Production of isobutyric acid from methanol by: Clostridium luticellarii

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DOI: 10.1039/D0GC02700F

ARTICLE

Production of isobutyric acid from methanol by Clostridium

luticellarii

Camille Petrognani^a^,^b^*, Nico Boon , Ramon Ganigué *

a,b

Received 00th January 20xx,

Accepted 00th January 20xx

Renewable methanol can be used as a feedstock to generate value-added multicarbon components through fermentation

technologies. Recently, researchers reported the production of isobutyric acid using methanol as an electron donor with

open culture systems dominated by Eubacterium sp. and Clostridium sp. Here we report the ability of Clostridium luticellarii

DOI: 10.1039/x0xx00000x

2 2

wild-type strain to produce isobutyric acid from methanol and CO & H . When growing on methanol, the supplementation

of acetic and butyric acid enhanced isobutyric acid final concentration, selectivity and production rate. A maximum of 5.04

0.08 g.L⁻¹isobutyric acid was produced at a rate of 0.420 ± 0.012 g.L .d and selectivity of 0.70 electron per electron of

−1

±

total products in batch with acetic and butyric acid as electron acceptors. The pH was also found to be a major factor

influencing isobutyric acid formation with maximal production at pH 6.50. Finally, in addition to its ability to produce isomers,

C. luticellarii was able to perform C2-unit elongation from methanol. Overall, this study positions C. luticellarii as a promising

platform for the production of isocarboxylic acids.

1

. Introduction

catalytic hydrogenation of captured waste-CO

2

and hydrogen

generated from renewable electricity, or of syngas obtained by

Transitioning from a petrochemical manufacturing industry to a

bio-based economy is cornerstone to a more sustainable and

circular society. The replacement of chemicals and materials

derived from fossil fuels by alternative counterparts with lower

greenhouse gas emissions is supposed to help mitigate climate

change and stimulate the bio-based and industrial

2

,4,6

waste biomass gasification.

It is forecasted that renewable

methanol will play a critical role in the transition towards a

2

,12

sustainable economy.

Large-scale renewable methanol

synthesis plants are already in operation in Iceland (Carbon

Recycling International, 4 kilotons per year), the Netherlands

(BioMCN, 20 kilotons per year), Canada (Enerkem, 38 million

litres per year) and Japan (Mitsui Chemical Inc., 100 tons per

1

biotechnology sectors. To date, the production of bio-based

chemicals, fuels and materials relies on the use of biomass-

derived feedstocks (in most cases sugars from sugar-crops such

as corn, sugarcane or sugar beet) which require the use of

arable land and raise ethical dilemmas. In the quest for

alternative feedstocks to avoid the competition between the

use of sugar-crops as food or fuel, methanol, a cheap and

6

,13

year).

currently aiming to build a renewable methanol pilot-plant with

a capacity of 8 kilotons per year from captured CO and

in the port of Antwerp. For all these reasons,

In Belgium a pilot-project, “Power-to-methanol”, is

2

1

4

renewable H

2

methanol has emerged as a promising alternative feedstock for

the production of bio-based chemicals and materials via

industrial fermentations.

2

–4

abundant chemical, has been brought into focus.

Methanol is an already widely used building block in the

chemical industry with a world production capacity of about

10 million metric tons per years. It has a lower price than

sugars (200-400 EUR per metric ton of methanol vs 300-600 EUR

per metric ton of sugar in Europe over the last 10 years) , it is

a more reduced carbon source (degree of reduction of 6 vs 4)

and has a higher energy density (22.7 vs 15.5 MJ kg )

Although today methanol is primarily produced on an industrial

scale from fossil fuel sources, i.e. natural gas and coal, it can also

be generated from renewable resources. Renewable methanol

is an ultra-low carbon chemical that can be obtained via

The use of methanol as a feedstock for bioproduction processes

has already been established for over 50 years, but it has for

now been limited at industrial scale to the production of single-

cell protein for feed and food. Methylotrophs are a group of

diverse organisms which includes yeasts and (an)aerobic

bacteria with various metabolisms, which opens up the

5

,6

1

7

,8

9

-

1 10,11

.

2

,4

potential for the production of various value-added products.

Acetogens are strictly anaerobic bacteria that employ the

Wood-Ljungdahl pathway as their main mechanism for energy

conservation and synthesis of acetyl-CoA and cell carbon from

1

5

2 2 2 2

CO and H . Syngas ( CO/CO /H -containing gas) fermentation

harnessing the metabolic capabilities of the acetogen

Clostridium autoethanogenum is currently carried out to

produce ethanol at commercial-scale with high productivities

^a^.Center for Microbiology Ecology and Technology (CMET), Ghent University,

Coupure Links 653, 9000 Ghent, Belgium

b.

-1

-

CAPTURE, www.capture-resources.be

(

volumetric productivity of Lanzatech syngas process, 10 g.L .h

c.

*Corresponding author

1

16,17

).

Some

acetogens

such

as

Butyribacterium

Electronic Supplementary Information (ESI) available: [details of any supplementary

information available should be included here]. See DOI: 10.1039/x0xx00000x

1

8

19

methylotrophicum , Sporomusa ovata , Eubacterium

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limosum²⁰and Acetobacterium woodii²¹can also use methanol isolation work was carried out under strictly anaerobic

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DOI: 10.1039/D0GC02700F

as an alternative electron donor and carbon source, with the conditions in an anaerobic glove box with a gas phase of N /CO

2 2

additional advantage that these are CO

2

-negative (80: 20, v/v). Two different media were used for isolation by

sinks . When grown on streak plating. The first medium was a defined synthetic

methanol, most methylotrophic acetogens present a product methanol medium. This medium consisted of a basal medium

2

fermentations, acting as extra CO

2

1

5,16

spectrum dominated by acetic and butyric acid.

2

However, prepared prior to autoclaving, followed by addition of N -

Eubacterium limosum was proven to be able to produce longer- gassed, sterile stock solutions of heat-labile components and

chain carboxylic acids, namely caproic and valeric acid when substrates. The basal medium contained the following (per

2

3

supplemented with butyric and propionic acid, respectively.

Recently, the product portfolio of anaerobic methanol 0.2 g CaCl

fermentation has been expanded by the observation that ethanesulfonic acid (MES) and 0.001 g resazurin. The other

liter): 0.36 g NH

4

H

2

PO

4 2 2 4 2

, 0.33 g MgCl .6H O, 0.2 g MgSO .7H O,

2

.2H

2

O, 0.15 g KCl, 4.88 g 2-(N-morpholino)

-1

isobutyric acid was produced (2.0 g.L .d ) by a methanol- medium was an enriched Reinforced Clostridial Medium (RCM)

2

4

fermenting mixed culture. This is a rather unique observation containing the following (per litre): 5 g glucose, 10 g tryptone, 3

since isobutyric acid is a rarely detected fermentation g yeast extract, 5 g beef extract, 1 g soluble starch, 5 g NaCl, 3 g

metabolite, mostly derived from the fermentation of amino sodium acetic acid, 5 g L-cysteine, 1 mg resazurin. Gellan gum

acids at concentrations below 0.1 g.L . Chen and co-workers 1% (w/v) was added to both media as a solidifying agent. The

hypothesised that isobutyric acid is produced through bio- pH was adjusted to 6.30 before autoclaving. After autoclaving

isomerisation as a detoxifying mechanism, by converting butyric and before pouring the plates, the following stock solutions

-1 25

3

acid to its less toxic branched-chain equivalent. However, there were added to the basal medium: 0.3 mL of a NaHCO (1.0 M),

2

4

was no evidence provided to support this hypothesis. A recent trace elements and vitamins solution, 0.3 mL of an

study of the ecology of a similar system to that of Chen and co- iron(II)chloride solution, 0.45 mL of a reducing agents solution

-1

2 2

workers revealed that the microbiome was dominated by (5.5 g·L NaS.9H O and 5.5 g·L L-cysteine-HCl.H O), 0.2 mL of

Eubacterium and Clostridium spp., and that isobutyric acid a 10.0 M methanol solution, 0.2 mL of a 5.0 M potassium

production was closely linked to the abundance of the acetate solution and 0.2 mL of a 1.14 M sodium butyrate

Clostridium sp.²

6

solution. Full details on trace elements and vitamins solutions

composition can be found in Supporting Information (Table S1).

After pouring, the plates were allowed to solidify and then were

stored at 4°C until use. In the anaerobic glove box, the mixed

culture was streaked onto solid medium plates. Plates were

then left to incubate in the anaerobic glove box at 37 °C. To

check for contamination, two plates without inoculum addition

were also incubated. Growth was checked daily, and when

visible growth appeared, single colonies were picked with a

disposable sterile inoculating loop and streaked on fresh plates

of the same medium for purification. Growth on subculture

plates was monitored, and if uniform colonies were observed, a

single colony was picked and transferred to a sterile Hungate

tube containing 10 mL of liquid isolation medium. Hungate

tubes were then incubated at 37 °C in a static incubator outside

of the anaerobic glove box. The purity of the cultures was

checked by microscopic examination and later confirmed by

Sanger sequencing of the 16S rRNA gene.

Isobutyric acid is a versatile platform chemical that can be used

for a broad range of applications, e.g. as a precursor for methyl

methacrylic acid (MMA) for the production of transparent

thermoplastics (PMMA) with an annual market size of up to 2.7

_m_i_l_l_i_o_n_t_o_n_n_e_s₂7,28, the synthesis of sucrose acetic acid, a

broadly used emulsifier in many commodities with an annual

market volume of 100,000 tonnes

acid is produced via the acid-catalysed Koch carbonylation

reaction of propene, which presents three major concerns: it is

derived from fossil resources, it is energy intensive and it

requires the use of environmentally harmful compounds such

2

8,29

, etc. Currently, isobutyric

2

8,30

as sulfuric acid, hydrogen fluoride and boron fluoride.

Thus

the development of a biological isobutyric acid production

route from renewable methanol appears as a promising

alternative in a sustainable economy context. In this study, we

identified C. luticellarii as the organism responsible for

isobutyric acid production from an in-house methylotrophic

open culture, explored its capacity to produce isomers from a

broad range of substrates and investigated the metabolic- 2.1.2.

Characterisation of the isolates production profile

triggers to isomerisation, such as pH and electron acceptor

After isolation, all isolates were grown in the same synthetic

methanol medium as the one used for streak plating isolation

without the addition of gellan gum. Basal medium was first

availability. The findings of this work position C. luticellarii as a

promising platform for the production of isocarboxylic acids

from renewable methanol.

2

boiled and cooled to room temperature under N atmosphere.

After cooling, 7.4 mL of basal medium was dispensed under N

2

gassing into Hungate tubes. All tubes were sealed with butyl

rubber stoppers and aluminium caps. The headspace of these

tubes was flushed with N :CO (80:20, v/v) with a gas exchange

apparatus and subsequently, the tubes were autoclaved for 20

minutes at 121 C. After autoclaving, the heat-labile

components and substrates stock solutions were added as

described in section 2.3. The same procedure for oxygen

2

. Materials and Methods

2

.1. Isolation of organisms from a methylotrophic open culture

producing isobutyric acid

2

.1.1.

Streak plating isolation

Bacterial strains were isolated from the isobutyric acid-

producing mixed-culture of the CSTR by streak plating. All

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removal and sterilisation was employed for all following batch package to generate consensus sequences which _V w _i _e e _w r _A e _r _t _i b _c _l l _e a _O s _n t _l e _i _n d _e

experiments. After addition of all stock solutions, 1 mL of each in NCBI against the 16S rRNA gene sequen^Dc^Oe^I^:d¹⁰a^.t¹a⁰b³⁹a^/s^De⁰(^GM^C⁰a²d⁷d⁰e⁰n^F

isolate culture was inoculated in duplicate, and all tubes were 2013; Zhang et al. 1997).

incubated for 10 days at 37 °C in a static incubator. Liquid

samples (1.0 mL) were taken after inoculation and at the end of 2.2. Characterisation of isobutyric acid production by Clostridium

incubation for measurement of pH, OD600, and alcohol and

carboxylic acid analysis.

luticellarii

2

.2.1. Inoculum

To ensure the characterisation could be linked to a culture

collection type strain, C. luticellarii DSM 29923 was purchased

from the German Collection of Microorganisms and Cell

Cultures (DSMZ, Braunschweig, Germany) and was routinely

cultivated on the same synthetic methanol medium

supplemented with methanol as electron donor (200 mM) and

potassium acetate (100 mM) and sodium butyrate (23 mM) as

electron acceptors. Cultures were always transferred at their

mid-exponential phase.

2

.1.3.

Sanger sequencing

DNA extraction was performed by means of bead beating with

a PowerLyzer instrument (Qiagen, Venlo, Netherlands) and

phenol/chloroform extraction. The 16S rRNA gene of isolate

cultures was amplified by PCR using 27F_LGC (5’-

GAGTTTGATCMTGGCTCAG341F5’-) and 1492R_LGC (5’-

GGYTACCTTGTTACGACTT-3’) primer pair. Each PCR

amplification mixture was prepared using a Fermentas PCR Kit,

according to the manufacturers’ specifications (Thermo Fisher

Scientific, Waltham, MA, USA), and PCR was carried out in a

BioRad T100TM Thermal Cycler (Applied Biosystems, Foster

City, CA, USA). PCR-products were purified with the QIAquick

PCR Purification Kit (Qiagen, Venlo, Netherlands). The quality of

the obtained purified PCR-products was checked by agarose gel

electrophoresis (2% agarose gel for 30 minutes at 100 V).

Subsequently, purified PCR-products were sent for molecular

identification by bi-directional Sanger sequencing to LGC

Genomics GmbH (Berlin, Germany). Forward and reverse 16S

rRNA gene Sanger reads were aligned using the Bioedit v7.0

2

.2.2.

Production of isobutyric acid from various electron

donors & acceptors

Batch experiments were conducted in 120 mL penicillin bottles

with 40 mL working volume to assess the substrate range of C.

luticellarii. C. luticellarii was tested on common fermentation

carbon & electron donors (i.e. glucose, glycerol and carbon

dioxide & hydrogen), common chain elongation carbon &

electron donors (methanol, ethanol, lactic acid), with acetic acid

(100 mM) as electron acceptor.

Table 1 Carbon source concentrations used in different batch experiments studying the substrate range of C. luticellarii

−1

Carbon source (mmol.L )

Experiment

Lactic

acid

Acetic

acid

Propionic

acid

Butyric

acid

Valeric

acid

Caproic

acid

Methanol

Ethanol

Glucose

Glycerol

Batch 1

MA

200

‒

100

‒

100

‒

MP

MB

‒

100

‒

MV

‒

50

‒

MC

‒

50

‒

MAB

MPV

100

‒

25

‒

100

25

‒

Batch 2

MA

200

‒

200

‒

100

‒

LA

EA

‒

200

Batch 3

Glu

‒

25

‒

Gly

‒

25

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Table 2 Composition of carbon source in different batch experiments studying the effect of substrate concentration on isobutyric acid production by C.

luticellarii

−1

Substrate concentration (mmol.L )

Experiment

Methanol

Acetic acid

Butyric acid

Fixed acetic acid

M1A

100

200

400

50

‒

25

‒

M2A

M4A

Fixed methanol

MA0

200

‒

MA25

25

MA50

MA100

50

‒

100

Fixed methanol+butyric acid

MA0B

200

‒

23

MA25B

25

MA50B

50

MA100B

100

A second batch experiment explored the growth of C. luticellarii Liquid samples (2.0 mL) were taken at the start and at the end

on methanol (200 mM) as electron donor in combination with of incubation for measurement of pH, OD600, and alcohol and

different carboxylic acids as electron acceptors. The exact carboxylic acid analysis. At the end of incubation, the pressure

composition of carbon source in the different batch was measured again to quantify gas consumption, after which

experiments is shown in Table 1. All bottles except the set gas and liquid samples were taken for analyses.

testing growth on CO

CO . For the test with carbon dioxide and hydrogen, the bottles

were flushed with 80% H and 20% CO

After autoclaving, carbon sources were added from anaerobic

2 2 2

& H were flushed with 80% N and 20%

2

.2.3.

Effect of fermentation pH on isobutyric acid production

2

.

A batch experiment was performed to study the impact of pH on

isobutyric acid selectivity when C. luticellarii is grown on methanol

filter-sterilised stock solutions to the penicillin bottles and CO with the additional supplementation of acetic and butyric

2

containing basal medium identical to that described in section acid as electron acceptors. C. luticellarii was inoculated in the

2

.2.1. Additionally, NaHCO

3

, vitamins, trace elements, and synthetic MES-buffered medium containing 200 mM methanol,

1

00 mM potassium acetate, 23 mM sodium butyrate and 30

reducing agents were also amended to the bottles to reach the

concentrations abovementioned in section 2.1.1. Initial pH of

the fermentation medium was set to 6.3 before inoculation.

Experiments were performed in triplicate and inoculated with

mM sodium bicarbonate at varying pH values. The pH was

adjusted before inoculation to 5.0, 5.25, 5.50, 6.0, 6.25, 6.50

and 7.0, respectively, by adding 2.0 M sterile NaOH. After

inoculation, all bottles were statically incubated at 37 °C. All pH

conditions were tested in triplicate. Liquid samples (2.0 mL)

were taken after inoculation and at the end of incubation for

measurement of pH, OD600, and alcohol and carboxylic acid

analysis.

4

.0 mL of an actively growing C. luticellarii culture. The initial

pressure of all tests was adjusted to ca. 110-120 hPa, except for

the test with carbon dioxide and hydrogen, in which the

headspace pressure was corrected to 250 hPa after initial liquid

and gas sampling. Afterwards, all bottles were statically

incubated at 37 °C.

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2

.2.4.

Effect of substrate concentration on isobutyric acid

production

In the time-course experiment, methanol consumption and

product formation rates during batch incubation were

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calculated as the slope of a linear regression over the linear

phase of batch incubations.

To investigate whether isobutyric acid production was affected

by the availability of methanol, acetic acid, and butyric acid C.

luticellarii was grown at fixed acetic acid concentration (50 mM)

with varying methanol concentrations of 100 mM, 200 mM

Two stoichiometric reactions representing the conditions with

net consumption of acetic acid (M1A, M2A, M4A, MA25,MA50,

MA100, MA25B,MA50B and MA100B) and the conditions with

no net consumption or production of acetic acid (MA0 and

MA0B) can be written:

and400 mM (M1A, M2A and M4A), and at fixed methanol

concentration (200 mM) with varying concentrations of acetic

acid (0-100 mM), with (MA0B, MA25B, MA50B and MA100B)

and without (MA0, MA25, MA50 and MA100) supplementation

of butyric acid (23 mM). This approach allowed investigation of

the effect of methanol to acetic acid ratio as well as the effect

of initial substrate concentrations on isobutyric acid production.

(1) a

1

CH

3

OH + b

(CH

1

CO

2

+ c

1

C

H + f

2

H

4

O

2

→ d

1

CH

3

(CH

H 0

1 2

2

)

2

CO

2

H + e

1

3 2

) CHCO

2

1

C

6 12

H O

2

+ g

The exact substrate concentrations in the different conditions (2) a

tested are reported in Table 2. All experiments were run in

duplicate. Liquid samples (2.0 mL) were taken after inoculation

2

CH

3

OH + b

2

CO

2

→ c

+ e

2

CH (CH

3

2

)

+ f

2

CO

2 2 3 2 2

H + d (CH ) CHCO H

2

C H O

6 12 2

2

H

2

0

and at the end of incubation at 37 °C for measurement of pH, For both stoichiometric reactions, the coefficient of methanol

OD600, and alcohol and carboxylic acid analysis. (a and a ) is fixed to 10. Six and five additional linear equations

1

2

Additionally, to gain deeper insight into how C. luticellarii are necessary to calculate the other stoichiometric coefficients

consumes methanol to produce isobutyric acid and the effects for the conditions represented by reaction (1) and (2),

of supplementing acetic acid and butyric acid as electron respectively. The elemental balances (C, H and O) make up

acceptors, substrate consumption and product formation was three of those equations, and the remaining equations are

tracked throughout growth in batch mode.

obtained from the experimental product molar yields (YC4/MeOH

,

Y

iC4/MeOH and YC6/MeOH). The following systems of linear equations

C. luticellarii was grown under three different conditions (M,

MA and MAB), in medium containing 200 mM methanol and 30

mM sodium bicarbonate, with potassium acetate (100 mM)

supplementation for the MA experiment and a combination of

potassium acetate (100 mM) and sodium butyrate (23 mM) for

MAB. Cultures were incubated in 250 mL penicillin bottles with

a working volume of 85 mL for 15 days at 37 °C, during which

liquid samples (0.7 mL per sampling point) were taken once a

day until day 8, and subsequently on days 10, 12 and 15. Initial

pH of the fermentation medium was adjusted to 6.5 before

inoculation, and all conditions were tested in triplicate.

are obtained and used to calculate the experimental

stoichiometric coefficients reported in Table 3:

(

1) C Balance: -a

H Balance: -4a

O Balance: -a

Butyric acid Yield: d

Isobutyric acid Yield: e

Caproic acid Yield: f

Fixed Coefficient: a

1

-b

-4c

-2b

1

-2c

+8d

-2c +2d

1

+4d

+8e

+2e

1

+4e

+12f

+ 2f

= 0

1

+6f

1

= 0

+2g

+g

1

= 0

1

-YC4/MeOH

-YiC4/MeOH

–YC6/MeOH

a

1

a = 0

= 0

1

a

1

–10 = 0

(

2) C Balance: -a

H Balance: -4a

O Balance: -a

Isobutyric acid Yield: d

Caproic acid Yield: E

Fixed Coefficient: a

2

-b

+8c

-2b +2c

2

+4c

2

+4d

2

+6e

2

= 0

2

.3. Analytical methods

2

+8d

2

+12e

2

+2f

+ 2e +f

= 0

2

= 0

The pH and optical density (OD600) of liquid samples was

measured after collection. The remainder of the samples was

then filtered over a 0.20 µm filter and stored at -20 °C for later

analysis of carboxylic acids and alcohols.

2

+2d

2

= 0

2

-YiC4/MeOH

-YC6/MeOH

a

2

a

2

–10 = 0

Analysis of C3 to C6 carboxylic acids (including isoforms of C4 to

C6) was done by gas chromatography with a flame ionisation

3

. Results

3

1

detector (FID) after extraction of acids into diethylether.

Concentrations of acetic acid and lactic acid were analysed by

3

1

ion chromatography with a conductivity detector. Alcohols 3.1. Isolation of microorganisms from an isobutyric acid producing

(

methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 1-

reactor: morphology, phylogeny and product spectrum

3

2

pentanol and 1-hexanol) were measured by GC-FID. Due to

technical issues methanol had to be analysed using an HPLC

method for a batch of samples. A full description of the method

is presented in Supplementary Information.

Isolation of the organism(s) able to produce isomers was

conducted from a mixed-culture CSTR (Figure S1) fermenting

methanol and acetic acid to a mixture of butyric and isobutyric

acids (Figure S2). The average isobutyric acid and butyric acid

-1

production rates in the reactor were 221.3 ± 29.8 mMe.d and

160.2 ± 22.2 mMe.d-1, respectively. The highest isobutyric acid

2

.4. Calculations

-1

concentration was 3.1 g.L .

All errors reported in this study are standard deviations over all

biological replicates (n=2 or 3) for all batch experiment.

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Following isolation by streak plating, twenty-eight isolates were rRNA gene sequences, all isobutyric acid-produ _V c _i _e in _w g _A _r _t i _i _c s _l o _e l _O a _n t _l _i e _n _e s

obtained from the isobutyric acid-producing microbiome of the shared over 99.7% similarity with C. l^Du^Oti^Ic^:e¹⁰ll^.a¹⁰r³ii⁹^/D^DS⁰M^G^C⁰2²9⁷9⁰2⁰3^F

continuous reactor. After seven days of incubation at 37ꢀ°C (NCBI Reference: NR_145907.1), an organism recently isolated

isolates formed circular, convex, ivory colonies with entire from a mud cellar used for the production of strong and

3

5

boundaries, and had a diameter ranging from 0.5 to 2.0ꢁmm. In aromatic Chinese liquors (Figure S5). The isolated strains

liquid culture, microscopic observation of isolates (R4, R8, M10 exhibiting isobutyric acid production ability were deposited in

and M16) revealed straight or slightly curved rods 4-8 μm long the Belgian Coordinated Collections of Microorganisms (BCCM)

and 1-2 μm wide (Figure S3). The twenty-eight isolates were with accession numbers pending. To confirm that C. luticellarii

tested for isobutyric acid production in batch culture using is the organism responsible for isobutyric acid production,

synthetic methanol fermentation medium supplemented with further experiments in this article were conducted with the

acetic and butyric acid. All isolates except isolates R2, R5, R11, wild-type strain C. luticellarii DSM 29923 obtained from a

M2, and M1 were able to grow on methanol (Figure S4). The culture collection (DSMZ, Braunschweig, Germany).

isolates could be clustered into two groups based on their

product profiles, those that could produce isobutyric acid (R1,

3

.2. Assessment of electron donors for the production of

R3, R10, M3, M4, M6, and M16), with net production ranging

from 2.22 g.L⁻¹(25.2 mM) to 3.90 g.L (44.3 mM) and others

that could not. Other products formed by the isobutyric acid-

producing isolates included butyric and caproic acid with

isobutyric acid by Clostridium luticellarii

−1

To gain insight into C. luticellarii substrate range and their

potential to produce isobutyric acid, the wild-type strain C.

luticellarii DSM 29923 was grown with various electron donors

in a series of batch tests. First, the growth of C. luticellarii was

tested on common carbon sources, namely glucose and glycerol

−1

respective net production ranging from 0.12 g.L (1.4 mM) to

−1

1

.29 g.L⁻¹(9.7 mM) and from 0.18 g.L (1.6 mM) to 0.55 g.L

(4.7 mM). The product spectrum of the non-producing isolates

was dominated by butyric acid, and trace amounts of caproic

acid were also detected.

and CO

2

& H . Growth was supported on all carbon sources

(Figure 1).

The 16S rRNA gene sequence analysis of the non-isobutyric

acid-producing isolates (R4, R6, R9, M1, M8 and M10) revealed

a 99.5% similarity with E. callanderi DSM 3662 (NCBI Reference:

NR_026330.1) and E. limosum ATCC 8486 (NCBI Reference:

NR_044719.2). Though poorly studied, E. callanderi had been

previously reported to be unable to utilise one-carbon

−1

For glucose and glycerol, propionic (1.06 ± 0.10 g.L and 1.28 ±

−1

0

.07 g.L , respectively) and acetic acid (0.71 ± 0.07 g.L and

.19 ± 0.02 g.L , respectively) were the most abundant

fermentation products. Valeric acid was also formed on glucose

with an average net production of 0.17 ± 0.02 g.L . When

, C. luticellarii produced mainly acetic (1.43 ±

.03 g.L ) and butyric acid (0.47 ± 0.03 g.L ). Isobutyric acid

−1

grown on CO

0

2

& H

2

3

compounds, including methanol, as growth substrates. On the

contrary, E. limosum is a metabolically versatile acetogen that

can grow on a wide range of substrates including methanol.

Acetic and butyric acid are the main fermentation end-products

−1

was also coproduced in the CO

average net production of 0.12 ± 0.01 g.L .

2 2

& H experiment with an

−1

2

0,34

of E. limosum when grown on methanol.

Based on the 16S

Figure 1. Panel A: Electron balance of C. luticellarii DSM 29923 fed with methanol in combination with different electron acceptors Panel B: Net

production and consumption of C. luticellarii DSM 29923 grown on different electron acceptors.

B

A

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This observation is in-line with the detection of 0.3 up to 1.6 The growth of C. luticellarii DSM 29923 with methanol as an

−1

36

g.L isobutyric acid in an MES cell. In addition to these electron donor in combination with different electron acceptors

products, trace amounts of caproic acid were also detected. was further investigated. Among electron acceptors tested,

Second, three electron donors used in chain elongation, namely only acetic and propionic acid supported growth (Figure 1). For

methanol, ethanol and lactic acid, were tested with acetic acid the conditions with acetic acid as electron acceptor (MA and

as electron acceptor (Figure 1). When grown on methanol, C. MAB), isobutyric acid and butyric acid were the main

luticellarii produced isobutyric and butyric acid in nearly fermentation products.

equimolar amounts, 17.13 ± 0.8 mM and 15.53 ± 0.24 mM, Under the condition with acetic acid only (MA), the same

−1

respectively (1.51 ± 0.07 g.L and 1.37 ± 0.02 g.L ). product spectrum and titers were observed as in the previous

Additionally, caproic acid was also formed as a byproduct. experiment.

Under the lactic acid and acetic acid combination, propionic acid

The total amount of methanol converted into fermentation

was the most abundant fermentation product with an average

products was the same under both conditions MA and MAB;

−1

net production of 3.18 ± 0.08 g.L . Acetic, butyric and valeric

acid were also formed as secondary products with an average

however, the supplementation of butyric acid led to higher

proportions of isobutyric and caproic acid (71% and 11% in MAB

compared to 44% and 5% in MA, on an electron basis). Under

the conditions with propionic acid as electron acceptor (MP and

MPV), growth and total methanol consumption were

significantly lower than with acetic acid. Even-chain

carboxylates, acetic and butyric acid, represented the highest

proportion of the total products (77% in MP and 74% in MPV on

an electron basis). However, production of valeric acid

proportional to the amount of propionic acid consumed was

also observed with an average net production of 0.23 ± 0.01

−

1

−1

net production of 1.14 ± 0.06 g.L , 0.51 ± 0.07 g.L and 0.27 ±

0

−1

.05 g.L , respectively. C. luticellarii did not grow with ethanol

as electron donor. Thus, the present study provides the first

report of isobutyric acid production from a pure culture grown

on C carbon sources.

1

3

.3. Screening the capactity of Clostridium luticellarii to produce

other isocarboxylic acids from methanol

−1

g.L and 0.31 ± 0.03 g.L , respectively.

A

B

Figure 2 Results of C. luticellarii DSM 29923 batch experiments at varying pH. Electron balance and increase in optical density (Panel A); final

isobutyric acid concentration and final pH (Panel B).

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3

.4. Effect of pH on isobutyric acid titer and selectivity

The effect of pH on isobutyric acid production by C. luticellarii

DSM 29923 was investigated during batch growth on methanol C. luticellarii was incubated in batch experiments at varying

200 mM) with initial supplementation of acetic acid (100 mM) initial methanol and acetic acid concentrations with and

3.5. Influence of substrate concentration on isobutyric acid

production

(

and butyric acid (23 mM). As observed in Figure 2, acidic pH without butyric acid supplementation to assess the impact of

conditions (below pH 5.50) inhibited C. luticellarii growth. Over those parameters on isobutyric acid production (Figure 3). At a

the range of pH 5.50 to 6.50, increasing pH led to higher cell fixed initial acetic acid concentration of 50 mM, higher cell

densities and increased the total amount of methanol densities were obtained with increasing initial methanol

converted (from 15.35 ± 2.11 mM to 167.91 ± 4.15 mM) and net concentrations. The percentage of methanol converted to

−1

isobutyric acid production (from 0.37 ± 0.02 g.L to 3.91 ± 0.06 carboxylic acids decreased from 100% in condition M1A to 89%

−1

g.L ). In terms of product speciation, isobutyric acid was the in condition M2A and 50% in condition M4A. Net production of

−1

dominant end-product under all pH conditions that supported isobutyric acid nearly doubled (from 1.47 ± 0.06 g.L to 2.66±

−1

growth, although the highest selectivity was achieved at pH 0.13 g.L ) with the doubling of initial methanol concentration

.00 (83% on an electron basis). In comparison, the selectivity from 100 mM to 200 mM while the selectivity remained the

7

at maximum isobutyric acid production (pH 6.5) was 69%.

same. Further increase in initial methanol concentration to 400

mM resulted in a decrease of both isobutyric acid final

concentration and selectivity.

A

B

C

D

E

F

Figure 3 Electron balance and final product concentration of C. luticellarii DSM 29923 grown under varying initial methanol to acetic acid ratios at

a fixed initial acetic acid concentration of 50 mM (Panel A and D) and at a fixed initial methanol concentration of 200 mM with (Panel C and F) and

without butyric acid supplementation (Panel B and E) .

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Table 3 Stoichiometric fermentation equations obtained under varying initial methanol to acetic acid ratios at a fixed initial acetic acid

concentration of 50 mM and at a fixed initial methanol concentration of 200 mM with and without butyric acid supplementation.

Experiment

Stoichiometric Equations

Fixed acetic acid

M1A

10 CH

3

OH + 0.96 CO

OH + 0.84 CO

2

+ 2.35 CH

+ 2.38 CH

3

CO

2

H → 1.82 CH

H → 1.46 CH

3

(CH

2

)

2

CO

2

H + 1.70 (CH

H + 1.66 (CH

3

)

2

CHCO

2

H + 0.26 CH

H + 0.52 CH

3

(CH

2

)

4

CO

2

H + 9.04 H

H + 9.16 H

2

O

M2A

3

2

3

2

3

2

3

2

3

2

4

2

M4A

10 CH

3

OH + 0.89 CO

2

3

+ 2.41 CH CO

2 3

H → 2.02 CH (CH

2

)

2

CO

2

H + 1.36 (CH

3

)

2

CHCO

2 3

H + 0.36 CH (CH

2

)

4

CO

2

H + 9.11 H

2

O

Fixed methanol

MA0

10 CH

3

OH + 1.86 CO

2

3

→ 1.43 CH (CH

2

)

2

CO

2

H + 1.01 (CH

3

)

2

CHCO

2 3

H + 0.35 CH (CH

2

)

4

CO

2

H + 8.14 H

2

O

MA25

10 CH

3

OH + 1.09 CO

2

+1.73 CH

3

CO H → 1.43 CH

2

3

(CH

2

)

2

CO H + 1.39 (CH

2

3

)

2

CHCO H + 0.55 CH

2

3

(CH

2

)

4

CO H + 8.91 H

2

O

MA50

MA100

10 CH

3

OH + 0.84 CO

OH + 0.62 CO

2

+ 2.38 CH

+ 3.10 CH

3

CO

2

H → 1.46 CH

H → 1.55 CH

3

(CH

2

)

2

CO

2

H + 1.66 (CH

H + 2.14 (CH

3

)

2

CHCO

2

H + 0.52 CH

H + 0.34 CH

3

(CH

2

)

4

CO

2

H + 9.16 H

2

O

3

2

3

2

3

2

)

2

3

)

2

3

2

)

4

2

H + 9.38 H

2

Fixed methanol+

butyric acid

MA0B

3 2 3 2 2 2 3 2 2 3 2 4 2 2

10 CH OH + 1.77 CO → 0.85 CH (CH ) CO H + 1.23 (CH ) CHCO H + 0.57 CH (CH ) CO H + 8.23 H O

MA25B

MA50B

MA100B

10 CH

3

OH + 1.14 CO

2

+1.54 CH

OH + 0.73 CO + 2.13 CH

2

OH + 0.78 CO + 2.50 CH

3

CO

3

CO

3

CO

2

H → 0.88 CH

H → 0.74 CH

3

(CH

3

(CH

3

(CH

2

)

2

)

2

)

2

CO

2

CO

2

CO

2

H + 1.77 (CH

2

H + 2.13 (CH

2

H + 2.36 (CH

3

)

3

)

3

)

2

CHCO

2

CHCO

2

CHCO

2

H + 0.61 CH

H + 0.62 CH

H + 0.56 CH

3

(CH

3

(CH

3

(CH

2

)

2

)

2

)

4

CO

4

CO

4

CO

2

H + 8.86H

H + 9.10 H

H + 9.22 H

2

O

10 CH

3

2

O

3

2

At a fixed initial methanol concentration of 200 mM with acetic of isobutyric acid per mol of methanol) were attained at initial

acid supplementation (M2A0, M2A25, M2A50, M2A100), C. methanol to acetic acid ratio of 2.0 with supplementation of

luticellarii grew well within the range of acetic acid butyric acid (MA100B). Finally, the production of caproic acid

concentrations tested (Final OD600=1.45± 0.01 for condition was also detected under all tested conditions and was the

MA0 and Final OD600=2.11± 0.23 for conditions MA25 to MA10). highest at initial methanol to acetic acid ratio of 4.0 regardless

−1

Increasing initial acetic acid concentrations led to an increase in of butyric acid supplementation (1.11 ± 0.04 g.L in condition

−1

the percentage of methanol converted from 51% in condition MA50 and 1.31 ± 0.07 g.L in condition MA50B).

MA0 up to complete depletion in condition MA100. Higher

To further investigate the effect of substrate composition, the

initial acetic acid concentrations also led to higher net

evolution of the product spectrum was monitored throughout

−1

production of isobutyric acid from 0.71 ± 0.05 g.L in condition

the complete batch incubation under three selected conditions

M, MA, MAB, Figure 4). During growth on methanol (200 mM)

with CO as the only available electron acceptor, acetic and

−1

MA0 to 3.64 ± 0.04 g.L in condition MA100. This isobutyric acid

production increase was accompanied by an increase in

selectivity and yield from 35% (MA0) to 51% (MA100) (on an

electron basis) and from 0.10 mol of isobutyric acid per mol of

methanol up to 0.21 (Table 3). In the absence of acetic acid

(

2

butyric acid production started immediately (Figure 4.A). On

day six, acetic acid concentration reached a maximum of 1.24

−1

±

0.05 g.L , after which C. luticellarii started consuming the

(MA0), butyric acid was the most abundant product (48% on an

acetic acid produced in-situ. In parallel, butyric acid production

electron basis). However, even though the net production of

−1 −1

continued at a production rate of 0.120 ± 0.024 g.L .d , and

−1

butyric acid increased from 1.76 ± 0.05 g.L in condition MA25

to 2.64 ± 0.01 g.L⁻¹in condition MA100, butyric acid selectivity

remained stable regardless of initial acetic acid concentration.

isobutyric acid started to accumulate from the same day at a

similar production rate. A nearly linear methanol consumption

was observed until day 12 when methanol concentration

−1

When comparing conditions with and without butyric acid levelled off. As a result, 45% (3.0 g.L ) of the methanol initially

supplementation, similar cell densities were achieved at fed was left unconverted. When acetic acid (100 mM) was fed

equivalent methanol to acetic acid initial ratios. Although the as an electron acceptor in condition MA, butyric and isobutyric

same amount of methanol was consumed at equivalent initial acid were initially coproduced at an equivalent production rate

−1

methanol to acetic acid ratio, butyric acid supplementation of about 0.260 ± 0.003 g.L .d (Figure 4.B). This drop in

resulted in higher isobutyric acid net production, selectivity, and butyric acid production rate coincided with a drop in acetic

yields. The highest isobutyric acid net production (4.50 ± 0.07 acid consumption rate. The addition of acetic acid as electron

−1

g.L ), selectivity (64% on an electron basis) and yield (0.24 mol acceptor resulted in a two-fold increase in methanol

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ARTICLE

A

B

C

D

Figure 4 Substrate and product concentration profile during batch growth of C. luticellarii DSM 29923 on methanol (Panel A; M), and methanol

and acetic acid with (Panel C; MAB) and without (Panel B; MA) butyric acid supplementation. Panel D represents the electron balance for all tested

conditions

consumption rate and the complete depletion of the methanol

4

. Discussion

initially fed. Under the condition with butyric acid

supplementation (MAB), only isobutyric acid net production

occurred during the first six days (Figure 4.C). Following day six,

butyric acid started to accumulate. Nearly the same methanol

consumption rate was observed compared to the condition MA

4

.1. C. luticellarii can utilise C1 carbon sources to produce

isobutyric acid

Firstly, a stable isobutyric acid-producing mixed culture was

obtained in a CSTR fed with methanol and acetic acid. Seven

methanol-utilising and isobutyric acid-producing bacterial

strains were isolated from the microbiome of the continuous

reactor through streak plating. The 16S rRNA gene sequence

−1

−1 −1

(0.632 ± 0.019

g.L .d and 0.655 ± 0.030 g.L .d ,

respectively), whereas a higher isobutyric acid production rate

−1

of 0.420 ± 0.012 g.L .d was observed. Additionally, the

highest final isobutyric acid concentration and selectivity were analysis of these strains revealed that they all shared high

also obtained when butyric acid was initially supplemented similarity with C. luticellarii DSM 29923 (>99.7%). C. luticellarii

−

1

DSM 29923 is a recently isolated organism whose only

metabolic feature reported to date is its ability to produce

butyric acid when cultured on RCM (Q. Wang et al., 2015). A

shotgun genome analysis of C. luticellarii DSM 29923 revealed

the presence of the acs gene cluster, which is specific to the

along with acetic acid (5.04 ± 0.08 g.L and 70%, respectively).

Finally, regardless of the substrate composition, caproic acid

started accumulating after day seven, and its highest

production rate was also achieved under the condition with

both acetic acid and butyric acid supplementation (0.108 ±

37

organisms that use the Wood-Ljungdahl pathway. Acetogenic

−1

0

.014 g.L .d ).

bacteria that use this pathway can grow on CO and H as well

2

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ARTICLE

as other one-carbon compounds including CO, formate and isobutyric acid selectivity (65%). When the continuous reactor

View Article Online

1

5,38

methanol.

However, the ability of C. luticellarii DSM 29923

DOI: 10.1039/D0GC02700F

was operated at pH 6.75, closer to C. luticellarii optimum pH

6.50), isobutyric acid production dropped considerably (below

%) due to a shift in microbial community composition. Shifting

to grow on methanol has not yet been described. During batch

experiments screening different electron donors and carbon

sources, C. luticellarii DSM 29923 was shown to produce

isobutyric acid when growing on methanol and acetic acid, and

(

3

the pH from 5.20 to 6.75 led to the dominance of an unknown

Eubacterium that resembled Eubacterium limosum which is not

-1

2 2

CO and H (1.69 ± 0.07 g.L and 0.24 ± 0.01 g.L , respectively).

2

4

26

able to produce isobutyric acid. Chen et al. and Huang et al.

Thus, the present study provides the first report of isobutyric

acid production from a pure culture grown on C1 carbon

sources.

both hypothesised that isobutyric acid was most likely produced

via bio-isomerisation of butyric acid and that bio-isomerisation

would serve as a detoxifying mechanism by converting butyric

acid into its less toxic branched equivalent. This could explain

the increase in isobutyric acid selectivity observed with pH

below the optimum (i.e. 78% at pH 5.75). However, a similar

trend was observed here with increasing pH (i.e. 83% at pH

4

.2. Acetic and butyric acid supplementation improves isobutyric

acid production

C. luticellarii was capable of producing isobutyric acid from

methanol (200 mM) and CO as the sole source of carbon and

energy. The time-course experiment showed that isobutyric

acid production only started once the acetic acid produced in-

situ started getting consumed. The incomplete conversion of

2

7

.00), which is contrary to this theory. It could be that isobutyric

acid production is favoured under diverse stress conditions,

which would make interesting to investigate how other

environmental stress conditions such as salt stress and

suboptimal temperature affect isobutyric acid titer and

methanol observed with methanol and CO

by either a substrate limitation, in this case CO

inhibitory effect of fermentation products. The former seems

more likely as pH did not significantly change during the batches selectivity.

Figure S6) and adding another electron acceptor and source of

carbon in the form of acetic acid improved methanol conversion 4.4. Caproic and valeric acid production by C. luticellarii during

2

could be explained

2

, or by the

(

up to complete consumption and led to higher concentrations

of (potentially toxic) carboxylic acid end products.

Overall, it was observed that the supplementation of acetic and

butyric acid enhanced isobutyric acid production in terms of

final concentration, selectivity and production rate. In the first

methylotrophic acidogenesis

C. luticellarii is the second organism, together with Eubacterium

limosum, shown to be able to synthesise valeric and caproic acid

with methanol as electron donor. C. luticellarii produced caproic

study that achieved isobutyric acid production with an open acid during growth on methanol under all tested conditions.

culture, the addition of 200 mM exogenous butyric acid Lindley and colleagues reported that E. limosum started

2

4

triggered isobutyric acid production. In comparison, in the producing caproic acid when butyric acid concentration (either

time-course experiment with acetic acid supplementation (100

mM) isobutyric and butyric acid were immediately coproduced.

This indicates that preliminary accumulation of butyric acid is

not necessary to trigger isobutyric acid production by C.

luticellarii, which goes against the hypothesis presenting

isomerisation as a detoxifying mechanism as initially presented

41

supplemented or produced in-situ) reached 150 mM. For C.

luticellarii, caproic acid production was not triggered by a

specific butyric acid concentration (similar to the case of

isobutyric acid). Caproic acid started accumulating after seven

days in batch cultures regardless of the butyric acid

concentration present in the broth. Although, the

supplementation of acetic and butyric acid did improve caproic

acid production both in terms of final concentration and

2

4

by Chen and co-workers.

4

.3. pH affects growth, product titer and selectivity

On RCM medium, C. luticellarii was previously shown to grow production rate.

within the pH range of 5.0 to 8.0 with an optimum growth at

When the medium was supplemented with propionic acid

instead of acetic acid, the global consumption of methanol was

lowered, and the spectrum of organic acids produced was

significantly altered. Acetic acid became the dominant product,

and valeric acid production occurred at levels proportional to

the amount of propionic acid consumed. The production of

valeric acid during methanol fermentation with propionic acid

supplementation has previously been shown in a bath culture

3

5

6

.50. On methanol, final isobutyric acid concentrations and

OD values obtained at varying initial fermentation pH (from pH

.00 to 7.00) showed that pH was a key factor determining

5

isobutyric acid production and C. luticellarii growth, with the

highest product concentration and cell density obtained at pH

6

.5. Despite the same initial substrate concentrations, the lower

final product concentrations and cell densities observed at

more acidic (≤5.75) and neutral (≥6.75) pH seem to indicate that

pH inhibited cell growth and activity. Product toxicity could

have also played a role under more acidic pH conditions and

prevented further product accumulation due to a higher

4

1

with Eubacterium limosum , and in continuous and batch

systems with an open culture dominated by a Clostridium spp.

4

2

OTU . Overall, these results suggest the ability of C. luticellarii

and potentially other close relatives) to perform C2-unit

elongation similarly to Eubacterium limosum when growing on

(

3

9

proportion of the more toxic undissociated acid forms.

Although lower isobutyric acid concentrations were produced

under more acidic (≤5.75) and neutral (≥6.75) pH conditions,

higher selectivities were achieved. In comparison, de Leeuw et

4

1

methanol.

The production of other isocarboxylic acids or higher n-alcohols

was not detected in our samples, but this study does not rule out the

possibility they are produced by C. luticellarii under the presently

tested or other conditions.

4

0

al. reported that with an open culture and continuous system,

maintaining a low pH of 5.20 was necessary to achieve high

J. Name., 2013, 00, 1-3 | 11

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Journal Name

.5. Hypothetical isobutyric acid biosynthesis pathway

ARTICLE

4

previously, a genome analysis of C. luticellarii_V _i D _e _w SM _A _r _t _i _c _l 2 _e 9 _O 9 _n _l 2 _i _n 3 _e

revealed the presence of an acs gene clu

homologues of genes encoding for the enzymes catalysing the

oxidation of methyl-THF to CO , the small and the large subunits

D

O

s

I

t

:

e

10

r

.1

w

03

h

9

ic/D

h

0

r

G

e

C

g

0

r

2

o

7

ups

0

0F

The hypothetical isobutyric acid biosynthesis pathway from

methanol of C. luticellarii is shown in Figure 5. Methanol is a

known electron donor of acetogenic bacteria, which is

metabolised via the Wood–Ljungdahl pathway. To enter the

Eastern branch of the WLP, the methyl group of methanol is

transferred to tetrahydrofolate (THF) by a methyltransferase

_s_y_s_t_e_m_y_i_e_l_d_i_n_g_m_e_t_h_y_l_-_T_H_F_.₃,5,38 The cobalamin-dependent

methyltransferase that catalyses the transfer of the methyl

2

of the Co/Fe-S, and the ACS and CODH of the CODH/ACS

complex. C. luticellarii genome also harbours genes encoding

for a methanol-cobalamin methyltransferase B subunit and a

methyl-THF:CoP methyltransferase A subunit suggesting its use

of a methanol methyltransferase system (MtaABC).³⁷

group of methanol to THF present in methylotrophic acetogens Acetyl-CoA can then be either incorporated into cell carbon

is composed of three subunits MtaA, MtaB, and MtaC (anabolism) or further converted into acetic, iso-/n-butyric and

(MtaABC). MtaB catalyses the transfer of the methyl group from caproic acid (as part of the organisms catabolism). Acetogens

methanol to the corrinoid cofactor of MtaC subunit. MtaA generate acetic acid from acetyl-CoA via the ATP-yielding

catalyses the transfer of the methyl group from the corrinoid of phosphotransacetylase /acetate kinase pathway (Pta-Ack). The

MtaC to THF. The methyl group of methyl-THF is then first step in butyric acid synthesis from acetyl-CoA is the

transferred onto the cobalt atom of a corrinoid iron sulfur condensation of two acetyl-CoA molecules to acetoacetyl-CoA

protein (Co/Fe-S) forming a Co-methylcorrinoid. ⁵^,³⁸^,⁴³One mol catalysed by acetyl-CoA-acetyltransferase (thiolase, Thl).

of methyl-THF is also oxidised to CO

that are used by CO dehydrogenase (CODH) to reduce three mol the action of 3-hydroxybutyryl-CoA dehydrogenase (Hbd) using

of CO to CO. Finally, acetyl-CoA is formed by condensation of NADH as cofactor. Crotonase (Crt), next, catalyses the

2

to generate six electrons Acetoacetyl-CoA is then reduced to 3-hydroxybutyryl-CoA via

2

the methylcorrinoid, coenzyme A (CoA) and CO by acetyl-CoA dehydration of 3-hydroxybutyryl-CoA to crotonyl-CoA.

synthase (ACS) of the CODH/ACS complex.⁵^,³⁸As stated

Figure 5 Hypothetical methanol metabolism and carboxylic acids biosynthesis pathways in C. luticellarii. The blue-highlighted and grey-highlighted pathways show the

Wood−Ljungdahl pathway and methanol methyltansferase sytem for ﬁxation of CO and synthesis of acetyl-CoA. Acetyl-CoA can then be converted to acetic acid (green-

2

highlighted), n-butyric acid (yellow-highlighted), iso-butyric acid (orange-highlighted) and caproic acid (pink-highlighted). Enzymes abbreviations: ACD, acyl-CoA

dehydrogenase; ACK, acetate kinase; ACS, acetylCoA synthase; BCD, butyryl-CoA dehydrogenase; BUK, butyrate kinase; CoAT, acetate CoA-transferase; CODH, carbon

monoxide dehydrogenase; CRT, crotonase; FDH, formate dehydrogenase; FTL, formyl-THF ligase; HAD, hydroxyacyl-CoA dehydrogenase; HBD, hydroxybutyryl-CoA

dehydrogenase; ICM, isobutyryl-CoA mutase; MET, methylTHF:corrinoid/iron-sulfur protein co-methyltransferase ; MTaA,; methyl-THF:CoPmethyltransferase; MTaB,

methanol:cobalamin methyltransferase, subunit B; MTaC, methanol-corrinoid protein; MTC, methenyl-THF cyclohydrolase; MTD, methylene-THF dehydrogenase; MTR,

methylene-THF reductase; PTA, phosphotransacetylase; THL, thiolase

J. Name., 2013, 00, 1-3 | 12

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DOI: 10.1039/D0GC02700F

ARTICLE

Finally, crotonyl-CoA is reduced to butyryl-CoA by the action of acid by an acyl CoA-transferase.¹⁵^,³⁶Additionally, we propose

a

ferredoxin-reducing, electron-bifurcating butyryl-CoA that exogenously supplied acetic acid and butyric acid during

dehydrogenase (Bcd).³^,¹⁵^,³⁸All genes coding for acetic acid growth on methanol could be converted to isobutyric acid via

formation from acetyl-CoA and butyryl-CoA formation from metabolic switch mechanisms which would reverse their flux via

the reverse action of Pta-Ack and CoAT generating acetyl-CoA

and butyryl-Coa which could then be directed towards

isobutyric acid production.

acetyl-CoA were found in the genome of C. luticellarii DSM

3

7

2

9923. Butyryl-CoA can then be directed towards butyric acid

formation. Two different pathways for the synthesis of butyric

acid from butyryl-CoA have been characterised in acetogens. 4.6. Outlook and future work

The

first

pathway

involves

the

enzymes

In this study, we provide hard evidence on the role of C.

luticellarii in isobutyric acid production, which was only

putatively attributed in previous studies based on 16S rRNA

_c_o_m_m_u_n_i_t_y_a_n_a_l_y_s_i_s_d_a_t_a_a_n_d_c_o_r_r_e_l_a_t_i_o_n_s_.24,26,40 This brings C.

luticellarii into the light as a potential new platform to produce

iso-carboxylic acids and iso-alcohols. In the MAB time-course

phosphotransbutyrylase (Ptb) and butyrate kinase (Buk), with

butyryl-CoA converted to butyric acid through the formation of

a butyryl-phosphate intermediate. In the second pathway,

butyryl-CoA:acetate CoA-transferase transfers the CoA moiety

from butyryl-CoA to acetic acid, resulting in the formation of

acetyl-CoA and butyric acid (CoAT).³^,¹⁵^,³⁸Genes encoding a

butyrate kinase and phosphotransbutyrylase were not found in

C. luticellarii DSM 29923 genome. However, the presence of

genes encoding acetate CoA- and acetoacetate CoA-

transferases indicates that C. luticellarii DSM 29923 probably

uses the alternative pathway that transfers the CoA moiety

from butyryl-CoA onto acetic acid or acetoacetic acid as the final

step in butyric acid formation.³⁷Even though acetoacetic acid

was not measured in the present study, based on the almost-

closed electron balances (90-95% closure), the involvement of

acetoacetate-CoA transferase compared to acetate-CoA

transferase seems unlikely.

experiment, the highest concentration of 5.04 ± 0.08 g

_i_s_o_b_u_t_y_r_i_c_a_c_i_d_._L-1 ₍₄_.₇₁_±₀_.₀₇_g_._L-1 net production) was

-1 -1

achieved with a productivity of 0.420 ± 0.012 g.L .d and a

selectivity toward isobutyric acid of 0.70 electron per electron

of total products. This concentration was 20% higher than the

one achieved in a continuous reactor with an open culture with

-1

1

50 mM (8.9 g L ) acetic acid and 300 mM (9.6 g L ) methanol.

de Leuuw and colleagues obtained a final isobutyric acid

-1

concentration of around 100 Mm (8.8 g L ) in batch incubation;

however, the net production was only around 50 mM (4.4 g L^-¹)

_a_s₅₀_m_M_o_f_i_s_o_b_u_t_y_r_i_c_a_c_i_d_w_a_s_a_m_e_n_d_e_d_f_r_o_m_t_h_e_s_t_a_r_t_.40

These results suggest that higher isobutyric acid concentrations

can potentially be achieved within a continuous or fed-batch

Alternatively, we hypothesise that butyryl-CoA can be directed

toward isobutyric and caproic acid formation. Isobutyryl CoA- system with C. luticellarii. This is an important point in view of

mutase (ICM), a methylmalonyl CoA-mutase-like protein, has the development of an industrial bioprocess, as high final

been identified as the key enzyme for isobutyrate formation in product titers are essential to cost-effective downstream

4

5

several sulfate-reducing bacteria by catalysing the processing. Given the fact that C. luticellarii is a newly isolated

4

isomerisation of butyryl-CoA to isobutyryl-CoA. Genes organism, there is currently no biokinetic data available, which

encoding for the large and small subunit of an MCM-like enzyme

is also key for bioprocess development and optimisation. Future

work should undertake a thorough kinetic characterisation of

this microorganism.

3

7

were annotated in the genome of C. luticellarii DSM 29923. A

database search revealed that the encoded proteins (NCBI

Reference Sequence: WP_106007777.1 and WP_106008210.1)

were part of the isobutyryl-CoA mutase (ICM)-like subfamily,

which corroborates that isobutyric acid synthesis in C. luticellarii

most likely occurs via butyryl-CoA to isobutyryl-CoA

isomerisation. The last step of isobutyric acid formation is

probably catalysed by an acetate CoA-transferase similarly to

In the present study as well as in recent mixed culture studies,

the supplementation of acetic and/or butyric acid was essential

to achieve high isobutyric acid titer, selectivity and

productivity.²⁴^,²⁶^,⁴⁰Unless acetic and/or butyric acid can be

supplied from a relatively cheap and renewable source, it is

butyric acid. The pathway of caproic acid formation from essential for the viability of the process to determine under

butyryl-CoA probably starts with a two-carbon chain elongation which operational conditions (e.g. pH, temperature, reactor

of butyryl-CoA with acetyl-CoA by the action of a thiolase. The operation/feeding regime, etc.) isobutyric acid production can

resulting 3-ketohexanoyl-CoA can subsequently be converted in be achieved on methanol and CO as sole electron donor and

2

two further steps to caproyl-CoA by enzymes that are assumed acceptor. One possible option here is the use of organic acids

to perform analogous reactions as for butyric acid formation.

Finally, caproyl-CoA is also supposed to be converted to caproic

derived from waste acidification as supplements, as proposed

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Page 14 of 15

Journal Name

ARTICLE

by Chen and co-workers.²⁴Another alternative could be the

development of selective isobutyric acid in-situ product

recovery strategies, leaving acetic and n-butyric acid in the

fermentation broth. This avenue would maximise the product

yield, as eventually, all methanol fed would end up being

recovered as isobutyric acid.

3

4

V. Müller, Trends in Biotechnology, 2019, 37, 1344–1354.

View Article Online

DOI: 10.1039/D0GC02700F

C. A. Cotton, N. J. Claassens, S. Benito-Vaquerizo and A. Bar-

Even, Current Opinion in Biotechnology, 2020, 62, 168–180.

5

F. Kremp and V. Müller, FEMS Microbiol Rev, ,

DOI:10.1093/femsre/fuaa040.

Last but not least, the metabolic pathway by which C. luticellarii

produces isobutyric acid is not yet known. Here we propose a

6

Renewable Methanol|Methanol Institute|www.methanol.org,

tentative pathway based on existing knowledge and genomic https://www.methanol.org/renewable-methanol/, (accessed 18

data, but it is purely hypothetical. A better understanding of the September 2020).

pathway would allow to better tune process design and

7

Pricing | Methanex Corporation,

conditions to its production. Further investigation through

transcriptomic analysis, isotope-labelled flux analysis, and

enzymatic assays are necessary to verify the proposed pathway,

establish if exogenously supplied acetic and butyric acid are

incorporated in the produced isobutyric acid and unravel the

full potential of C. luticellarii as a host for the production of

isomers.⁴⁶^–⁴⁹

https://www.methanex.com/our-business/pricing, (accessed 19

September 2020).

8

Sugar, https://ec.europa.eu/info/food-farming-

fisheries/farming/facts-and-figures/markets/prices/price-

monitoring-sector/plant-products/sugar_en, (accessed 19

September 2020).

5

. Conclusion

9

W. Zhang, M. Song, Q. Yang, Z. Dai, S. Zhang, F. Xin, W. Dong, J.

Here we revealed the ability of C. luticellarii to produce

isobutyric acid from C carbon sources, including methanol. The

Ma and M. Jiang, Biotechnol Biofuels, 2018, 11, 260.

1

wild-type strain grows on methanol and generates isobutyric

acid without the external addition of acetic and butyric acid as

electron acceptors, although their presence steered isobutyric

acid production in terms of final titer, selectivity, and

1

0 H. Offermanns, L. Plass and M. Bertau, in Methanol: The Basic

Chemical and Energy Feedstock of the Future: Asinger’s Vision

Today, eds. M. Bertau, H. Offermanns, L. Plass, F. Schmidt and H.-J.

Wernicke, Springer, Berlin, Heidelberg, 2014, pp. 1–22.

−1

productivity. A maximum concentration of 5.04 ± 0.08 g.L

isobutyric acid was produced in batch at a maximum

1

1 Z. Zhu, T. Kin Tam, F. Sun, C. You and Y.-H. Percival Zhang,

−1

productivity of 0.420 ± 0.012 g.L .d and a selectivity of 70%

on an electron basis. pH was shown to significantly influence

isobutyric acid production with the highest production obtained

at pH 6.50; however, higher selectivities were achieved under

less favourable pH conditions (≤5.75 and ≥6.75). C. luticellarii

was also shown to be able to produce valeric and caproic acid

Nature Communications, 2014, 5, 3026.

1

2

2 G. A. Olah, Angewandte Chemie International Edition, 2005, 44,

636–2639.

1

3 M. Bertau, H. J. Wernicke, F. Schmidt, U.-D. Standt, F. Seyfried,

2

by C -unit elongation. Overall given the high isobutyric acid

production capability of C. luticellarii, it can be considered as a

promising workhorse for producing value-added isocarboxylic

2

acids from CO via methanol.

S. Buchholz, G. Busch, M. Winterberg, L. Reichelt, C. Pätzold, S. Pohl,

L. Plass, J. Roes, M. Steffen, G. Sandstede, A. Heinzel, S. Hippmann,

D. Holtmann, F. Sonntag, T. Veith and J. Schrader, in Methanol: The

Basic Chemical and Energy Feedstock of the Future: Asinger’s Vision

Today, eds. M. Bertau, H. Offermanns, L. Plass, F. Schmidt and H.-J.

Wernicke, Springer, Berlin, Heidelberg, 2014, pp. 327–601.

Conflicts of interest

There are no conflicts to declare.

1

4 Port of Antwerp brings different players together to produce

sustainable methanol,

https://www.portofantwerp.com/en/news/port-antwerp-brings-

different-players-together-produce-sustainable-methanol,

Acknowledgements

C.P and R.G gratefully acknowledge support from BOF

startkrediet (BOF19/STA/044).

(accessed 21 September 2020).

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Article Doi

Production of isobutyric acid from methanol by: Clostridium luticellarii

DOI: 10.1039/d0gc02700f

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Authors:

Article abstract of DOI:10.1039/d0gc02700f

Full text of DOI:10.1039/d0gc02700f

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