Effect of n-Caproate Concentration on Chain Elongation and

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Effect of n-caproate concentration on chain elongation and competing processes Mark Roghair, Yuchen Liu, Julius Adiatma, Ruud A. Weusthuis, Marieke E. Bruins, Cees J.N. Buisman, and David P.B.T.B. Strik ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00200 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 17, 2018

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ACS Sustainable Chemistry & Engineering

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Effect of n-caproate concentration on chain elongation and

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competing processes

3 4

Mark Roghair a, Yuchen Liu a, Julius C. Adiatma a, Ruud A. Weusthuis b, Marieke E. Bruins c,

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Cees J.N. Buisman a, David P.B.T.B. Strik a,*

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a

7

Weilanden 9, 6708 WG, Wageningen, the Netherlands

8

b

9

PB, Wageningen, the Netherlands

Sub-department of Environmental Technology, Wageningen University & Research, Bornse

Bioprocess Engineering, Wageningen University & Research, Droevendaalsesteeg 1, 6708

10

c

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Weilanden 9, 6708 WG, Wageningen, the Netherlands

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* Corresponding author: E-mail: [email protected]; tel +31 317 483 447

Wageningen Food & Biobased Research, Wageningen University & Research, Bornse

13 14 15

KEYWORDS

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Chain elongation, n-caproate, inhibition, competing processes, adapted sludge

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ABSTRACT

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Chain elongation is an open-culture fermentation process that facilitates conversion of organic

20

residues with an additional electron donor, such as ethanol, into valuable n-caproate. Open-

21

culture processes are catalyzed by an undefined consortium of microorganisms which

22

typically also bring undesired (competing) processes. Inhibition of competing processes, such

23

as syntrophic ethanol oxidation, will lead to a more selective n-caproate production process.

24

In this study, we investigated the effect of n-caproate concentration on the specific activity of

25

chain elongation and competing processes using batch inhibition assays. With ‘synthetic

26

medium sludge’ (originally operating at 3.4 g/L n-caproate), syntrophic ethanol oxidation was

27

proportionally inhibited by n-caproate until 45% inhibition at 20 g/L n-caproate.

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Hydrogenotrophic methanogenesis was for 58% inhibited at 20 g/L n-caproate. Chain

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elongation of volatile fatty acids (VFA upgrading; the desired process), was completely

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inhibited at 20 g/L n-caproate with all tested sludge types. ‘Adapted sludge’ (operating at 23.2

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g/L n-caproate) showed a 10 times higher VFA upgrading activity at 15 g/L n-caproate

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compared to ‘non-adapted sludge’ (operating at 7.1 g/L n-caproate). This shows that

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microbiomes do adapt to perform chain elongation at high n-caproate concentrations which

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likely inhibits syntrophic ethanol oxidation through hydrogenotrophic methanogenesis. As

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such, we provide supporting evidence that the formation of n-caproate inhibits syntrophic

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ethanol oxidation which leads to a more selective MCFA production process.

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INTRODUCTION

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Organic residual streams, such as food waste, have great potential as alternative resource for

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production of fuels and chemicals. These residues are conventionally anaerobically digested

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to produce methane for electric power and/or heat production 1. Methane, however, has a low

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monetary value. Producing higher-value products than methane, therefore, is gaining

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increasing interest. Among these higher-value products, particularly medium chain fatty acids

47

(MCFAs), such as caproic acid and heptanoic acid are interesting because MCFAs can be

48

used for a wide variety of applications (e.g. aviation fuels, lubricants, feed additives).

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Moreover, like methane, MCFAs are also produced through open-culture anaerobic reactor

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microbiomes. Open-culture processes are catalysed by an undefined consortium of

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microorganisms. This is different from pure culture or defined co-culture processes, which are

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catalysed by only one (or more) known species with known pathways. The advantage of

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open-culture processes is that they handle a mixture of residual streams without the need for

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sterilisation. The technological challenge is to control the mixed microbial population within

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the microbiome to produce MCFAs selectively from the substrates.

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MCFA production from organic residues by open-cultures occurs via two subsequent

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processes which can be combined or performed separately. First, organic residues are

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hydrolysed by hydrolytic enzymes and acidified by acidogenic bacteria, resulting in

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production of volatile fatty acids (VFAs) such as acetate, propionate and butyrate. Second,

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these VFAs are converted together with an electron donor, such as ethanol, into MCFAs

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through chain elongation. This conversion is performed by chain elongating micro-organisms

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(e.g. Clostridium kluyveri) that use the reverse β-oxidation pathway (eq 1) 2. Chain elongation



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with open cultures is an emerging application that can handle many organic feedstocks at

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various conditions and reactor configurations 3.

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Reverse β-oxidation pathway

5 + 6 → 5 +

66

+ 4 + + 2

(eq 1)

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Here, we focus on ethanol-based chain elongation which is catalyzed by anaerobic

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open-cultures. These open-cultures include not only chain elongating microorganisms but also

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other functional groups of microorganisms. These other functional groups can degrade the

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substrates and products into undesired metabolites. This makes their presence and activity

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detrimental to the MCFA selectivity of chain elongation processes. The challenge, therefore,

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is to find process conditions that reduce their activity through a selection pressure like

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inhibition. Inhibition is preferably performed without the use of bioactive chemicals (e.g.

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2-bromoethanesulfonate or iodoform as methanogenic inhibitor) so that the non-converted

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organics from the process can be used as soil fertilizer upon composting. We recognize the

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following competing processes in chain elongation 4 5:

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Acetotrophic methanogenesis

+ → +

(eq 2)

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Anaerobic acetate oxidation

+ + 2 → 4 + 2

(eq 3)

79

Excessive ethanol oxidation (EEO) + → + + 2

(eq 4)

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Hydrogenotrophic methanogenesis 2 + 0.5 → 0.5 +

(eq 5)

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Syntrophic ethanol oxidation

+ 0.5 → + + 0.5 (eq 6)

82 83

Understanding the role of competing processes and how they can be suppressed is essential to

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establish a selective MCFA production process. For example, acetotrophic methanogenesis 4 ACS Paragon Plus Environment

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degrades acetate into methane and CO2 (eq 2). This process is performed by acetotrophic

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methanogens. Acetotrophic methanogenesis was shown to be largely inhibited at slightly

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acidic pH (5.5) in chain elongating processes 6. When working at near-neutral pH, which is

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more favorable for these methanogens, they could be outselected by applying a combination

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of sufficient hydraulic shear force and a low hydraulic retention time (HRT) to respectively

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detach and wash out these slow-growing methanogens 7. Anaerobic oxidation of fatty acids

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occurs though β-oxidation of fatty acids into acetate and hydrogen (and also CO2 in the case

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of propionate oxidation). Acetate can be further oxidized into CO2 and hydrogen (eq 3).

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Anaerobic oxidation of fatty acids is performed by acetogenic bacteria and can be

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thermodynamically inhibited at a hydrogen partial pressure (pH2) of >0.007 % at standard

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conditions 5. Excessive ethanol oxidation (EEO) occurs through direct anaerobic oxidation of

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ethanol into acetate and hydrogen (eq 4). This process is considered to be performed by

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ethanol-oxidizing microorganisms which do not perform chain elongation. Suppression of

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EEO is important not only because this process leads to an inefficient use of ethanol but also

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because it acidifies the fermentation broth. Because of this acidifying effect, EEO requires

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extra base addition which increases operating expenses 8. EEO can be thermodynamically

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inhibited at a hydrogen partial pressure (pH2) of ≥ 14% at standard conditions 5. Such high

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pH2 pressures, however, are not common in chain elongation processes because hydrogen is

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typically consumed through hydrogenotrophic methanogenesis (eq 5). In our previous study 5,

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we showed that EEO and hydrogenotrophic methanogenesis are coupled processes. The

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overall reaction can be referred to as syntrophic ethanol oxidation (eq 6). Syntrophic ethanol

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oxidation can be limited by reducing the available amount of CO2. This has been shown to

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limit hydrogenotrophic methanogenic activity and the resulting higher pH2

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thermodynamically inhibits EEO 4 5 9. CO2 loading rate may be difficult to control, however,

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when working with (hydrolyzed and acidified) organic residues. This is because CO2 is



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produced during acidification of hydrolyzed residues and the presence of this CO2 in the feed

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may complicate the actual CO2 supply to the chain elongation process. Even though

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acidification and chain elongation can be separated, dissolved CO2 could still complicate the

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control over the actual CO2 supply. As such, alternative methods to limit EEO in chain

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elongation are needed.

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In a previous study, an alternative method to inhibit EEO may have been shown; by operating

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a continuous chain elongation process at long HRT (4 d), EEO was limited to 0.9 g/L/d (14.7

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% of total ethanol consumption) while n-caproate was produced at a high concentration (23.4

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g/L) 8. In contrast, at short HRT (1 d), EEO occurred at a higher rate (5.6 g/L/d; 45.0 % of

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total ethanol consumption) while n-caproate was produced at a lower concentration (7.1 g/L).

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A hypothesis is that the high n-caproate concentration may have had a selective inhibitory

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effect on one of the competing syntrophs (ethanol oxidizers or hydrogenotrophic

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methanogens). Note that we refer to n-caproate as both forms together, undissociated

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n-caproic acid and dissociated n-caproate, and that we refer to each specific form when

124

appropriate.

125

It is well known that MCFAs are toxic to microorganisms because they can damage the cell

126

membrane 10. Gram-positive bacteria and methanogens tend to be more easily inhibited by

127

long chain fatty acids (LCFAs) than Gram-negative bacteria 11. Selective inhibition of

128

MCFAs on competing functional groups of microorganisms may be an alternative strategy to

129

suppress competing processes to enhance the MCFA selectivity. High MCFAs concentrations,

130

however, may also inhibit chain elongation itself. To circumvent potential MCFA toxicity, in

131

situ MCFA extraction was applied which showed to enhance n-caproate production 12. These

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results were used to clarify that MCFAs inhibit chain elongating microorganisms. A

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systematic investigation of the inhibitory effect of n-caproate on chain elongation and

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competing processes in chain elongation microbiomes has not been reported to date. 6 ACS Paragon Plus Environment

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In this study, we investigated the effect of n-caproate concentration on the specific activities

136

of chain elongation microbiomes in two experiments. Firstly, we studied the effect of

137

n-caproate on the specific activity of five processes: 1) chain elongation (VFA upgrading), 2)

138

syntrophic ethanol oxidation, 3) hydrogenotrophic methanogenesis, 4) acetotrophic

139

methanogenesis, and 5) anaerobic acetate oxidation. This was performed using sludge from a

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continuous chain elongation process that operated at 3.4 g/L n-caproate. Secondly, we

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extended the experiment for chain elongation comparing two types of sludge to study the

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potential adaptation of chain elongation microbiomes to high n-caproate concentrations. One

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sludge type originally operated at 7.1 g/L caproate (non-adapted sludge) whereas the other

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sludge type originally operated at 23.2 g/L n-caproate (adapted sludge)



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EXPERIMENTAL SECTION

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Experimental design of batch inhibition assays

147

An overview of the experimental design is reported in Table 1.

148

Media preparation

149

All liquid media were initially prepared the same way by adding salts, vitamins, trace

150

elements and yeast extract to deoxygenated water 13. For NH4(H2)PO4, we used 1.8 g/L

151

instead of 3.6 g/L. Buffering agents were also added: 200 mM MES (VWR, the Netherlands),

152

200 mM BISTRIS (Sigma-Aldrich, the Netherlands) and 10 mM PIPPS (98.4%, Merck

153

Millipore, USA). For the first experiment, five different media were prepared. Thus, after

154

buffers were added, the mixture was divided into five fractions. The fraction used to monitor

155

chain elongation (VFA upgrading) was supplemented with 11.5 g/L ethanol (Absolute, VWR,

156

France) and 3 g/L propionic acid (≥ 99.5%, Sigma-Aldrich). With this ethanol concentration,

157

ethanol toxicity is avoided 14. The fraction used to monitor syntrophic ethanol oxidation was

158

supplemented with 11.5 g/L ethanol. The fraction used to monitor acetotrophic

159

methanogenesis and anaerobic acetate oxidation was supplemented with 2 g/L acetic acid

160

(99.9%, VWR, France). The fraction used to monitor hydrogenotrophic methanogenesis was

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not supplemented with a substrate. Each fraction used to monitor a specific process was again

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divided into 4 smaller fractions to add n-caproic acid (≥ 98%, Sigma-Aldrich) to

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concentrations of 0 (control), 10, 15 and 20 g/L. For the second experiment, we added one

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assay in which n-caproic acid was added up to 25 g/L. Finally, the pH of all assays were

165

adjusted to 6.8 with 5M NaOH.

166

Methods


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Batch inhibition assays were performed in 125 or 250 mL serum bottles. The bottles were

168

filled with 45.0 g medium under anaerobic conditions. After filling, bottles were closed with a

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rubber stopper and an aluminium crimp seal. The headspaces of the bottles for chain

170

elongation (VFA upgrading), syntrophic ethanol oxidation, acetotrophic methanogenesis and

171

anaerobic acetate oxidation were flushed and pressurized to 1.5 bar with a mixture of N2/CO2

172

(80/20%). Likewise, the headspaces of the bottles for hydrogenotrophic methanogenesis were

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flushed and pressurized to 1.5 bar with a mixture of H2/CO2 (80/20%). After preparation,

174

bottles were inoculated with a fresh inoculum (5 mL) using a needle and syringe.

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In the first experiment, bottles were inoculated with ‘synthetic medium sludge’. Synthetic

176

medium sludge was directly derived from a running continuous chain elongation process that

177

converted synthetic substrates (propionate and ethanol) into MCFAs at 3.4 g/L n-caproate 13.

178

Based on a carbon flux analysis (e.g. ref 5), we determined that synthetic medium sludge

179

contained chain elongating microorganisms, ethanol oxidizers and hydrogenotrophic

180

methanogens.

181

In the second experiment, bottles were inoculated with ‘non-adapted sludge’ or ‘adapted

182

sludge’. Both sludge types were directly derived from a running continuous chain elongation

183

process that converted acidified food waste and ethanol into MCFAs 8. Whereas non-adapted

184

sludge operated at 7.1 g/L n-caproate (day 103), adapted sludge operated at 23.2 g/L

185

n-caproate (day 124) 8. Properties of the used inocula, including VSS concentrations, sodium

186

concentrations, n-caproate concentrations and original average specific activities in reactor,

187

are reported in Table 2. Specifications on process conditions that conditioned the inocula are

188

reported in the supporting information.

189

After inoculation, bottles were incubated at 30 °C at 100 rpm in a rotary shaker (New

190

Brunswick Scientific Innova 44). Contents of the bottles were daily analysed on headspace



191

pressure, headspace composition, fatty acids and alcohols. All assays were conducted in

192

duplicate.

193

Determination of specific activity

194

The activity of syntrophic ethanol oxidation, acetotrophic methanogenesis and

195

hydrogenotrophic methanogenesis was based on the maximum slope of methane production

196

vs. time divided by the initial VSS concentration.

197

The activity of anaerobic acetate oxidation was based on the maximum slope of hydrogen

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production vs. time divided by the initial VSS concentration.

199

The activity of chain elongation was based on the maximum slope of n-valerate plus

200

n-heptanoate production vs. time divided by the initial VSS concentration. Chain elongation

201

activity was based on the production of odd-numbered fatty acids and not on production of

202

even-numbered fatty acids. This was performed to determine a measure of chain elongation

203

activity that is independent of other active functional groups of microorganisms. Whereas

204

odd-numbered fatty acids can only be produced through VFA upgrading (chain elongation of

205

added VFAs), even-numbered fatty acids can also be produced through ethanol upgrading (in

206

situ ethanol oxidation into acetate and subsequent chain elongation into even-numbered fatty

207

acids). Because ethanol upgrading is not only mediated by chain elongating microorganisms

208

but also by ethanol oxidizers and hydrogenotrophic methanogens 5, we quantified only the

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odd numbered chain elongation products (valerate and heptanoate) by VFA upgrading as a

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measure for chain elongation activity.

211

Undissociated n-caproic acid concentrations were calculated from the observed n-caproate

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concentrations and pH values (during the maximum specific activity) using the

213

Henderson-Hasselbalch equation.

214 10 ACS Paragon Plus Environment

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Analytical techniques

216 217

Alcohols (C2-C6) and fatty acids (C2-C8) were analyzed by gas chromatography 15. Gaseous

218

compounds were determined by gas chromatography 16. Sodium was measured by ion

219

chromatography 17. VSS measurements were performed in the same way as before 13.

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Table 1. Experimental design of batch inhibition assays Process studied in inhibition assay

Initial aqueous

Initial headspace

Activity based on

substrate(s)

composition

production of

[g/L]

[vol %]

Inoculum

Initial n-caproate

Working

concentration

volume/ Total

[g/L]

bottle volume [mL]

VFA Upgrading

Ethanol [11.5]

N2/CO2 [80/20]

Valerate and heptanoate

Synthetic medium sludge

iii

N2/CO2 [80/20]

Methane


iii

0, 10, 15, 20

50 / 125


iii

0, 10, 15, 20

50 / 250

0, 10, 15, 20

50 / 125

0, 10, 15, 20

50 / 125

0, 10, 15, 20, 25

50 / 125

0, 10, 15, 20, 25

50 / 125

0, 10, 15, 20

50 / 125

Propionate [3]

Syntrophic ethanol oxidation Hydrogenotrophic methanogenesis

Acetotrophic methanogenesis Anaerobic acetate oxidation VFA Upgrading

Ethanol [11.5] None

Acetate [2] Acetate [2] Ethanol [11.5]

H2/CO2 [80/20]

N2/CO2 [80/20] N2/CO2 [80/20]

Methane

Methane

i

Hydrogen

Synthetic medium sludge ii


N2/CO2 [80/20]


Non-adapted sludge

N2/CO2 [80/20]


Adapted sludge v

iv

iii iii

Propionate [3] VFA Upgrading

Ethanol [11.5] Propionate [3]

222 223

i

Can only be quantified when anaerobic acetate oxidation and hydrogenotrophic methanogenesis do not occur.

224

ii

Can only be quantified when acetotrophic methanogenesis and hydrogenotrophic methanogenesis do not occur.

225

iii

Synthetic medium sludge was grown converting ethanol and propionate into MCFAs at 3.4 g/L caproate.

226

iv

Non-adapted sludge was grown converting acidified food waste and ethanol into MCFAs at 7.1 g/L n-caproate.

12

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227


v

Adapted sludge was grown converting acidified food waste and ethanol into MCFAs at 23.2 g/L n-caproate.

13



228

RESULTS

229 230

n-Caproate concentration affects chain elongation and competing processes using

231

‘synthetic medium sludge’

232 233

The effect of n-caproate concentration on the maximum specific activities of various

234

processes was investigated with ‘synthetic medium sludge’ as inoculum. Processes that were

235

investigated were: VFA upgrading, syntrophic ethanol oxidation, hydrogenotrophic

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methanogenesis, acetotrophic methanogenesis and anaerobic acetate oxidation. Hereby we

237

note that VFA upgrading is the desired chain elongation process that leads to a selective

238

MCFA production process. Results of this batch inhibition assay that showed an effect of

239

n-caproate are graphically summarized in Figure 1a and 1b. Numerical values of average

240

specific activities are reported in Table 2 and profiles of product production are presented in

241

the supporting information (Figure S1-S3).

242 243

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Figure 1. Results of batch inhibition assays; initial n-caproate concentration vs. observed

245

activity (a) and relative activity (b) of various processes using synthetic medium sludge as

246

inoculum. Synthetic medium sludge was grown converting synthetic substrates (propionate

247

and ethanol) at 3.4 g/L n-caproate. Values indicate averages of duplicates and bars indicate

248

range of duplicates (often too small to be visual). T = 30 °C, pH = 6.8 (buffered).

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The effect of n-caproate on both acetotrophic methanogenesis and anaerobic acetate oxidation

251

could not be determined because these processes did not occur in the controls (0 g/L

252

n-caproate) or in the experimental assays. Likely, acetotrophic methanogens and acetogenic

253

bacteria were not substantially present in the sludge.

254

Evidently, n-caproate inhibited all other quantified processes in the tested range (Figure 1).

255

The activity of VFA upgrading (i.e. specific valerate plus heptanoate activity) in the control

256

was 798 mmol C/gVSS/d and was for 90 % inhibited at 10 g/L n-caproate. Remarkably, at 15

257

and 20 g/L n-caproate, VFA upgrading was completely inhibited. VFA upgrading was the

258

most sensitive process for n-caproate inhibition compared to syntrophic ethanol oxidation and

259

hydrogenotrophic methanogenesis. The activity of syntrophic ethanol oxidation in the control

260

was 97 mmol C/gVSS/d and was proportionally inhibited by n-caproate until 45% inhibition

261

at 20 g/L n-caproate. Syntrophic ethanol oxidation was the least sensitive process to

262

n-caproate inhibition. Hydrogenotrophic methanogenic activity in the control was 295 mmol

263

C/gVSS/d and was not inhibited at 10 and 15 g/L n-caproate. However, at a higher 20 g/L

264

n-caproate, hydrogenotrophic methanogenesis was indeed inhibited with 58 %.

265

Hydrogenotrophic methanogenic activity was always higher than the activity of syntrophic

266

ethanol oxidation.

267

Up to 15 g/L n-caproate, the relative order of inhibition was VFA upgrading > syntrophic

268

ethanol oxidation with no inhibition for hydrogenotrophic methanogenesis. Within this

269

n-caproate concentration range, syntrophic ethanol oxidation was not limited by

270

hydrogenotrophic methanogenesis. At 20 g/L n-caproate, the relative order of inhibition was

271

VFA upgrading > hydrogenotrophic methanogenesis > syntrophic ethanol oxidation. At this

272

n-caproate concentration, hydrogenotrophic methanogenesis was severely suppressed by

273

n-caproate inhibition. This is in line with earlier work by Hajarnis et al. (1994) who found that

274

pure cultures of hydrogenotrophic methanogens, Methanobacterium bryantii and 15 ACS Paragon Plus Environment


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Methanobacterium formicium, were inhibited for 96 and 95% respectively by 20 g/L

276

n-caproate at pH 7.0 18. By extrapolating our data, we estimate that, at 25 g/L n-caproate,

277

syntrophic ethanol oxidation may become rate-limited by hydrogenotrophic methanogenesis.

278

This extrapolation provides supportive evidence for the hypothesis that a high n-caproate

279

concentration is a control strategy to suppress syntrophic ethanol oxidation through

280

hydrogenotrophic methanogenesis in chain elongation processes. At high n-caproate

281

concentrations, however, chain elongation itself may also be inhibited; though ethanol may be

282

used more efficiently, as was shown in our previous study 8.

283

It was remarkable that at 15 and 20 g/L n-caproate, VFA upgrading was not observed, which

284

shows that chain elongating microorganisms were completely inhibited by n-caproate.

285

Previous studies, however, reported chain elongation activity up to considerably higher

286

n-caproate concentrations (i.e. 21 g/L 19, 23 g/L 20 and even up to 25 g/L 8 at similar pH).

287

Probably, the sludge that we used as inoculum was not adapted to such high n-caproate

288

concentrations. The actual inoculum, ‘synthetic medium sludge’, was indeed derived from a

289

running chain elongation process with a lower n-caproate concentration (3.4 g/L) than that

290

was present in the experimental assays at 15 and 20 g/L n-caproate. Therefore, the sudden

291

increase of n-caproate during inoculation may have resulted in an acute toxic effect to the

292

chain elongating microorganisms.

293

Comparing VFA upgrading using non-adapted sludge and adapted sludge

294

To investigate whether sludge can be adapted to perform VFA upgrading (i.e. chain

295

elongation) at high n-caproate concentrations, a follow-up experiment was performed. In this

296

experiment, VFA upgrading activity was compared using an inoculum that operated at low

297

n-caproate concentration (7.1 g/L; non-adapted sludge) with an inoculum that operated at high

298

n-caproate concentration (23.2 g/L; adapted sludge). Results of this experiment are

299

graphically summarized in Figure 2. Numerical values on average specific activities are 16 ACS Paragon Plus Environment

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300

reported in Table 2 and profiles of product production are reported in the supporting

301

information (Figure S4 and S5). At 10 g/L n-caproate, VFA upgrading activity was a factor 14

302

higher with adapted sludge (411 mmol C/gVSS/d) than with non-adapted sludge. At 15 g/L,

303

VFA upgrading activity was a factor 10 higher with adapted sludge (181 mmol C/gVSS/d)

304

than with non-adapted sludge. These figures show that the n-caproate tolerance of VFA

305

upgrading activity was clearly increased with adapted sludge. However, at 20 g/L n-caproate

306

the VFA upgrading process was completely inhibited with both adapted sludge and

307

non-adapted sludge.

308 309

310

Figure 2. Results of batch inhibition assays; initial n-caproate concentration vs. observed

311

activity (a) and relative activity (b) of VFA upgrading using non-adapted and adapted sludge

312

as inoculum. Non-adapted sludge was grown converting acidified food waste and ethanol into

313

MCFAs at 7.1 g/L n-caproate. Adapted sludge was grown converting acidified food waste and

314

ethanol into MCFAs at 23.2 g/L n-caproate. Values indicate averages of duplicates and bars

315

indicate range of duplicates (often too small to be visual). T = 30 °C, pH = 6.8 (buffered).

316 317


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318

Table 2. Inoculum specifications, average specific activities in batch assays and observed sodium and undissociated n-caproic acid

319

concentrations in assays. Inoculum name, compounds in inoculum [g/L] and average specific activity of

Average specific activity in batch assays [mmol C/gVSS/d]

inoculum in reactor [mmol C/gVSS/d]

at different initial n-caproate concentrations [g/L].

Process

Inoculum name

VSS

Sodium

n-Caproate

Activity

VFA Upgrading


0.6

6.1 ± 0.5

3.4 ± 0.3

Syntrophic ethanol oxidation


0.6

6.1 ± 0.5

Hydrogenotrophic methanogenesis


0.6

Acetotrophic methanogenesis


Anaerobic acetate oxidation

iii

0

10

15

20

25

1362 ± 106

798

77

0

0

ND

3.4 ± 0.3

59 ± 11

97

82

68

53

ND

6.1 ± 0.5

3.4 ± 0.3

59 ± 11

295

319

301

124

ND

0.6

6.1 ± 0.5

3.4 ± 0.3

ND

0

0

0

0

0


0.6

6.1 ± 0.5

3.4 ± 0.3

ND

0

0

0

0

0

VFA Upgrading

Non-adapted sludge

0.4

7.0 ± 0.0

7.1 ± 0.9

220 ± 95

1476

29

18

17

0

VFA Upgrading

Adapted sludge

0.5

9.1 ± 0.5

23.2 ± 1.9

386 ± 76

1056

411

181

0

0

2.3 - 3.2

4.0 - 5.0

4.7 - 5.6

5.6 - 6.4

7.1 - 7.1

0 - 0.1

0.1 - 0.2

0.1 - 0.3

0.2 - 0.4

0.3 - 0.4

Solutes in batch assays [g/L] Sodium concentration i Undissociated n-caproic acid concentration

320 321 322 323 324 325

ii

i

Observed range of initial sodium concentrations in batch assays for different initial n-caproate concentrations.

ii

Observed range of undissociated n-caproic acid concentrations during batch inhibition assays for different initial n-caproate concentrations.

iii

Original average reactor activity of inoculum; calculated from reactor data (see supporting information)

ND = Not determined. ± = Standard deviation based on 3 or more measurements.

326

18


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327


DISCUSSION

328 329

n-Caproate inhibits the active functional groups in chain elongation microbiomes

330

In this study, the effect of n-caproate concentration on the specific activity of chain elongation

331

and competing processes was investigated. The specific activity of all active processes

332

decreased with an increasing n-caproate concentration, though the degree in which these

333

processes were inhibited was different for each process. The most plausible explanation for

334

these decreased activities is that n-caproate is inhibitory to the active microorganisms. Such

335

kind of inhibition depends on the MCFA concentration (in this case n-caproate), the chain

336

length, the pH 21 and also on the identity and the degree of adaptation of the microorganism 10.

337

Evenly, in theory, n-caproate can thermodynamically inhibit its own production. However, we

338

calculated that under the actual conditions, n-caproate did not induce a thermodynamic

339

bottleneck for chain elongation 5.

340 341

Both undissociated n-caproic acid and dissociated n-caproate are inhibitory

342

Evidently, fatty acids in water can appear in their undissociated form or in their dissociated

343

form. The distribution of these two forms depends on the pH and the pKa of the fatty acid. A

344

low (i.e. acidic) pH yields a high fraction of undissociated fatty acids whereas a neutral and a

345

high pH yields a low fraction of undissociated fatty acids. Undissociated MCFAs are

346

considered to be more inhibitory to microorganisms than dissociated MCFAs 10. This seems

347

also true for chain elongating microorganisms. Chain elongation studies that operate at

348

slightly acidic pH, therefore, typically report lower MCFA production rates (9 12) compared to

349

studies that operate at near-neutral pH (13, 22, 23). For details on mechanisms of (un)dissociated 19 ACS Paragon Plus Environment


350

MCFA inhibition, we refer the interested reader to a review article 24.

351

In this study, we were interested in the inhibitory effect of both n-caproate forms together at

352

constant pH. As such, even though EEO and the reverse β-oxidation pathway release a proton

353

(i.e. acidify the media), we had to minimize undissociation of n-caproate by keeping the pH

354

constant during the assays. This was performed through the use of various buffers.

355

The assays in our study had an initial pH of 6.8; meaning that 1.1% of total n-caproate, with a

356

pKa of 4.85, was undissociated. The lowest observed pH at the end of an assay was 6.5 (data

357

not shown); meaning that, at most, 2% of total n-caproate was undissociated. Therefore, we

358

conclude that the buffers worked well and the protocol used in this study could thus be used

359

for other batch inhibition assays that require constant pH. We also conclude that VFA

360

upgrading in underlying study was likely not solely inhibited by undissociated n-caproic acid

361

alone. Rather, this process was likely inhibited by a combination of both forms together. This

362

is because the highest observed undissociated n-caproic acid concentration in our study (~0.4

363

g/L; Table 2) was always below the proposed toxic limit of undissociated n-caproic acid for

364

chain elongation (~0.87 g/L 12). This would mean that, although dissociated MCFAs are

365

considered less toxic than undissociated MCFAs, they are inhibitory too.

366 367 368

The role of exogenous n-caproate and sodium

369

Before adapted sludge was used as inoculum in the batch inhibition assays, it was active in

370

VFA upgrading (386 mmolC/gVSS/d) at an average n-caproate concentration of 23.2 g/L

371

(Table 2). In the batch assays at 20 and 25 g/L n-caproate, however, this sludge showed no

372

VFA upgrading activity (Figure 2). We have no mechanistic explanation for this. Possibly,

373

exogenous (i.e. supplied) n-caproate may impose a different inhibitory effect than endogenous 20 ACS Paragon Plus Environment

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374

(i.e. produced) n-caproate. The exogenous n-caproate in the batch inhibition assays may thus

375

be more toxic to chain elongating microorganisms than endogenous produced n-caproate in

376

the continuous chain elongation process. Earlier work demonstrated a similar, but opposite

377

effect; endogenous ethanol exerted a greater impact on yeast performance than exogenous

378

ethanol 25. Another example is that Escherichia coli was associated with fewer membrane

379

damage with exogenously supplied styrene than with endogenously produced styrene 26.

380

Hence, these authors stressed the importance of considering the difference between

381

exogenous and endogenous compounds when characterizing the effects of product inhibition.

382

All assays started with the same initial pH (6.8). Because n-caproic acid was added at

383

different concentrations in the preparation of the media, however, also different amounts of

384

sodium hydroxide were added to adjust the pH. This means that, besides n-caproate, also the

385

sodium concentrations varied among the assays. Indeed, observed initial sodium

386

concentrations were proportional to the initial n-caproate concentrations (Table 2). Sodium

387

can be inhibitory to microorganisms through an increase of osmotic pressure or complete

388

dehydration of microorganisms 27. To determine whether sodium was inhibitory, observed

389

sodium concentrations were compared (Table 2).

390

Sodium had likely no substantial inhibitory effect on VFA upgrading in all assays. The used

391

inocula were derived from environments with a similar, but often higher, sodium

392

concentration than that was present in the assays. For example, adapted sludge was active in

393

VFA upgrading in the reactor (386 mmolC/gVSS/d) at a high sodium concentration (9.1 g/L)

394

but was completely inhibited in the 20 g/L n-caproate batch assay that contained a lower

395

sodium concentration (6.4 g/L). Some strains of the chain elongating bacterium C. kluyveri

396

were derived from brackish sediments 28, underlining that the active chain elongating

397

microorganisms in our batch inhibition assays were likely not rigorously inhibited by sodium

398

but by n-caproate. Also, the synthetic medium sludge was active in hydrogenotrophic 21 ACS Paragon Plus Environment


399

methanogenesis in the reactor (59 mmolC/gVSS/d) at a sodium concentration of 6.1 g/L

400

whereas sodium concentrations in the batch inhibition assays were lower or similar (2.3 – 7.1

401

g/L). Likewise, earlier work showed that hydrogenotrophic methanogens can remain active

402

even up to high sodium chloride concentrations of 1.25M (29 g/L sodium) 29. This shows that

403

hydrogenotrophic methanogenesis was likely also not rigorously inhibited by sodium but by

404

n-caproate in our batch inhibition assays.

405 406

n-Caproate formation may adapt the microbial sludge to perform chain elongation at

407

higher concentrations

408

In an effective chain elongation process, chain elongation is stimulated while competing

409

processes are suppressed. Whereas chain elongation can be stimulated by adding C. kluyveri

410

30

411

hydraulic shear force in combination with low hydraulic retention time 7 and CO2 loading rate

412

5

413

itself may have an effect since earlier work stated that undissociated n-caproic acid does have

414

a microbial toxicity (at pH 5.5) which warranted the need of an continuous extraction unit 12.

415

In our systematic study, it was shown that exogenous n-caproate does affect the different

416

functional groups of microorganisms which may also be the case with endogenous n-caproate

417

within bioreactors. With the activity tests, we show that adapted sludge is less inhibited than

418

non-adapted sludge at higher n-caproate concentrations. This difference in n-caproate

419

tolerance between adapted sludge and non-adapted sludge can be explained by a change in

420

physiology of chain elongating microorganisms (e.g. membrane composition, fluidity,

421

integrity, and hydrophobicity) 24. Another explanation is that the higher caproate

422

concentration in the reactors selected for more n-caproate-tolerant chain elongating

, competing processes can be suppressed in various ways. Past research explained that pH 9,

are control parameters to suppress competing processes. Also, the n-caproate concentration


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423

microorganisms. Our explanation (i.e. hypothesis) is that the formation of n-caproate in

424

reactor microbiomes does acts as an in situ stimulatory agent to form higher n-caproate

425

concentrations which leads towards a further accumulation and possible more selective

426

production of n-caproate.

427 428

CONCLUSIONS

429

In this study, we showed that n-caproate inhibits chain elongation and competing processes in

430

chain elongation microbiomes. Hydrogenotrophic methanogenesis was severely inhibited at

431

20 g/L n-caproate in batch. This inhibition probably rate-limits syntrophic ethanol oxidation at

432

25 g/L n-caproate. VFA upgrading was the most sensitive process to n-caproate inhibition but

433

we demonstrated that the n-caproate tolerance of this process is higher with adapted sludge

434

than with non-adapted sludge. Thus, microbiomes can be adapted to perform VFA upgrading

435

at high n-caproate concentrations which likely limits syntrophic ethanol oxidation through

436

hydrogenotrophic methanogenesis. n-Caproate formation is consequently another control

437

parameter to inhibit competing processes to steer to higher n-caproate concentrations.

438



439

ASSOCIATED CONTENT

440

Profiles of product production and specifications on used inocula (pdf)

441 442

AUTHOR INFORMATION

443

* Corresponding author: E-mail: [email protected]; tel +31 317 483 447

444 445

ACKNOWLEDGEMENTS

446

This work has been carried out with a grant from the BE-BASIC program FS 01.006

447

(www.be-basic.org)

448 449

REFERENCES

450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466

1. Weiland, P., Biogas production: current state and perspectives. Appl. Microbiol. Biotechnol. 2010, 85 (4), 849-860. 2. Seedorf, H.; Fricke, W. F.; Veith, B.; Bruggemann, H.; Liesegang, H.; Strittmatter, A.; Miethke, M.; Buckel, W.; Hinderberger, J.; Li, F.; Hagemeier, C.; Thauer, R. K.; Gottschalk, G., The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (6), 2128-2133. 3. Angenent, L. T.; Richter, H.; Buckel, W.; Spirito, C. M.; Steinbusch, K. J. J.; Plugge, C. M.; Strik, D. P. B. T. B.; Grootscholten, T. I. M.; Buisman, C. J. N.; Hamelers, H. V. M., Chain Elongation with Reactor Microbiomes: Open-Culture Biotechnology To Produce Biochemicals. Environ. Sci. Technol. 2016, 50 (6), 2796-2810. 4. Grootscholten, T. I. M.; Strik, D. P. B. T. B.; Steinbusch, K. J. J.; Buisman, C. J. N.; Hamelers, H. V. M., Two-stage medium chain fatty acid (MCFA) production from municipal solid waste and ethanol. Appl. Energy 2014, 116, 223-229. 5. Roghair, M.; Hoogstad, T.; Strik, D. P. B. T. B.; Plugge, C. M.; Timmers, P. H. A.; Weusthuis, R. A.; Bruins, M. E.; Buisman, C. J. N., Controlling ethanol use in chain elongation by CO2 loading rate. Environ. Sci. Technol. 2018, 52 (3), 1496-1505.


Page 24 of 29

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516


6. Agler, M. T.; Spirito, C. M.; Usack, J. G.; Werner, J. J.; Angenent, L. T., Chain elongation with reactor microbiomes: Upgrading dilute ethanol to medium-chain carboxylates. Energ Environ Sci 2012, 5 (8), 8189-8192. 7. Grootscholten, T. I. M.; Steinbusch, K. J. J.; Hamelers, H. V. M.; Buisman, C. J. N., Chain elongation of acetate and ethanol in an upflow anaerobic filter for high rate MCFA production. Bioresour. Technol. 2012, 135, 440-445. 8. Roghair, M.; Liu, Y.; Strik, D. P. B. T. B.; Weusthuis, R. A.; Bruins, M. E.; Buisman, C. J. N., Development of an effective chain elongation process from acidified food waste and ethanol into n-caproate. Front. Bioeng. Biotechnol. 2018. 9. Agler, M. T.; Spirito, C. M.; Usack, J. G.; Werner, J. J.; Angenent, L. T., Development of a highly specific and productive process for n-caproic acid production: applying lessons from methanogenic microbiomes. Water Sci. Technol. 2014, 69 (1), 62-68. 10. Royce, L. A.; Liu, P.; Stebbins, M. J.; Hanson, B. C.; Jarboe, L. R., The damaging effects of short chain fatty acids on Escherichia coli membranes. Appl. Microbiol. Biotechnol. 2013, 97 (18), 8317-8327. 11. Roy, F.; Albagnac, G.; Samain, E., Influence of Calcium Addition on Growth of Highly Purified Syntrophic Cultures Degrading Long-Chain Fatty Acids. Appl. Environ. Microbiol. 1985, 49 (3), 702-705. 12. Ge, S.; Usack, J. G.; Spirito, C. M.; Angenent, L. T., Long-term n-caproic acid production from yeast-fermentation beer in an anaerobic bioreactor with continuous product extraction. Environ. Sci. Technol. 2015, 49 (13), 8012-8021. 13. Roghair, M.; Strik, D. P. B. T. B.; Steinbusch, K. J. J.; Weusthuis, R. A.; Bruins, M. E.; Buisman, C. J. N., Granular sludge formation and characterization in a chain elongation process. Process Biochem. 2016, 51 (10), 1594-1598. 14. Lonkar, S.; Fu, Z.; Holtzapple, M., Optimum alcohol concentration for chain elongation in mixed-culture fermentation of cellulosic substrate. Biotechnol. Bioeng. 2016, 113 (12), 2597-2604. 15. Sudmalis, D.; Gagliano, M. C.; Pei, R.; Grolle, K.; Plugge, C. M.; Rijnaarts, H. H. M.; Zeeman, G.; Temmink, H., Fast anaerobic sludge granulation at elevated salinity. Water Res. 2017, 128, 293-303. 16. Roman, P.; Bijmans, M. F. M.; Janssen, A. J. H., Influence of methanethiol on biological sulphide oxidation in gas treatment system. Environ. Technol. 2016, 37 (13), 16931703. 17. Rodríguez Arredondo, M.; Kuntke, P.; ter Heijne, A.; Hamelers, H. V. M.; Buisman, C. J. N., Load ratio determines the ammonia recovery and energy input of an electrochemical system. Water Res. 2017, 111, 330-337. 18. Hajarnis, S. R.; Ranade, D. R., Inhibition of methanogens by n- and iso-volatile fatty acids. World J. Microbiol. Biotechnol. 1994, 10 (3), 350-351. 19. Liu, Y.; He, P.; Shao, L.; Zhang, H.; Lü, F., Significant enhancement by biochar of caproate production via chain elongation. Water Res. 2017, 119, 150-159. 20. Zhu, X.; Tao, Y.; Liang, C.; Li, X.; Wei, N.; Zhang, W.; Zhou, Y.; Yang, Y.; Bo, T., The synthesis of n-caproate from lactate: A new efficient process for medium-chain carboxylates production. Sci. Rep. 2015, 5, 14360. 21. Liu, P.; Chernyshov, A.; Najdi, T.; Fu, Y.; Dickerson, J.; Sandmeyer, S.; Jarboe, L., Membrane stress caused by octanoic acid in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 2013, 97 (7), 3239-3251. 22. Grootscholten, T. I. M.; Steinbusch, K. J. J.; Hamelers, H. V. M.; Buisman, C. J. N., Improving medium chain fatty acid productivity using chain elongation by reducing the hydraulic retention time in an upflow anaerobic filter. Bioresour. Technol. 2013, 136, 735738. 25 ACS Paragon Plus Environment


517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536

23. Grootscholten, T. I. M.; Steinbusch, K. J. J.; Hamelers, H. V. M.; Buisman, C. J. N., High rate heptanoate production from propionate and ethanol using chain elongation. Bioresour. Technol. 2013, 136 (0), 715-718. 24. Jarboe, L. R.; Royce, L. A.; Liu, P., Understanding biocatalyst inhibition by carboxylic acids. Front. Microbiol. 2013, 4, 272. 25. Zhang, Q.; Wu, D.; Lin, Y.; Wang, X.; Kong, H.; Tanaka, S., Substrate and Product Inhibition on Yeast Performance in Ethanol Fermentation. Energy Fuels 2015, 29 (2), 10191027. 26. Lian, J.; McKenna, R.; Rover, M. R.; Nielsen, D. R.; Wen, Z.; Jarboe, L. R., Production of biorenewable styrene: utilization of biomass-derived sugars and insights into toxicity. J. Ind. Microbiol. Biotechnol. 2016, 43 (5), 595-604. 27. Hierholtzer, A.; Akunna, J. C., Modelling sodium inhibition on the anaerobic digestion process. Water Sci. Technol. 2012, 66 (7), 1565-1573. 28. Kenealy, W. R.; Waselefsky, D. M., Studies on the substrate range of Clostridium kluyveri; the use of propanol and succinate. Arch. Microbiol. 1985, 141 (3), 187-194. 29. Liu, Y.; Boone, D. R., Effects of salinity on methanogenic decomposition. Bioresour. Technol. 1991, 35 (3), 271-273. 30. Reddy, M. V.; Hayashi, S.; Choi, D.; Cho, H.; Chang, Y.-C., Short chain and medium chain fatty acids production using food waste under non-augmented and bio-augmented conditions. Journal of Cleaner Production 2018, 176, 645-653.

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539 540

TOC / ABSTRACT GRAPHICS

541 542 543

SYNOPSIS

544

Chain elongation with open-cultures is more effective at high n-caproate concentrations

545

because this inhibits competitive processes.


a)

b)

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VFA upgrading Hydrogenotrophic methanogenesis Syntrophic ethanol oxidation

Relative activity [%]

Activity [mmol C/gVSS/d]

1 2 3 750 4 5 6 7 8 500 9 10 11 12 250 13 14 15 0 16 0 17 18

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5 10 15 20 25 Initial n-caproate concentration [g/L]

b)

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1 2 3 4 1500 5 6 7 8 1000 9 10 11 12 500 13 14 15 16 0 17 0 18

Non-adapted sludge Adapted sludge

Relative VFA upgrading activity [%]

VFA upgrading activity [mmol C/gVSS/d]

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Effect of n-Caproate Concentration on Chain Elongation and

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