Fumaric

acid

production

by

fermentation

The

potential

of

fumaric

acid

as

a

raw

material

in

the

polymer

industry

and

the

increment

of

cost

of

petroleum-based

fumaric

acid

raises

interest

in

fermentation

processes

for

production

of

this

compound

from

renewable

resources

Although

the

chemical

process

yields

w/w

fumaric

acid

from

maleic

anhydride

and

the

fermentation

process

yields

only

w/w

from

glucose

the

latter

raw

material

is

three

times

cheaper

Besides

the

fermentation

fixes

Production

of

fumaric

acid

by

Rhizopus

species

and

the

involved

metabolic

pathways

are

reviewed

Submerged

fermentation

systems

coupled

with

product

recovery

techniques

seem

to

have

achieved

economically

attractive

yields

and

productivities

Future

prospects

for

improvement

of

fumaric

acid

production

include

metabolic

engineering

approaches

to

achieve

low

pH

fermentations

Introduction

Fumaric

acid

-LRB-

Fig.

-RRB-

is

a

naturally

occurring

organic

acid

It

was

first

isolated

from

the

plant

Fumaria

officinalis

from

which

it

derives

its

name

Many

microorganisms

produce

fumaric

acid

in

small

amounts

as

it

is

a

key

intermediate

in

the

citrate

cycle

Fumaric

acid

is

also

known

as

-LRB-

E

-RRB-

acid

or

acid

Sometimes

the

term

fumarates

is

also

used

In

this

review

this

general

term

is

not

used

to

describe

fumaric

acid

esters

like

dimethyl

fumarate

but

only

to

describe

the

salts

of

fumaric

acid

-LRB-

sodium

fumarate

calcium

fumarate

-RRB-

Fig.

applications

of

fumaric

acid

production

Miscellaneous

include

lubricating

oil

inks

lacquers

carboxylating

agent

for

styrenebutadiene

rubber

personal

care

additives

a

Anonymous

b

Willke

and

Vorlop

Currently

fumaric

acid

is

produced

chemically

from

maleic

anhydride

which

in

turn

is

produced

from

butane

However

as

petroleum

prices

are

rising

rather

quickly

maleic

anhydride

as

a

petroleum

derivative

has

increased

in

price

as

well

-LRB-

Anonymous

-RRB-

This

situation

has

caused

a

renewed

interest

in

the

fumaric

acid

production

by

fermentation

that

was

operational

during

the

-LRB-

Goldberg

et

al.

-RRB-

but

was

discontinued

and

replaced

by

petrochemical

processes

The

fermentation

process

is

also

interesting

because

it

involves

carbon

dioxide

fixation

as

will

be

discussed

later

Fungi

are

known

for

their

organic

acid-producing

capability

and

have

been

used

in

fermentation

processes

for

fumaric

acid

production

Production

by

filamentous

fungi

of

organic

acids

including

fumaric

acid

has

recently

been

reviewed

-LRB-

Magnuson

and

Lasure

Goldberg

et

al.

-RRB-

These

reviews

focused

on

the

microorganisms

with

their

associated

metabolic

pathways

In

contrast

the

current

review

focuses

on

development

of

fumaric

acid

production

processes

After

summarizing

fumaric

acid

properties

applications

and

production

metabolic

pathways

for

fumaric

acid

production

will

be

discussed

only

briefly

before

turning

to

fermentation

performance

of

the

most

prominent

fumaric

acid-producing

strains

Furthermore

different

methods

that

have

been

studied

to

optimize

fermentation

processes

will

be

mentioned

and

future

prospects

for

process

development

will

be

discussed

Properties

and

applications

of

fumaric

acid

Because

of

its

structure

-LRB-

a

carbon

carbon

double

bond

and

two

carboxylic

acid

groups

-RRB-

fumaric

acid

has

many

potential

industrial

applications

-LRB-

Fig.

-RRB-

It

can

act

as

starting

material

for

polymerization

and

esterification

reactions

-LRB-

-RRB-

As

raw

material

for

polymerization

especially

in

the

production

of

unsaturated

polyester

resins

maleic

anhydride

is

currently

preferred

to

fumaric

acid

because

maleic

anhydride

-LSB-

kg

-LRB-

Anonymous

-RRB-

-RSB-

is

cheaper

than

fumaric

acid

the

latter

historically

being

around

more

expensive

than

maleic

anhydride

-LRB-

-RRB-

However

fumaric

acid

could

be

a

better

option

for

the

polymer

industry

among

other

carboxylic

acids

or

their

derivatives

because

of

its

nontoxic

nature

In

addition

special

properties

like

greater

hardness

in

the

polymer

structure

can

be

achieved

when

fumaric

acid

is

used

-LRB-

-RRB-

In

addition

to

polymerization

there

are

two

potentially

new

applications

for

fumaric

acid

and

both

require

a

different

grade

of

purity

of

it

The

first

is

as

a

medicine

to

treat

psoriasis

a

skin

condition

-LRB-

Altmeyer

et

al.

Mrowietz

et

al.

-RRB-

Psoriatic

individuals

are

unable

to

produce

fumaric

acid

in

their

body

-LRB-

which

is

not

the

case

in

normal

individuals

-RRB-

due

to

a

certain

biochemical

defect

that

interferes

with

adequate

fumaric

acid

production

in

the

skin

Therefore

psoriatic

individuals

need

to

take

orally

fumaric

acid

in

the

form

of

fumaric

acid

monoethyl

or

dimethyl

ester

to

treat

their

disease

The

second

potential

application

of

fumaric

acid

is

as

supplement

in

cattle

feed

Recent

studies

indicate

that

a

large

reduction

in

the

methane

emissions

of

cattle

can

be

achieved

-LRB-

up

to

-RRB-

if

this

cattle

receives

fumaric

acid-based

additive

as

a

supplement

in

their

diet

-LRB-

Mcginn

et

al.

-RRB-

This

could

greatly

reduce

the

total

emission

of

methane

as

farm

animals

are

responsible

for

of

the

methane

emission

caused

by

human

activity

Fumaric

acid

together

with

the

related

succinic

and

malic

acids

has

been

identified

as

one

of

the

top

ten

building

block

chemicals

that

can

be

produced

from

sugars

via

biological

or

chemical

conversion

-LRB-

Werpy

and

Petersen

-RRB-

Compared

to

these

other

dicarboxylic

acids

fumaric

acid

has

a

low

aqueous

solubility

-LSB-

g/kg

at

°C

g/kg

at

°C

-LRB-

Stephen

-RRB-

-RSB-

and

low

pKa

values

-LSB-

and

-LRB-

Lohbeck

et

al.

-RRB-

-RSB-

which

are

properties

that

can

be

exploited

for

product

recovery

Fumaric

acid

production

by

petrochemical

routes

Fumaric

acid

is

currently

produced

by

isomerization

of

maleic

acid

which

is

produced

from

maleic

anhydride

Maleic

anhydride

in

turn

is

industrially

produced

by

catalytic

oxidation

of

suitable

hydrocarbons

in

the

gas

phase

Benzene

used

to

be

the

predominant

starting

material

but

oxidation

of

n-butane

or

n-butane

n-butene

mixtures

has

become

more

important

in

recent

years

-LRB-

Lohbeck

et

al.

-RRB-

The

butane

oxidation

reaction

equation

to

maleic

anhydride

is

In

the

most

common

maleic

anhydride

process

the

catalyst

is

embedded

in

fixed-bed

tubular

reactors

The

catalysts

applied

in

this

process

are

based

on

vanadium

and

phosphorus

oxides

In

this

process

water

is

formed

as

a

by-product

and

a

fairly

small

amount

of

it

can

be

directly

liquefied

from

the

reaction

gas

by

partial

condensation

Organic

solvents

absorb

maleic

anhydride

contained

in

the

reaction

gas

More

than

of

the

anhydride

can

be

absorbed

in

this

way

The

solvent

anhydride

mixture

is

then

subjected

to

fractional

distillation

to

separate

maleic

anhydride

from

the

solvent

and

the

latter

is

returned

to

the

absorption

column

-LRB-

Lohbeck

et

al.

-RRB-

The

thus

obtained

pure

maleic

anhydride

is

then

hydrolyzed

into

maleic

acid

according

to

the

reaction

equation

Subsequently

the

maleic

acid

is

converted

almost

quantitatively

by

thermal

or

catalytic

cis

trans

isomerization

into

fumaric

acid

-LRB-

Lohbeck

et

al.

-RRB-

The

most

common

catalysts

used

are

mineral

acids

peroxy

compounds

and

thiourea

The

crude

fumaric

acid

obtained

in

this

way

is

purified

by

crystallization

from

water

-LRB-

Fig.

-RRB-

As

will

be

shown

later

a

comparable

purification

may

be

used

for

fermentative

fumaric

acid

production

Fig.

for

fumaric

acid

production

from

malic

acid

a

Isomerization

vessel

b

Centrifuge

c

Dissolving

tank

d

Filter

e

Crystallizer

f

Dryer

-LRB-

adapted

from

Felthouse

et

al.

-RRB-

Production

capacity

of

maleic

anhydride

in

was

about

ton/a

-LRB-

Anonymous

-RRB-

with

around

of

this

being

used

for

fumaric

acid

production

-LRB-

liArticleID

-RRB-

corresponding

to

ton/a

Enzymatic

conversion

of

maleic

acid

into

fumaric

acid

In

the

aforementioned

chemical

isomerization

of

maleic

acid

into

fumaric

acid

the

conversion

yield

is

restricted

by

the

reaction

equilibrium

This

chemical

conversion

is

occurring

at

high

temperatures

that

causes

formation

of

by-products

from

maleic

and

fumaric

acids

and

as

a

consequence

yields

are

below

the

equilibrium

yields

-LRB-

Goto

et

al.

-RRB-

This

problem

was

the

main

motivation

to

look

for

a

good

enzyme

that

would

facilitate

isomerization

so

that

the

equilibrium

mixture

would

be

obtained

without

formation

of

by-products

It

is

known

that

the

enzyme

maleate

isomerase

-LRB-

maleate

cis-trans-isomerase

-RRB-

isomerizes

maleic

acid

into

fumaric

acid

Microorganisms

that

produce

maleate

isomerase

are

Pseudomonas

species

-LRB-

Otsuka

-RRB-

Alcaligenes

faecalis

-LRB-

Takamura

et

al.

-RRB-

and

Pseudomonas

fluorescens

-LRB-

Scher

and

Lennarz

-RRB-

However

maleate

isomerase

is

unstable

even

at

moderate

temperatures

-LRB-

Takamura

et

al.

-RRB-

and

therefore

a

heat-stable

maleate

isomerase

is

desired

Thermo-stable

maleate

isomerases

derived

from

Bacillus

stearothermophilus

Bacillus

brevis

and

Bacillus

sp.

were

reported

by

Goto

et

al.

-LRB-

-RRB-

and

according

to

the

authors

the

use

of

enzymes

from

these

bacteria

can

improve

the

fumaric

acid

production

process

Furthermore

in

a

more

recent

study

using

Pseudomonas

alcaligenes

high

rates

of

conversion

of

maleic

acid

into

fumaric

acid

-LRB-

g

L

h

-RRB-

were

reported

-LRB-

NakajimaKambe

et

al.

-RRB-

This

organism

does

not

normally

accumulate

fumaric

acid

but

by

heat

treatment

of

the

cells

-LRB-

°C

for

h

-RRB-

they

lost

their

fumarase

activity

which

otherwise

would

be

responsible

for

a

conversion

of

fumaric

acid

into

l-malic

acid

This

heat

treatment

did

not

affect

the

maleate

isomerase

activity

In

addition

when

calcium

ions

were

added

during

the

conversion

step

the

thermal

stability

of

maleate

isomerase

was

increased

With

the

use

of

P.

alcaligenes

the

highest

reported

conversion

yield

was

achieved

in

the

conversion

of

maleic

acid

to

fumaric

acid

-LRB-

Ichikawa

et

al.

-RRB-

Immobilization

of

the

heat-treated

cells

is

currently

under

investigation

Microbial

production

of

fumaric

acid

Fumaric

acid

production

by

fermentation

was

operated

in

the

United

States

during

the

but

later

this

process

was

discontinued

and

replaced

by

chemical

synthesis

from

petrochemical

feedstocks

the

latter

being

explained

in

a

previous

section

Nevertheless

the

continuous

increase

of

the

petroleum

prices

has

brought

back

the

interest

in

fumaric

acid

production

by

submerged

fermentation

-LRB-

Goldberg

et

al.

-RRB-

Fumaric

acid

production

by

fermentation

using

Rhizopus

species

has

been

patented

occasionally

-LRB-

Waksman

Kane

Lubowitz

and

La

Roe

La

Roe

Goldberg

and

Stieglitz

-RRB-

In

DuPont

patented

an

improved

fermentation

process

producing

carboxylic

acids

-LRB-

fumaric

succinic

malic

and

mixtures

thereof

-RRB-

by

controlling

dissolved

oxygen

levels

between

and

-LRB-

Ling

and

Ng

-RRB-

In

the

field

of

fumaric

acid

production

by

fermentation

there

are

many

aspects

determining

the

productivity

of

the

fermentation

process

such

as

the

applied

microbial

strain

and

its

morphology

the

use

of

a

neutralizing

agent

and

the

applied

feedstock

Those

aspects

are

reviewed

and

analyzed

in

more

detail

in

the

following

sections

Fumaric

acid-producing

strains

After

the

discovery

of

fumaric

acid

production

in

Rhizopus

nigricans

by

Felix

Ehrlich

in

Foster

and

Waksman

-LRB-

-RRB-

screened

strains

from

eight

different

genera

to

identify

high

fumarate

producing

strains

The

fumarate-producing

genera

identified

were

Rhizopus

Mucor

Cunninghamella

and

Circinella

species

Among

these

strains

Rhizopus

species

-LRB-

nigricans

arrhizus

oryzae

and

formosa

-RRB-

were

the

best-producing

ones

yielding

fumaric

acid

under

aerobic

and

anaerobic

conditions

-LRB-

Foster

and

Waksman

Rhodes

et

al

Kenealy

et

al.

Cao

et

al.

Carta

et

al.

-RRB-

Table

lists

these

fungi

and

as

is

shown

the

R.

arrhizus

NRRL

and

R.

oryzae

ATCC

gave

the

highest

volumetric

productivity

product

titer

and

product

yield

values

-LRB-

Gangl

et

al.

Cao

et

al.

-RRB-

Table

data

on

fumaric

acid

production

by

different

Rhizopus

generaStrainFermenterSubstrateProduct

Titer

-LRB-

g

l

-RRB-

Yield

-LRB-

g

g

-RRB-

Vol

Prod

-LRB-

g

l

h

-RRB-

Time

-LRB-

h

-RRB-

Final

pHReferenceR

nigricans

Foster

Waksman

Romano

et

al.

arrhizus

NRRL

Rhodes

et

al.

Ng

et

al.

Gangl

et

al.

arrhizus

NRRL

Kenealy

et

al.

Petruccioli

et

al.

Riscaldati

et

al.

oryzae

ATCC

plus

Cao

et

al.

Cao

et

al.

Cao

et

al.

air

Du

et

al.

Zhou

Zhou

et

al.

formosa

MUCL

tankCassava

Carta

et

al.

biofilm

contactor

Despite

the

fact

that

the

aforementioned

experimental

studies

involve

fungi

the

use

of

bacteria

has

been

considered

as

well

Donnelly

et

al.

-LRB-

-RRB-

have

suggested

using

a

Lactobacillus

host

strain

that

lacks

the

malolactate

enzyme

fumarase

and

fumarate

dehydrogenase

Introducing

the

maeA

gene

for

the

malic

enzyme

from

Escherichia

coli

would

result

in

a

pathway

from

pyruvate

to

malate

and

hence

in

a

malic

acid-producing

mutant

Additional

genetic

engineering

would

result

in

fumaric

acid

production

Genetic

modification

of

microorganisms

has

hardly

been

explored

for

fumaric

acid

production

but

offers

a

potentially

useful

approach

solution

for

improving

yields

and

rates

in

fermentation

Metabolic

pathways

to

fumaric

acid

Fumaric

acid

is

primarily

an

intermediate

of

the

citrate

cycle

but

is

also

involved

in

other

metabolic

pathways

In

it

was

suggested

that

fumarate

biosynthesis

involved

a

plus

condensation

in

Rhizopus

species

-LRB-

Foster

et

al.

-RRB-

The

reactions

in

this

pathway

seemed

to

be

similar

to

those

of

the

glyoxylate

bypass

-LRB-

Foster

et

al.

-RRB-

Years

after

the

glyoxylate

bypass

mechanism

was

ruled

out

because

the

theoretical

molar

yield

for

this

pathway

of

was

not

in

agreement

with

the

experimental

yield

of

-LRB-

Rhodes

et

al.

Romano

et

al.

-RRB-

However

the

main

evidence

for

rejecting

the

glyoxylate

bypass

mechanism

is

that

the

key

enzyme

of

the

glyoxylate

pathway

isocitrate-glyoxylate

lyase

was

repressed

when

high

glucose

concentrations

were

present

like

in

the

experiments

used

for

fumaric

acid

production

-LRB-

Romano

et

al.

-RRB-

It

was

discovered

that

a

plus

mechanism

involving

fixation

catalyzed

by

pyruvate

carboxylase

under

aerobic

conditions

explained

the

high

molar

yields

in

fumarate

production

-LRB-

Overman

and

Romano

-RRB-

This

fixation

leads

to

oxaloacetic

acid

formation

-LRB-

Osmani

and

Scrutton

-RRB-

so

that

citrate

cycle

intermediates

can

be

withdrawn

for

biosynthesis

during

the

growth

phase

under

aerobic

conditions

When

nitrogen

becomes

limiting

and

the

growth

phase

stops

the

metabolism

of

glucose

and

fixation

could

continue

and

lead

to

an

accumulation

of

acids

-LRB-

Romano

et

al.

-RRB-

The

maximal

theoretical

yield

in

a

nongrowth

situation

is

mol

of

fumaric

acid

per

mole

of

glucose

consumed

upon

fixation

of

mol

of

via

reductive

pyruvate

carboxylation

However

if

the

reductive

pyruvate

carboxylation

would

be

the

sole

pathway

no

ATP

would

be

produced

for

maintenance

or

transport

purposes

Therefore

the

citrate

cycle

is

also

active

during

fumaric

acid

production

-LRB-

Rhodes

et

al.

Kenealy

et

al.

-RRB-

The

carboxylation

enzyme

pyruvate

carboxylase

is

known

to

be

localized

exclusively

in

the

cytosol

together

with

NAD-malate

dehydrogenase

and

fumarase

-LRB-

that

are

present

in

the

cytosol

and

in

the

mitochondria

-RRB-

leading

to

fumaric

acid

synthesis

in

this

cell

compartment

-LRB-

Osmani

and

Scrutton

-RRB-

Studies

performed

by

Peleg

et

al.

-LRB-

-RRB-

indicated

higher

activities

of

these

enzymes

-LRB-

especially

the

cytosolic

isoenzymes

-RRB-

during

fumaric

acid

production

Kenealy

et

al.

-LRB-

-RRB-

used

mitochondrial

inhibitors

to

investigate

their

role

in

fumarate

accumulation

and

found

no

direct

involvement

of

such

inhibitors

of

the

citrate

cycle

in

fumarate

production

However

carbon-labeling

studies

have

demonstrated

the

simultaneous

utilization

of

both

the

citrate

cycle

and

the

reductive

pyruvate

carboxylation

pathways

under

aerobic

conditions

-LRB-

Fig.

-RRB-

Besides

the

localization

of

fumarase

isoenzymes

it

was

also

found

that

addition

of

cycloheximide

virtually

eliminated

the

cytosolic

isoenzyme

and

therefore

caused

a

large

decrease

in

the

amount

of

fumaric

acid

produced

by

R.

oryzae

-LRB-

Peleg

et

al.

-RRB-

Fig.

cycle

pathway

and

reductive

carboxylation

pathway

leading

to

fumaric

acid

accumulation

-LRB-

adapted

from

Kenealy

et

al.

-RRB-

The

ratio

between

the

two

pathways

is

not

as

suggested

by

this

figure

The

carboxylation

of

pyruvate

has

been

studied

in

more

detail

for

the

analogous

microbial

succinate

production

but

the

understanding

of

the

different

metabolic

fluxes

involved

is

still

incomplete

-LRB-

McKinlay

et

al.

-RRB-

High-producing

fumaric

acid

strains

of

Rhizopus

not

only

produce

fumaric

acid

but

also

other

carboxylic

acids

like

malic

lactic

acetic

succinic

and

citric

in

smaller

amounts

than

fumaric

acid

production

-LRB-

Rhodes

et

al.

Carta

et

al.

-RRB-

Sometimes

ethanol

is

also

produced

like

in

the

case

of

the

high-producing

strain

R.

oryzae

ATCC

-LRB-

Cao

et

al.

-RRB-

However

it

was

found

that

ethanol

production

can

be

reduced

by

a

sufficient

supply

of

oxygen

to

the

culture

-LRB-

Cao

et

al.

-RRB-

Fumaric

acid

transport

across

the

cell

membrane

The

transport

mechanism

of

fumaric

acid

in

fungi

has

not

been

studied

yet

However

transport

of

l-malic

acid

and

other

dicarboxylic

acids

including

fumaric

acid

has

been

studied

in

yeasts

like

Schizosaccharomyces

pombe

Candida

utilis

Candida

sphaerica

and

Hansenula

anomala

-LRB-

Corte-Real

et

al.

Corte-Real

and

Leao

Saayman

et

al.

-RRB-

These

microorganisms

may

be

comparable

to

fumaric-producing

fungi

These

studies

showed

that

the

initial

uptake

of

malic

acid

was

accompanied

by

disappearance

of

extracellular

protons

suggesting

that

the

anionic

form

of

the

acid

was

transported

by

an

accumulative

dicarboxylate

proton

symporter

As

fumaric

acid

seems

to

be

a

competitive

inhibitor

of

l-malic

acid

uptake

it

was

suggested

that

fumaric

acid

uses

the

same

import

system

On

the

other

hand

these

studies

showed

that

undissociated

dicarboxylic

acid

entered

the

cells

slowly

by

simple

diffusion

In

addition

it

was

revealed

that

the

rate

of

diffusion

of

the

undissociated

acid

across

the

plasma

membrane

is

subjected

to

opposite

pH

influences

an

increase

due

to

the

relative

increase

of

undissociated

acid

with

decreasing

pH

and

a

decrease

due

to

a

decrease

of

the

permeability

of

the

cell

membrane

for

the

undissociated

acid

at

decreasing

pH

-LRB-

Corte-Real

and

Leao

-RRB-

Increasing

the

number

or

activity

of

the

dicarboxylic

acid

transporters

could

lower

the

intracellular

fumarate

concentration

and

could

therefore

have

a

positive

effect

on

the

production

yield

-LRB-

van

Maris

et

al.

-RRB-

Alternative

fermentation

feedstocks

Glucose

has

not

been

the

only

carbon

source

used

for

fumaric

acid-producing

strains

The

use

of

xylose

with

immobilized

R.

arrhizus

was

studied

but

the

highest

obtained

volumetric

productivity

was

only

g

l

h

-LRB-

Kautola

and

Linko

-RRB-

Also

sucrose

has

been

considered

as

a

raw

material

in

fumaric

acid-production

processes

-LRB-

Kautola

and

Linko

Zhang

et

al.

-RRB-

but

sucrose

has

the

disadvantage

of

being

poorly

metabolized

by

R.

oryzae

compared

to

glucose

-LRB-

Bulut

et

al.

-RRB-

Starch-containing

materials

were

also

screened

as

feedstock

for

the

fumaric

acid

production

Potato

flour

was

used

as

feedstock

for

R.

arrhizus

and

although

fumaric

acid

was

the

main

metabolic

product

a

volumetric

productivity

of

only

g

l

h

was

achieved

-LRB-

Moresi

et

al.

-RRB-

On

the

other

hand

it

was

found

that

the

highest

yields

of

fumaric

acid

were

obtained

from

acid

hydrolysates

of

starch-based

materials

-LRB-

Moresi

et

al.

-RRB-

The

same

feedstock

was

used

by

Federici

et

al.

-LRB-

-RRB-

in

studies

on

the

effect

of

agitation

speed

and

applied

neutralizing

agent

The

tested

neutralizing

agent

was

a

combination

of

and

and

it

was

concluded

that

comparable

fumaric

acid

yields

were

obtained

using

these

neutralizing

agents

and

starch-based

materials

-LRB-

Federici

et

al.

-RRB-

Carta

et

al.

-LRB-

-RRB-

optimized

the

fumaric

acid

production

by

R.

formosa

MUCL

by

using

enzymatic

hydrolysates

of

cassava

bagasse

a

high-starch-containing

waste

product

The

productivity

was

not

improved

with

respect

to

a

fermentation

where

glucose

was

used

as

a

feedstock

but

the

fact

that

these

feedstocks

are

cheap

and

abundant

makes

these

optimized

fermentations

economically

attractive

Nutrient

requirements

Physical

and

nutritional

requirements

for

fumaric

acid

fermentation

have

been

studied

in

Rhizopus

arrhizus

-LRB-

Rhodes

et

al.

Rhodes

et

al.

-RRB-

As

Rhizopus

species

should

enter

a

phase

of

limited

growth

during

the

fermentation

which

can

be

achieved

by

nitrogen

limitation

these

studies

showed

that

to

achieve

high

yields

during

fumaric

acid

fermentation

the

most

critical

parameter

is

the

ratio

of

glucose

to

nitrogen

For

example

a

yield

of

fumaric

acid

on

glucose

of

-LRB-

w/w

-RRB-

was

obtained

using

an

initial

C

N

molar

ratio

of

for

R.

arrhizus

When

nitrogen

limitation

is

not

desired

phosphorus

limitation

can

be

used

instead

-LRB-

Riscaldati

et

al.

-RRB-

Trace

metal

concentrations

of

ppm

ppm

and

ppb

for

Mg

Zn

and

Fe

respectively

were

found

optimal

for

the

formation

of

small

-LRB-

mm

-RRB-

spherical

pellets

that

produced

high

concentrations

of

fumaric

acid

-LRB-

g

L

Zhou

-RRB-

Because

is

needed

for

the

oxaloacetate

formation

from

pyruvate

by

pyruvate

carboxylase

the

addition

of

calcium

carbonate

-LRB-

-RRB-

which

is

used

in

many

cases

as

a

neutralizing

agent

seems

also

important

as

a

source

during

the

production

phase

of

the

fermentation

However

in

case

that

no

or

carbonate

is

added

the

complete

catabolism

of

a

mole

of

glucose

via

the

citrate

cycle

provides

mol

of

that

may

be

used

for

pyruvate

carboxylation

-LRB-

see

Fig.

-RRB-

In

this

case

the

maximum

theoretical

yield

would

be

mol

of

fumaric

acid

per

mole

of

glucose

This

theoretical

value

is

close

to

the

value

of

mol

of

fumaric

acid

per

mole

of

glucose

reported

by

Cao

et

al.

-LRB-

-RRB-

who

optimized

a

fermentation

process

without

or

carbonate

feeding

Neutralizing

agents

Continuous

neutralization

of

the

pH

has

been

necessary

to

obtain

optimal

yields

in

fumaric

acid

production

by

fermentation

Preferably

has

been

used

as

a

neutralizing

agent

but

at

the

same

time

this

compound

is

causing

viscosity

problems

due

to

the

low

aqueous

solubility

of

calcium

fumarate

-LSB-

g/l

at

°C

-LRB-

Gangl

et

al.

-RRB-

-RSB-

that

precipitates

as

a

fermentation

product

Furthermore

the

cells

can

interact

with

the

precipitated

product

as

has

been

found

for

R.

oryzae

resulting

in

a

highly

viscous

suspension

This

has

a

detrimental

effect

on

the

rate

of

oxygen

transfer

which

can

be

achieved

and

hence

the

fermentation

might

fail

due

to

oxygen-limitation

problems

Therefore

replacement

of

by

other

neutralizing

agents

like

Ca

-LRB-

OH

-RRB-

and

-LRB-

-RRB-

has

been

studied

by

different

authors

-LRB-

Gangl

et

al.

Riscaldati

et

al.

Zhou

et

al.

-RRB-

However

these

studies

showed

that

the

fumarate

production

rates

are

the

highest

when

is

used

as

a

neutralizing

agent

Because

of

the

high

solubility

of

sodium

fumarate

fermentative

production

of

fumarate

using

as

neutralizing

agent

leads

to

cheaper

downstream

processing

than

when

is

used

This

is

due

to

the

fact

that

the

fermentation

product

sodium

fumarate

has

a

higher

solubility

than

and

hence

there

is

no

need

of

heating

to

recover

the

fermentation

product

-LRB-

see

Fig.

-RRB-

In

addition

cells

can

be

reused

-LRB-

Gangl

et

al.

Zhou

et

al.

-RRB-

A

similar

situation

was

obtained

when

Rhizopus

growth

was

limited

by

phosphorus

so

that

-LRB-

-RRB-

could

be

used

as

neutralizing

agent

-LRB-

Riscaldati

et

al.

-RRB-

Nevertheless

it

has

been

argued

that

a

fermentation

process

without

the

use

of

neutralizing

agents

and

at

the

same

time

preventing

product

inhibition

will

improve

the

economics

of

the

general

process

-LRB-

Gangl

et

al.

-RRB-

However

when

a

high-yield

process

is

developed

without

a

carbonate

as

neutralizing

agent

the

required

must

be

supplied

by

other

sources

Fumaric

acid

fermentation

systems

without

the

use

of

neutralizing

agents

have

been

studied

in

the

past

and

will

be

discussed

in

the

following

sections

Fig.

for

fumaric

acid

production

via

fermentation

a

Formulation

tanks

containing

glucose

and

nutrients

b

Seed

fermentor

c

Production

fermentor

d

Filter

e

Acidification

tank

f

Filter

g

Rotary

dryer

-LRB-

adapted

from

Gangl

et

al.

-RRB-

With

respect

to

fumarate

salts

inhibition

Rhodes

et

al.

-LRB-

-RRB-

reported

that

production

of

soluble

sodium

or

potassium

fumarates

was

inhibited

when

the

concentration

of

fumarate

reached

values

of

g

L

using

R.

arrhizus

as

a

producer

strain

Nonetheless

Gangl

et

al.

-LRB-

-RRB-

found

that

addition

of

sodium

fumarate

-LRB-

g

L

-RRB-

was

not

inhibiting

the

same

strain

although

the

cells

needed

h

to

adapt

to

the

high

sodium

fumarate

concentrations

On

the

other

hand

the

free

fumaric

acid

does

inhibit

its

own

production

because

the

accumulated

protons

in

the

production

medium

decrease

the

pH

thus

exerting

a

progressive

inhibitory

effect

on

fumaric

acid

production

-LRB-

Riscaldati

et

al.

-RRB-

At

low

pH

excreted

fumaric

acid

will

passively

diffuse

back

through

the

plasma

membrane

of

the

fungus

decreasing

its

intracellular

pH

and

due

to

this

phenomenon

the

fermentation

fails

Proposed

methods

to

enable

carboxylic

acid

fermentation

at

low

pH

are

genetic

engineering

of

acid

tolerant

organisms

such

as

yeasts

and

in

situ

product

removal

techniques

-LRB-

Cao

et

al.

Schügerl

van

Maris

et

al.

-RRB-

Morphology

and

oxygen-transfer

problems

One

of

the

difficulties

of

fermenting

Rhizopus

species

is

the

morphology

of

these

fungi

Rhizopus

species

tend

to

grow

on

the

walls

and

on

the

stirrer

of

the

reactor

and

sometimes

clumps

are

formed

Therefore

the

fermentation

can

suffer

from

oxygen

limitation

in

particular

when

calcium

fumarate

is

present

One

way

to

solve

this

problem

is

to

control

the

growth

of

the

fungi

and

their

morphology

A

way

to

minimize

oxygen

mass

transfer

limitation

to

the

cells

is

to

stimulate

formation

of

small

spherical

cell

pellets

-LRB-

Zhou

-RRB-

Small

pellets

can

reduce

clump

formation

during

fermentation

and

even

if

is

present

the

viscosity

of

the

broth

can

be

reduced

Moreover

pellets

can

facilitate

the

performance

of

a

biomass

retention

system

For

R.

oryzae

low

initial

pH

values

for

the

cultivation

media

favored

pellet

formation

and

good

fumaric

acid

yields

have

been

reached

-LRB-

Zhou

et

al.

-RRB-

In

another

morphology

improvement

study

Cao

et

al.

-LRB-

-RRB-

used

a

rotary

biofilm

contactor

-LRB-

RBC

-RRB-

as

fermentor

with

immobilized

R.

oryzae

to

produce

fumaric

acid

was

used

as

neutralizing

agent

during

this

experiment

During

the

fermentation

the

discs

with

immobilized

cells

were

rotating

moving

the

cells

from

the

gas

phase

of

the

fermentor

to

the

liquid

phase

and

back

again

-LRB-

Cao

et

al.

-RRB-

When

the

cells

are

exposed

to

the

air

high

oxygen

transfer

rates

can

be

reached

while

the

cells

can

take

up

substrate

and

excrete

the

produced

fumaric

acid

when

they

are

submerged

In

this

system

additional

agitation

was

not

needed

The

volumetric

productivity

was

very

high

compared

to

the

volumetric

productivity

of

an

equivalent

stirred

vessel

fermentor

setup

-LRB-

see

Table

-RRB-

A

disadvantage

of

this

system

could

be

the

scalability

potential

of

the

RBC

fermentor

Immobilization

techniques

for

Rhizopus

species

have

been

investigated

to

open

the

possibility

of

a

continuous

operation

mode

for

fumaric

acid

production

and

to

reduce

oxygen

transfer

problems

as

well

Buzzini

et

al.

-LRB-

-RRB-

searched

for

the

most

suitable

support

for

cell

immobilization

in

fluidized-bed

reactors

during

fumaric

acid

fermentation

Cork

expanded

polystyrene

expanded

clay

and

wood

shavings

were

investigated

With

mm

pieces

of

cork

the

highest

titer

g

fumaric

acid

l

in

h

was

achieved

which

is

comparable

to

the

titer

of

g

l

after

h

under

conventional

submerged

conditions

-LRB-

Buzzini

et

al.

-RRB-

The

performance

of

a

semi-continuous

process

was

investigated

with

R.

arrhizus

immobilized

on

mm

cubic

particles

made

of

polyurethane

sponge

-LRB-

Petruccioli

et

al.

-RRB-

These

particles

were

used

in

repeated

batch

fermentations

in

a

fluidized-bed

reactor

-LRB-

h

eight

times

-RRB-

In

the

optimized

process

the

fumaric

acid

titer

was

g

l

and

the

volumetric

productivity

was

g

l

h

Furthermore

as

the

oxygen

mass

transfer

resistance

through

the

boundary

layer

on

the

liquid

side

of

the

gas

liquid

interface

can

affect

the

interfacial

oxygen

transfer

from

the

gas

phase

to

the

liquid

phase

pressure

pulsation

was

applied

in

a

stirred

tank

fermentor

using

R.

oryzae

-LRB-

Zhou

-RRB-

to

reduce

this

resistance

Mass

yield

and

volumetric

productivity

of

fumarate

were

and

g

L

h

respectively

which

were

higher

than

for

traditional

stirred

tank

fermentations

The

same

organism

was

studied

in

an

airlift

loop

reactor

as

a

fermentation

system

for

fumaric

acid

production

-LRB-

Du

et

al.

-RRB-

Here

the

airlift

loop

reactor

with

porous

sparger

produced

favorable

conditions

for

mass

transfer

and

also

higher

yields

and

productivities

were

reached

than

in

stirred

tank

fermentations

Integrated

fermentation

and

recovery

of

fumaric

acid

Figure

presents

a

flow

scheme

proposed

for

fumaric

acid

production

by

batch

fermentation

-LRB-

Gangl

et

al.

-RRB-

Glucose

and

mineral

salts

are

fed

to

the

fermentor

The

nitrogen

source

solution

-LRB-

-RRB-

is

sterilized

separately

and

fed

to

the

seed

fermentor

The

harvested

broth

containing

sodium

fumarate

-LRB-

cells

and

trace

amounts

of

-RRB-

is

filtered

to

remove

the

cells

and

then

acidified

by

to

pH

After

acidification

fumaric

acid

precipitates

out

of

the

solution

and

is

sent

to

a

rotary

dryer

to

be

completely

recovered

-LRB-

Gangl

et

al.

-RRB-

When

is

used

as

a

neutralizing

agent

instead

of

additional

heating

after

the

fermentation

was

supposed

to

be

required

to

dissolve

calcium

fumarate

and

the

excess

of

that

usually

stick

to

the

cells

This

problem

leads

to

a

tedious

and

expensive

downstream

processing

In

the

downstream

processing

field

recovery

techniques

have

not

so

much

been

studied

for

fumaric

acid

producers

in

submerged

cultivations

as

it

has

been

done

for

related

fermentation

products

like

succinic

acid

-LRB-

Zeikus

et

al.

-RRB-

citric

acid

-LRB-

Heinzle

et

al.

-RRB-

and

lactic

acid

-LRB-

Joglekar

et

al.

-RRB-

Recovery

systems

like

reactive

extraction

and

membrane

electrodialysis

have

not

been

studied

yet

However

simultaneous

fermentation

and

adsorption

have

been

studied

to

remove

fumaric

acid

during

its

formation

to

control

fermentation

pH

at

desired

values

and

avoid

product

inhibition

-LRB-

Cao

et

al.

Zhou

-RRB-

Cao

et

al.

-LRB-

-RRB-

used

a

rotary

biofilm

contactor

-LRB-

RBC

-RRB-

setup

as

fermentor

for

R.

oryzae

in

combination

with

an

adsorption

column

The

produced

fumaric

acid

was

removed

from

the

broth

by

the

adsorption

column

in

a

recycle

loop

reducing

product

inhibition

and

thus

increasing

the

production

rate

and

sustaining

cell

viability

Polyvinyl

pyridine

-LRB-

PVP

-RRB-

anion

exchange

resin

in

the

hydroxide

form

was

selected

as

adsorbent

because

it

yielded

the

highest

loading

capacity

for

fumaric

acid

-LRB-

g

g

dry

wt

-RRB-

The

RBC

coupled

with

the

adsorption

column

increased

the

fumaric

acid

productivity

significantly

to

g

l

h

because

the

total

fermentation

time

was

much

less

than

in

traditional

stirred

tank

fermentations

-LRB-

see

Table

-RRB-

This

volumetric

productivity

and

the

yield

are

the

highest

reported

in

literature

In

this

integrated

system

the

constant

removal

of

the

fumarate

produced

and

the

liberation

of

OH

from

the

adsorption

column

kept

the

fermentation

pH

at

The

fumarate

was

desorbed

from

the

adsorption

resin

by

using

M

NaOH

so

additional

processing

will

be

necessary

to

convert

the

sodium

fumarate

into

neutral

fumaric

acid

Addition

of

extra

was

not

necessary

due

to

the

fact

that

during

the

production

stage

the

biofilm

was

exposed

to

sterile

air

that

was

present

in

the

headspace

of

the

RBC

enhancing

the

opportunity

for

fixation

by

the

biofilm

A

novel

product

recovery

process

by

an

ion

exchanger

resin

was

developed

by

Zhou

-LRB-

-RRB-

Fumarate

was

recovered

from

the

fermentation

broth

without

the

use

of

neutralizing

agents

while

keeping

the

pH

at

by

cycling

the

broth

over

a

column

of

a

resin

-LRB-

Amberlite

with

OH

as

counterion

-RRB-

After

eluting

the

loaded

column

with

ammonium

hydroxide

ammonium

fumarate

solution

was

obtained

This

was

passed

through

a

Y-zeolite

column

that

retained

the

ammonium

and

liberated

the

fumaric

acid

The

ammonium

hydroxide

solution

can

be

recovered

and

recycled

by

thermal

regeneration

of

the

zeolite

-LRB-

Zhou

-RRB-

However

this

integrated

process

did

not

surpass

yield

values

obtained

in

a

stirred

tank

fermentor

when

fumaric

acid

fermentation

was

controlled

by

under

pressure

pulsation

-LRB-

see

previous

section

-RRB-

but

a

higher

productivity

value

of

g

L

h

was

reached

-LRB-

Zhou

et

al.

-RRB-

Economics

of

fumaric

acid

production

In

a

theoretical

study

Gangl

et

al.

-LRB-

-RRB-

compared

a

fermentation

and

a

benzene-based

petrochemical

process

with

respect

to

economy

The

fermentation

process

taken

for

this

has

been

described

in

the

beginning

of

the

previous

section

Although

was

assumed

as

a

neutralizing

agent

in

this

study

the

productivity

-LRB-

g

l

h

-RRB-

and

yield

-LRB-

g

g

-RRB-

of

the

fumaric

acid

fermentation

with

obtained

in

a

previous

study

were

used

-LRB-

Ng

et

al.

-RRB-

This

economic

evaluation

showed

that

the

fermentation

route

was

less

favorable

than

the

petrochemical

route

-LRB-

economic

evaluation

included

upstream

and

downstream

sections

-RRB-

In

particular

the

raw

material

costs

were

higher

for

the

fermentation

process

than

for

the

petrochemical

route

It

was

concluded

that

the

fermentation

route

could

become

competitive

with

the

petrochemical

route

if

oil

prices

were

around

barrel

The

latter

price

has

been

reached

in

the

recent

years

-LRB-

-RRB-

while

the

productivity

of

the

fermentation

process

has

been

improved

significantly

as

is

shown

in

Table

Besides

one

can

roughly

calculate

from

Gangl

's

study

that

the

assumed

sugar

price

was

around

kg

of

glucose

which

is

high

compared

to

the

current

price

-LRB-

see

Table

-RRB-

Therefore

if

the

current

glucose

price

is

assumed

in

the

calculations

of

Gangl

's

study

one

can

expect

lower

raw

material

costs

for

the

fermentation

route

On

the

other

hand

the

petrochemical

route

has

also

been

improved

significantly

now

using

butane

instead

of

benzene

This

will

be

reflected

in

the

current

maleic

anhydride

price

which

is

shown

in

Table

Table

between

petrochemical

and

fermentation

route

for

fumaric

acid

productionParameterPetrochemical

routeFermentation

routeRaw

materialMaleic

anhydrideGlucoseReaction

temperature

-LRB-

°C

-RRB-

material

price

-LRB-

kg

-RRB-

-LRB-

Anonymous

-RRB-

aProduct

yield

-LRB-

w/w

-RRB-

-LRB-

Lohbeck

et

al.

-RRB-

-LRB-

Cao

et

al.

-RRB-

ahttp

for

quarter

of

The

simple

comparison

of

petrochemical

and

fermentation

routes

for

fumaric

acid

production

given

in

Table

suggests

that

the

lower

raw

material

price

of

the

fermentative

production

might

compensate

the

higher

yields

of

the

petrochemical

production

from

maleic

anhydride

and

fermentation

may

become

an

economically

viable

alternative

Conclusions

and

future

prospects

Due

to

increasing

prices

of

fossil

feedstock

fermentatively

produced

fumaric

acid

could

become

a

cheaper

alternative

to

the

petrochemically

based

maleic

acid

as

unsaturated

dibasic

acid

in

polyester

resins

in

the

nearby

future

Based

on

the

available

literature

the

microorganism

with

the

highest

productivity

and

yield

of

fumaric

acid

appears

to

be

R.

oryzae

This

organism

produces

fumaric

acid

via

a

combination

of

the

citrate

cycle

and

reductive

pyruvate

carboxylation

In

addition

the

fumaric

acid

production

by

fermentation

can

be

improved

if

the

use

of

neutralizing

agents

is

reduced

or

avoided

and

the

morphology

of

the

fungi

is

optimized

However

avoiding

the

use

of

a

neutralizing

agent

will

lead

to

product

inhibition

therefore

it

would

be

useful

if

metabolic

engineering

is

applied

to

achieve

fumaric

acid

production

in

suspended

acid-resistant

microorganisms

such

as

yeast

Another

way

to

solve

product

inhibition

problems

is

applying

in

situ

removal

of

fumaric

acid

during

the

fermentation

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O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B I O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O B O

fumaric acid
fermentation
rhizopus species
maleate isomerase
product recovery