomyces cerevisiae 424A(LNH-ST). Proc. Natl. Acad. Sci. USA 2009, 106, (5),

371

1368-1373; DOI 10.1073/pnas.0812364106.

372

20.

373

Bothfeld, W.; Higbee, A.; Grass, J. A.; Cotten, C.; Reed, J. L.; Sousa, L. D.; Jin, M. J.; Balan,

374

V.; Ellinger, J.; Dale, B.; Kiley, P. J.; Landick, R., Complex Physiology and Compound Stress

375

Responses during Fermentation of Alkali-Pretreated Corn Stover Hydrolysate by an Escherichia

376

coli Ethanologen. Appl. Environ. Microbiol. 2012, 78, (9), 3442-3457; DOI 10.1128/aem.07329-

377

11.

378

21.

379

Pelletier, D. A.; Beatty, J. T.; Lang, A. S.; Tabita, F. R.; Gibson, J. L.; Hanson, T. E.; Bobst, C.;

Jonsson, L. J.; Palmqvist, E.; Nilvebrant, N. O.; Hahn-Hagerdal, B., Detoxification of

Parawira, W.; Tekere, M., Biotechnological strategies to overcome inhibitors in

Harwood, C. S.; Gibson, J., Anaerobic and aerobic metabolism of diverse aromatic

Lau, M. W.; Dale, B. E., Cellulosic ethanol production from AFEX-treated corn stover

Schwalbach, M. S.; Keating, D. H.; Tremaine, M.; Marner, W. D.; Zhang, Y. P.;

Larimer, F. W.; Chain, P.; Hauser, L.; Lamerdin, J.; Malfatti, S.; Do, L.; Land, M. L.;


Page 19 of 31


380

Torres, J.; Peres, C.; Harrison, F. H.; Gibson, J.; Harwood, C. S., Complete genome sequence of

381

the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris. Nat.

382

Biotechnol. 2004, 22, (1), 55-61; DOI 10.1038/nbt923.

383

22.

384

bacterial genes for anaerobic benzene ring biodegradation. Proc. Natl. Acad. Sci. USA 1997, 94,

385

(12), 6484-6489; DOI 10.1073/pnas.94.12.6484.

386

23.

387

(dehydroxylating) is required for anaerobic degradation of 4-hydroxybenzoate by

388

Rhodopseudomonas palustris and shares features with molybdenum-containing hydroxylases. J.

389

Bacteriol. 1997, 179, (3), 634-642.

390

24.

391

P.; Barber, R. D.; Donohue, T. J.; Hosler, J. P.; Newman, J. E.; Shapleigh, J. P.; Sockett, R. E.;

392

Zeilstra-Ryalls, J.; Kaplan, S., The home stretch, a first analysis of the nearly completed genome

393

of Rhodobacter sphaeroides 2.4.1. Photosynthesis Res. 2001, 70, (1), 19-41; DOI

394

10.1023/A:1013831823701.

395

25.

396

H.; Pennachio, C.; Sodergren, E.; Weinstock, G.; Noguera, D. R.; Donohue, T. J., Revised

397

sequence and annotation of the Rhodobacter sphaeroides 2.4.1 genome. Journal of Bacteriology

398

2012, 194, 7016-7017; DOI 10.1128/JB.01214-12.

399

26.

400

palustris. FEMS Microbiol. Lett. 1991, 83, (2), 199-203; DOI 10.1111/j.1574-

401

6968.1991.tb04441.x.

Egland, P. G.; Pelletier, D. A.; Dispensa, M.; Gibson, J.; Harwood, C. S., A cluster of

Gibson, J.; Dispensa, M.; Harwood, C. S., 4-hydroxybenzoyl coenzyme A reductase

Mackenzie, C.; Choudhary, M.; Larimer, F. W.; Predki, P. F.; Stilwagen, S.; Armitage, J.

Kontur, W. S.; Shackwitz, W.; Ivanova, N.; Martin, J.; LaButti, K.; Deshpande, S.; Tice,

Kim, M. K.; Harwood, C. S., Regulation of benzoate-CoA ligase in Rhodopseudomonas



Page 20 of 31

402

27.

Sistrom, W. R., The kinetics of the synthesis of photopigments in Rhodopseudomonas

403

spheroides. J Gen Microbiol 1962, 28, 607-16.

404

28.

405

Tremaine, M.; Bothfeld, W.; Higbee, A.; Ulbrich, A.; Balloon, A.; Westphall, M. S.; Aldrich, J.;

406

Lipton, M. S.; Kim, J.; Moskvin, O.; Bukhman, Y. V.; Coon, J.; Kiley, P. J.; Bates, D. M.;

407

Landick, R., Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder

408

bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and

409

detoxification. Frontiers in Microbiology 2014, 5, 1-17; DOI 10.3389/fmicb.2014.00402.

410

29.

411

for hydrogen production by photosynthetic bacteria. J. Biotechnol. 2001, 85, (1), 25-33; DOI

412

10.1016/s0168-1656(00)00368-0.

413

30.

414

Rhodopseudomonas palustris. Biochem. J. 1969, 113, 525.

415

31.

416

aromatic compounds via the benzoyl-CoA pathway. FEMS Microbiol. Rev. 1998, 22, (5), 439-

417

458; DOI 10.1111/j.1574-6976.1998.tb00380.x.

418

32.

419

palustris mediates dicarboxylic acid degradation and participates in anaerobic benzoate

420

degradation. Microbiology-Sgm 2005, 151, 727-736; DOI 10.1099/mic.0.27731-0.

421

33.

422

Degradation by Rhodopseudomonas palustris and Identification of CouR, a MarR Repressor

423

Protein That Binds p-Coumaroyl Coenzyme A. J. Bacteriol. 2012, 194, (8), 1960-1967; DOI

424

10.1128/jb.06817-11.

Keating, D. H.; Zhang, Y.; Ong, I. M.; McIlwain, S.; Morales, E. H.; Grass, J. A.;

Barbosa, M. J.; Rocha, J. M. S.; Tramper, J.; Wijffels, R. H., Acetate as a carbon source

Dutton, P. L.; Evans, W. C., The Metabolism of Aromatic Compounds by

Harwood, C. S.; Burchhardt, G.; Herrmann, H.; Fuchs, G., Anaerobic metabolism of

Harrison, F. H.; Harwood, C. S., The pimFABCDE operon from Rhodopseudomonas

Hirakawa, H.; Schaefer, A. L.; Greenberg, E. P.; Harwood, C. S., Anaerobic p-Coumarate


Page 21 of 31


425

34.

Pan, C.; Oda, Y.; Lankford, P. K.; Zhang, B.; Samatova, N. F.; Pelletier, D. A.; Harwood,

426

C. S.; Hettich, R. L., Characterization of anaerobic catabolism of p-coumarate in

427

Rhodopseudomonas palustris by integrating transcriptomics and quantitative proteomics. Mol.

428

Cell. Proteomics 2008, 7, (5), 938-948; DOI 10.1074/mcp.M700147-MCP200.

429

35.

430

J.; Harwood, C. S., Identification of a p-Coumarate Degradation Regulon in Rhodopseudomonas

431

palustris by Xpression, an Integrated Tool for Prokaryotic RNA-Seq Data Processing. Appl.

432

Environ. Microbiol. 2012, 78, (19), 6812-6818; DOI 10.1128/aem.01418-12.

433

36.

434

mediated metabolism of meta-hydroxy-aromatic acids in Rhodopseudomonas palustris. J

435

Bacteriol 2013, 195, (18), 4112-20; DOI 10.1128/JB.00634-13.

436

37.

437

Influence of lignocellulose-derived aromatic compounds on oxygen-limited growth and ethanolic

438

fermentation by Saccharomyces cerevisiae. Appl Biochem Biotechnol 2000, 84-86, 617-32; DOI

439

10.1385/ABAB:84-86:1-9:617.

440

38.

441

cerevisiae for simultaneous saccharification and ethanol fermentation from industrial waste

442

corncob residues. Bioresour. Technol. 2014, 157, 6-13; DOI 10.1016/j.biortech.2014.01.060.

443

39.

444

metabolic fluxes in yeasts - A continuous-culture study on the regulation of respiration and

445

alcoholic fermentation. Yeast 1992, 8, (7), 501-517; DOI 10.1002/yea.320080703.

Phattarasukol, S.; Radey, M. C.; Lappala, C. R.; Oda, Y.; Hirakawa, H.; Brittnacher, M.

Gall, D. L.; Ralph, J.; Donohue, T. J.; Noguera, D. R., Benzoyl coenzyme A pathway-

Larsson, S.; Quintana-Sainz, A.; Reimann, A.; Nilvebrant, N. O.; Jonsson, L. J.,

Gu, H. Q.; Zhang, J.; Bao, J., Inhibitor analysis and adaptive evolution of Saccharomyces

Verduyn, C.; Postma, E.; Scheffers, W. A.; Vandijken, J. P., Effect of benzoic acid on



Page 22 of 31

446

40.

Fitzgerald, D. J.; Stratford, M.; Gasson, M. J.; Ueckert, J.; Bos, A.; Narbad, A., Mode of

447

antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum and Listeria

448

innocua. J. Appl. Microbiol. 2004, 97, (1), 104-113; DOI 10.1111/j.1365-2672.2004.02275.x.

449

41.

450

Coon, J. J.; Donohue, T. J., Synthesis and scavenging role of furan fatty acids. Proc. Natl. Acad.

451

Sci. USA 2014, 111, (33), E3450-E3457; DOI 10.1073/pnas.1405520111.

452

42.

453

regulation of bacterial lipid production. J. Bacteriol. 2015, 197, (9), 1649-1658; DOI

454

10.1128/JB.02510-14.

455

43.

456

Rhodopseudomonas palustris under photoheterotrophic conditions. MS Thesis, University of

457

Wisconsin-Madison, Madison, WI, 2013.

458

44.

459

growth by acetic acid: a problem with methionine biosynthesis and homocysteine toxicity.

460

Microbiology-Sgm 2002, 148, 2215-2222.

461

45.

462

A.; Frederick, W. J.; Hallett, J. P.; Leak, D. J.; Liotta, C. L.; Mielenz, J. R.; Murphy, R.;

463

Templer, R.; Tschaplinski, T., The path forward for biofuels and biomaterials. Science 2006,

464

311, (5760), 484-489; DOI 10.1126/science.1114736.

Lemke, R. A. S.; Peterson, A. C.; Ziegelhoffer, E. C.; Westphall, M. S.; Tjellstrom, H.;

Lemmer, K.; Dohnalkova, A.; Noguera, D. R.; Donohue, T. J., Oxygen-dependent

Austin, S. Utilization of corn stover hydrolysates by Rhodobacter sphaeroides and

Roe, A. J.; O'Byrne, C.; McLaggan, D.; Booth, I. R., Inhibition of Escherichia coli

Ragauskas, A. J.; Williams, C. K.; Davison, B. H.; Britovsek, G.; Cairney, J.; Eckert, C.

465

466 467 468 21 ACS Paragon Plus Environment

Page 23 of 31

469


Table 1. Anaerobic transformation of aromatic compounds by R. palustris CGA009* Degradation as sole carbon source Transformed Ring fission

Compound Acetosyringone Acetovanillone Benzaldehyde Benzoic acid Benzoylformic acid Caffeic acid Cinnamaldehyde Cinnamic acid Coumaroyl amide p-Coumaric acid Cyclohexanecarboxylic acid A-1-cyclohexenecarboxylic acid A-3-cyclohexenecarboxylic acid Cyclohexanepropionic acid 3,4-dihydroxybenzoic acid (protocatechuic acid) Ferulic acid Feruloyl amide Hydrocaffeic acid Hydrocinnamaldehyde 4-Hydroxyacetophenone 4-Hydroxybenzamide 4-Hydroxybenzaldehyde 3-Hydroxybenzoic acid 4-Hydroxybenzoic acid 4-Hydroxybenzoylformic acid DL-mandelic acid 4-Phenylbutyric acid 3-Phenylpropionic acid (hydrocinnamic acid) 5-Phenylvaleric acid Syringaldehyde Syringamide Syringic acid Vanillamide Vanillic acid Vanillin

470 471 472 473 474 475 476 477 478 479 480 481

Y18 Y18 Y18 Y18 Y18 Y18

Y18 Y18 N18

Detected Y Y

Transformed N N

Y

Y

Y Y

Y Y

Y Y Y

Y** Y Y

Y Y Y

N N Y

Y

Y

Y Y Y Y Y Y

Y N N N N Y

Y18

Y18,34 Y18 Y18 Y18 Y18 N18,36 Y18

Y18,34

Y18 Y18

N18

Y18 Y18 Y18 Y18 Y18 Y18 Y18 Y18

Transformation in ACSH

N

18

Y18 Y18 Y18 Y18 N18 Y18 Y18

N18 N18 N18

*

Harwood et al.18 reports transformations in R. palustris CGA001, the parent strain of CGA009. Other compounds, not found in hydrolysates, and shown to not support anaerobic growth of R. palustris CGA001 are: 4-aminobenzoic acid, anthranilate (2-aminobenzoic acid), catechol, 3chlorobenzoic acid, coniferyl alcohol, 4-cresol, cyclohexanol, cyclohexanone, ethylvanillic acid, 2-fluorobenzoic acid, gallic acid (trihydroxybenzoic acid), gentisic acid, nicotinic acid, phenol, phenoxyacetic acid, 3-phenylbutyric acid, quinic acid, resorcinol, salicylic acid (2hydroxybenzoic acid), shikimic acid, trimethoxybenzoic acid, trimethoxycinnamic acid, 3toluic acid, 4-toluic acid, homogentisic acid, isovanillic acid, phenylacetic acid. Y = yes, N = no. ** Protocatechuic acid has been shown to be degraded if benzoic acid or p-hydroxybenzoic acid are present in the medium36 22 ACS Paragon Plus Environment


Page 24 of 31

482 483

FIGURE LEGENDS

484 485

Figure 1. Aromatics known to be degraded by R. palustris CGA009 through the benzoyl-CoA

486

pathway. The boxes indicate aromatics found in cellulosic biomass hydrolysates. References for

487

the reactions shown in this figure are included in Table 1.

488 489

Figure 2. Chemical structures of aromatic compounds found in lignocellulosic hydrolysates.

490

Concentrations in one batch of hydrolysate used in this study are indicated for each compound.

491

The greyed out aromatics, sinapic acid and sinapoyl amide, were not detected.

492 493

Figure 3. Transformation of aromatic compounds by R. palustris CGA009 in growth

494

experiments using ACSH. Aromatic amides present at (A) high and (B) low concentrations;

495

aromatic acids at (C) high and (D) low concentrations; (E) aromatic aldehydes; and (F)

496

phenones. The molecular structure of each compound is presented in Figure 2.

497 498

Figure 4. Growth (A), concentration of sugars (B), and concentration of short-chain organic acid

499

(C) during growth of R. palustris CGA009 in ACSH.

500 501

Figure 5. Concentrations of aromatic compounds in ACSH and extracellular concentrations of

502

the same aromatics after growth of R. palustris CGA009 (wild type), CGA606 (∆badE) and

503

CGA506 (∆hbaB).

504


Page 25 of 31


505

Figure 6. Growth of R. sphaeroides 241EDD (A) and sugar utilization (B) in ACSH that had

506

been biologically conditioned with R. palustris CGA009. Note that R. sphaeroides 241EDD did

507

not grow in ACSH that had not been conditioned with R. palustris CGA009 (see supporting

508

information).



Figure 1. Aromatics known to be degraded by R. palustris CGA009 through the benzoyl-CoA pathway. The boxes indicate aromatics found in cellulosic biomass hydrolysates. References for the reactions shown in this figure are included in Table 1. 152x114mm (300 x 300 DPI)


Page 26 of 31

Page 27 of 31


Figure 2. Chemical structures of aromatic compounds found in lignocellulosic hydrolysates. Concentrations in the hydrolysates used in this study are indicated for each compound. The greyed out aromatics, sinapic acid and sinapoyl amide, were not detected. 99x78mm (300 x 300 DPI)



Figure 3. Transformation of aromatic compounds by R. palustris CGA009 in growth experiments using ACSH. Aromatic amides present at (A) high and (B) low concentrations; aromatic acids at (C) high and (D) low concentrations; (E) aromatic aldehydes; and (F) phenones. The molecular structure of each compound is presented in Figure 2. 242x307mm (300 x 300 DPI)


Page 28 of 31

Page 29 of 31


Figure 4. Growth (A), concentration of sugars (B), and concentration of short-chain organic acid (C) during growth of R. palustris CGA009 in ACSH. 178x375mm (300 x 300 DPI)



Figure 5. Concentrations of aromatic compounds in ACSH and extracellular concentrations of the same aromatics after growth of R. palustris CGA009 (wild type), CGA606 (∆badE) and CGA506 (∆hbaB). 69x50mm (300 x 300 DPI)


Page 30 of 31

Page 31 of 31


Figure 6. Growth of R. sphaeroides 241EDD (A) and sugar utilization (B) in ACSH that had been biologically conditioned with R. palustris CGA009. Note that R. sphaeroides 241EDD did not grow in ACSH that had not been conditioned with R. palustris CGA009 (see supporting information). 120x161mm (300 x 300 DPI)


Metabolism of Multiple Aromatic Compounds in ... - ACS Publications

Recommend Documents