Expression

of

the

chemokine

receptor

in

psoriasis

and

results

of

a

randomized

placebo

controlled

trial

with

a

inhibitor

Several

reports

have

indicated

that

the

chemokine

receptor

and

its

ligands

especially

-LRB-

formerly

known

as

RANTES

-RRB-

may

play

a

role

in

the

pathogenesis

of

psoriasis

The

purpose

of

this

investigation

was

to

examine

the

expression

of

and

its

ligands

in

chronic

plaque

psoriasis

and

to

evaluate

the

clinical

and

immunohistochemical

effect

of

a

receptor

inhibitor

Immunohistochemical

analysis

showed

low

but

significant

increased

total

numbers

of

positive

cells

in

epidermis

and

dermis

of

lesional

skin

in

comparison

to

non-lesional

skin

However

relative

expression

of

proportional

to

the

cells

observed

revealed

that

the

difference

between

lesional

and

non-lesional

skin

was

only

statistically

significant

in

the

epidermis

for

positive

cells

and

in

the

dermis

for

positive

cells

Quantification

of

mRNA

by

reverse

transcriptase-polymerase

chain

reaction

only

showed

an

increased

expression

of

-LRB-

RANTES

-RRB-

in

lesional

skin

A

randomized

placebo-controlled

clinical

trial

in

psoriasis

patients

revealed

no

significant

clinical

effect

and

no

changes

at

the

immunohistochemical

level

comparing

patients

treated

with

placebo

or

a

inhibitor

We

conclude

that

although

expression

is

increased

in

psoriatic

lesions

this

receptor

does

not

play

a

crucial

role

in

the

pathogenesis

of

psoriasis

Introduction

Psoriasis

is

a

chronic

skin

disease

affecting

approximately

of

the

population

worldwide

Despite

its

common

occurrence

the

exact

pathogenesis

of

psoriasis

remains

unclear

and

adaptations

to

the

pathogenesis

of

this

inflammatory

disease

are

continuing

-LSB-

-RSB-

Although

the

specific

effector

cells

responsible

for

the

inflammatory

process

in

psoriasis

are

not

known

reported

beneficial

effects

of

specific

T

cell

targeted

therapies

such

as

cyclosporine

A

and

alefacept

support

a

central

role

for

T

cells

in

the

pathogenesis

of

psoriasis

-LSB-

-RSB-

Further

investigation

on

the

immunophenotype

and

cytokine

secretion

patterns

of

T

cells

have

indicated

that

specifically

are

involved

in

psoriasis

-LSB-

-RSB-

In

the

many

aspects

encompassing

T-cell

homeostasis

the

trafficking

of

T

cells

from

blood

to

tissues

is

thought

to

be

relevant

in

chronic

inflammatory

diseases

such

as

psoriasis

Key

factors

in

this

migration

are

chemo-attractant

cytokine

molecules

known

as

chemokines

and

their

receptors

-LSB-

-RSB-

The

predominant

chemokine

receptors

expressed

on

are

and

-LSB-

-RSB-

Besides

its

preferential

expression

on

cells

is

also

expressed

on

monocytes

macrophages

natural

killer

and

dendritic

cells

all

thought

to

be

significant

elements

in

the

pathogenesis

of

psoriasis

-LSB-

-RSB-

The

ligands

of

-LSB-

and

-LRB-

formerly

known

as

and

RANTES

respectively

-RRB-

-RSB-

are

highly

expressed

by

keratinocytes

in

psoriatic

tissue

-LSB-

-RSB-

Furthermore

it

has

been

demonstrated

that

the

proinflammatory

cytokines

IFN-γ

and

TNF-α

can

induce

the

expression

of

these

chemokines

-LSB-

-RSB-

and

that

treatment

of

psoriasis

resulted

in

a

significant

decrease

of

as

well

as

a

reduction

of

T

cells

in

the

skin

-LSB-

-RSB-

Several

animal

models

resembling

psoriasis

have

been

developed

yet

of

these

models

imitates

psoriasis

completely

hence

limiting

their

utility

-LSB-

-RSB-

Investigations

by

Mack

et

al.

-LSB-

-RSB-

showed

a

different

expression

pattern

of

in

mice

and

humans

Additionally

different

expression

of

a

single

amino

acid

in

the

molecule

between

rhesus

macaques

and

humans

resulted

in

a

different

response

to

inhibitors

of

the

receptor

in

the

species

-LSB-

-RSB-

Therefore

research

on

the

expression

of

in

psoriasis

as

well

as

clinical

efficacy

of

a

inhibitor

is

limited

to

humans

So

far

the

data

available

on

the

expression

of

in

psoriatic

skin

in

humans

are

not

univocal

-LRB-

varying

from

high

to

minimal

-RRB-

and

were

obtained

with

divergent

methods

in

investigations

in

which

was

never

the

main

focus

-LSB-

-RSB-

The

primary

purpose

of

this

study

was

to

determine

the

expression

of

and

its

ligands

in

chronic

plaque

psoriasis

in

situ

compared

to

non-lesional

skin

through

analysis

by

immunohistochemistry

and

quantitative

reverse

transcriptase-polymerase

chain

reaction

-LRB-

RT-PCR

-RRB-

In

order

to

examine

the

possibility

that

plays

a

functional

role

in

the

pathogenesis

of

psoriasis

we

also

analyzed

clinical

and

immunohistochemical

data

obtained

from

lesional

and

non-lesional

skin

biopsies

of

psoriasis

patients

before

and

after

treatment

with

a

inhibitor

Materials

and

methods

Study

design

and

patients

Lesional

and

non-lesional

skin

biopsies

were

obtained

from

nine

patients

with

moderate

to

severe

chronic

plaque

psoriasis

defined

by

the

psoriasis

area

severity

index

-LRB-

PASI

-RRB-

These

skin

biopsies

were

analyzed

by

manual

quantification

of

immunohistochemical

double-staining

and

quantitative

RT-PCR

In

order

to

get

insight

in

the

possibility

of

a

functional

role

of

in

the

pathogenesis

of

psoriasis

patients

including

the

previous

patients

participated

in

an

week

randomized

placebo-controlled

parallel

group

multi-centre

double-blind

clinical

trial

in

which

patients

received

either

mg

twice

daily

of

the

inhibitor

-LRB-

patients

-RRB-

or

matched

placebo

-LRB-

patients

-RRB-

orally

for

days

followed

by

a

follow-up

period

of

weeks

During

the

follow-up

period

patients

were

only

allowed

to

use

emollients

as

treatment

and

on

day

vital

signs

PASI

and

blood

were

assessed

Patients

were

included

at

the

dermatology

outpatient

departments

of

four

academic

hospitals

from

April

to

December

At

baseline

and

the

last

day

of

treatment

-LRB-

day

-RRB-

lesional

biopsies

were

taken

to

evaluate

the

immunohistochemical

effect

of

the

inhibitor

For

this

immunohistochemical

evaluation

single-stained

sections

were

analyzed

with

digital

image

analysis

semi-quantitative

analysis

-LRB-

SQA

-RRB-

and

confocal

scanning

microscopy

and

double-stained

sections

on

baseline

and

day

were

analyzed

by

manual

quantification

To

evaluate

the

clinical

effect

of

the

inhibitor

the

PASI

was

assessed

at

baseline

day

and

day

In

all

patients

psoriasis

was

diagnosed

at

least

months

prior

to

enrolment

and

patients

were

not

allowed

to

use

systemic

psoriasis

treatment

or

phototherapy

within

weeks

of

study

entry

Only

emollients

were

allowed

as

topical

treatment

All

other

topical

anti-psoriasis

therapy

-LRB-

e.g.

corticosteroids

vitamin

D

derivates

etc.

-RRB-

had

to

be

stopped

weeks

before

study

entry

The

protocol

was

reviewed

and

approved

by

the

medical

ethical

committees

of

all

participating

centres

and

all

patients

gave

their

written

informed

consent

before

enrolment

The

study

was

conducted

according

to

the

Declaration

of

Helsinki

principles

and

is

registered

at

the

ISRCT

register

-LRB-

-RRB-

Biopsies

Four-millimeter

biopsies

were

taken

from

the

inside

border

of

a

target

psoriatic

plaque

preferentially

from

a

non-sun-exposed

area

Lesional

biopsies

from

each

patient

were

obtained

from

the

same

target

lesion

separated

by

at

least

cm

The

biopsy

samples

were

randomly

coded

snap-frozen

in

Tissue-Tek

OCT

compound

-LRB-

Sakura

Finetek

Europe

Zoeterwoude

The

Netherlands

-RRB-

by

immersion

in

liquid

nitrogen

and

stored

at

°C

until

processing

Five-micrometer

cryostat

sections

were

cut

and

mounted

on

glass

slides

-LRB-

Star

Frost

adhesive

slides

Knittelgläser

Braunschweig

Germany

-RRB-

before

being

stored

at

°C

until

immunohistochemical

staining

For

each

staining

three

sections

of

each

biopsy

were

analyzed

to

minimize

random

variation

Immunohistochemistry

After

fixation

of

the

slides

the

endogenous

peroxide

activity

was

inhibited

with

sodium

azide

and

hydrogen

peroxide

in

Tris-buffered

saline

-LRB-

TBS

-RRB-

before

incubating

for

min

with

normal

goat

serum

in

TBS

Next

the

sections

were

incubated

for

h

at

room

temperature

-LRB-

overnight

at

°C

in

the

case

of

-RRB-

with

the

primary

antibody

in

bovine

serum

albumin

-LRB-

BSA

Sigma-Chemical

Co

St.

Louis

MO

USA

-RRB-

in

TBS

The

following

mouse

anti-human

monoclonal

antibodies

were

used

FITC-conjugated

-LRB-

BD

Pharmingen

San

Jose

CA

USA

-RRB-

FITC-conjugated-anti

-LRB-

clone

Dako

-RRB-

anti-human

neutrophil

elastase

-LRB-

Dako

Glostrup

Denmark

-RRB-

anti-cytokeratin

-LRB-

keratin

Sigma

Saint

Louis

MO

USA

-RRB-

-LRB-

NK-T

cells

BD

Pharmingen

-RRB-

and

-LRB-

BD

Pharmingen

-RRB-

After

rinsing

with

TBS

sections

were

further

incubated

with

biotin-conjugated

goat

anti-mouse

antibody

-LRB-

Dako

-RRB-

or

in

case

of

and

staining

with

rabbit

anti-FITC

-LRB-

Dako

-RRB-

in

normal

human

serum

-LRB-

NHS

-RRB-

in

TBS

for

min

Following

a

wash

step

with

TBS

sections

were

subsequently

incubated

with

horseradish

peroxidase

-LRB-

HPR

-RRB-

conjugated

streptavidin

-LRB-

Dako

-RRB-

or

in

case

of

staining

with

HRP-conjugated

goat

anti-rabbit

antibody

-LRB-

Dako

-RRB-

in

BSA

in

TBS

for

min

In

case

of

staining

an

amplification

step

was

performed

with

the

TSA

biotin

system

-LRB-

Perkin

Elmer

Boston

MA

USA

-RRB-

Sections

were

counterstained

with

Mayer

's

haematoxylin

-LRB-

Merck

Darmstadt

Germany

-RRB-

and

mounted

in

Kaiser

's

glycerol

gelatine

-LRB-

Merck

-RRB-

Skin

sections

were

double

stained

with

together

with

or

macrophage

marker

The

double

stained

sections

were

manually

counted

by

two

independent

observers

blinded

for

order

patient

and

clinical

data

Using

a

mm

ocular

grid

and

at

magnification

single

red

-LRB-

-RRB-

single

blue

-LRB-

or

-RRB-

and

purple

double-positive

cells

were

counted

in

the

entire

section

The

epidermal

and

dermal

regions

were

separately

counted

The

results

are

expressed

as

the

number

of

double-positive

RNA

analysis

RNA

was

extracted

from

frozen

skin

biopsies

using

the

RNeasy

mini

kit

-LRB-

Qiagen

-RRB-

RNA

quantity

was

assessed

by

OD

at

nm

and

RNA

quality

was

analyzed

by

measuring

the

ratio

of

and

rRNA

using

the

Agilent

bioanalyzer

Quantitative

PCR

Taqman

primers

and

probes

were

designed

with

Primer

Express

software

-LRB-

ABI

-RRB-

and

purchased

from

ABI

The

sequences

of

the

human

primers

and

probes

are

available

upon

request

For

the

human

skin

tissue

quantitative

PCR

was

carried

out

with

an

ABI

Prism

Sequence

Detection

System

-LRB-

Applied

Biosystems

Foster

City

CA

USA

-RRB-

The

PCR

reactions

were

prepared

using

the

components

from

the

Invitrogen

Platinum

Quantitative

RT-PCR

One-Step

kit

and

assembled

according

to

the

manufacturer

's

instructions

-LRB-

Invitrogen

Carlsbad

CA

USA

-RRB-

The

final

concentrations

of

the

primers

and

probe

in

the

PCR

reactions

were

and

nM

respectively

The

RT-PCR

reactions

for

each

gene

were

performed

in

a

single

plate

Separate

plates

of

the

same

RNAs

were

used

to

quantitate

RNA

as

an

internal

control

for

RNA

quality

and

a

primer/probe

set

for

the

promotor

was

used

to

check

the

RNAs

for

genomic

contamination

The

PCR

data

was

quantitated

based

on

a

standard

curve

generated

using

fourfold

serial

dilutions

of

the

target

genes

The

fourfold

dilutions

began

at

ng

and

eight

dilutions

were

used

to

generate

the

standard

curve

This

procedure

provides

an

absolute

quantitation

of

the

amount

of

mRNA

in

a

given

tissue

Data

were

analyzed

using

Sequence

Detection

Systems

software

version

-LRB-

Applied

Biosystems

Foster

City

CA

USA

-RRB-

Digital

image

analysis

Single

stained

sections

were

randomly

coded

and

analyzed

by

computer-assisted

image

analysis

as

described

previously

in

detail

-LSB-

-RSB-

In

short

images

were

acquired

and

analyzed

using

Syndia

algorithm

on

a

Qwin

based

analysis

system

-LRB-

Leica

Cambridge

UK

-RRB-

Twenty

high

power

fields

per

section

were

analyzed

Positive

staining

of

cellular

markers

was

expressed

as

positive

Semi-quantitative

analysis

For

keratinocyte

expression

of

keratin

a

semi-quantitative

score

was

done

by

two

independent

observers

blinded

for

order

patient

and

clinical

data

with

a

standard

binocular

light

microscope

-LRB-

Olympus

-RRB-

at

magnification

The

semi-quantitative

score

ranged

from

to

A

score

of

represented

no

expression

while

a

score

of

represented

abundant

expression

in

all

layers

of

the

epidermis

Confocal

scanning

microscope

Cryosections

of

μm

on

silanized

slides

were

fixed

with

acetone

and

dried

at

room

temperature

and

stored

at

°C

Sections

were

incubated

in

BSA

for

min

and

washed

with

PBS

before

all

steps

Next

monoclonal

anti-human

antibody

-LRB-

R&D

Systems

clone

-RRB-

Texas

red

immunoglobulins

diluted

in

BSA

-LRB-

-RRB-

was

applied

followed

by

application

of

the

primary

monoclonal

specific

antibodies

-LRB-

clone

BD

Biosciences

-RRB-

-LRB-

clone

BD

Biosciences

-RRB-

-LRB-

clone

DakoCytomation

-RRB-

or

-LRB-

clone

DakoCytomation

-RRB-

Next

FITC-conjugated

affinipure

rabbit

anti-mouse

IgG

-LRB-

Jackson

A

E

-RRB-

diluted

in

BSA

-LRB-

-RRB-

was

applied

and

sections

were

mounted

in

fluorescent

mounting

medium

-LRB-

DakoCytomation

-RRB-

Of

each

double

stained

slide

three

pictures

were

taken

with

an

image

definition

of

pixels

at

a

magnification

of

Sample

size

calculation

The

randomized

placebo

controlled

clinical

trial

was

targeted

to

randomize

a

total

of

subjects

-LRB-

on

active

treatment

and

on

placebo

-RRB-

With

this

sample

size

the

trials

would

be

able

to

detect

a

difference

of

in

the

response

rate

from

the

placebo

group

assuming

a

response

rate

in

the

placebo

group

with

power

at

an

alpha

level

of

-LRB-

two-sided

test

-RRB-

Randomization

Randomization

was

stratified

by

sites

Each

site

was

assigned

a

fixed

number

of

subjects

numbers

e.g.

site

would

get

numbers

and

so

on

Once

the

physician

of

the

study

site

would

enroll

a

subject

the

subject

would

be

assigned

the

next

available

subject

number

assigned

to

the

site

starting

with

the

bottom

of

the

list

e.g.

the

first

subject

enrolled

in

site

would

get

number

the

second

subject

would

get

number

and

so

on

Treatments

would

be

assigned

in

an

active

to

placebo

ratio

of

according

to

a

computer

generated

randomization

schedule

No

stratification

based

on

age

sex

or

other

characteristics

was

performed

Throughout

the

study

both

patient

and

treating

physician

were

blinded

to

the

group

assignment

Statistical

analysis

We

used

SPSS

for

Windows

-LRB-

SPSS

Chicago

IL

USA

-RRB-

for

the

statistical

analysis

of

clinical

data

The

Mann

Whitney

test

was

used

to

compare

lesional

and

non-lesional

skin

at

baseline

and

to

compare

skin

biopsies

before

and

after

treatment

All

statistical

tests

were

two-sided

P

values

less

than

were

considered

significant

The

results

are

expressed

as

median

standard

error

of

the

mean

To

evaluate

the

clinical

effect

of

treatment

with

a

inhibitor

an

intention

to

treat

analysis

was

performed

Quantitative

PCR

data

analysis

was

performed

by

two-sided

t

test

as

implemented

by

Graphpad

Prizm

-LRB-

version

Graphpad

Software

San

Diego

CA

USA

-RRB-

A

P

value

less

than

was

considered

significant

Results

Comparison

of

expression

in

lesional

versus

non-lesional

psoriatic

skin

The

expression

of

in

T

cells

-LRB-

-RRB-

and

macrophages

-LRB-

-RRB-

in

lesional

and

non-lesional

skin

of

nine

psoriasis

patients

was

compared

at

baseline

We

observed

a

clear

expression

of

which

was

primarily

present

in

the

dermis

In

absolute

numbers

approximately

half

of

the

T

cells

and

half

of

the

macrophages

co-expressed

-LRB-

Fig.

-RRB-

The

and

double

positive

cells

showed

a

low

but

statistically

significant

increased

expression

of

in

epidermis

and

dermis

of

lesional

skin

in

comparison

to

non-lesional

skin

as

shown

in

Fig.

Focussing

on

the

expression

of

as

the

percentage

of

all

T

cells

or

macrophages

present

in

the

sections

the

difference

between

lesional

and

non-lesional

skin

was

only

statistically

significant

in

the

epidermis

for

cells

-LRB-

P

-RRB-

and

in

the

dermis

for

cells

-LRB-

P

-RRB-

Fig.

analysis

of

in

lesional

versus

non-lesional

skin

Data

are

shown

as

mean

SD

ns

non-significant

P

P

P

Quantitative

RT-PCR

analysis

indicated

no

increased

expression

of

mRNA

for

and

-LRB-

-RRB-

in

lesional

skin

-LRB-

Fig.

-RRB-

only

the

expression

of

-LRB-

RANTES

-RRB-

and

was

significantly

increased

in

lesional

skin

-LRB-

P

and

P

-RRB-

The

well-known

enhanced

expression

of

in

psoriatic

skin

was

included

as

positive

control

Fig.

analysis

of

in

lesional

psoriatic

skin

in

comparison

with

non-lesional

skin

is

used

as

a

control

marker

P

P

Lack

of

clinical

efficacy

of

a

inhibitor

In

total

patients

were

randomized

as

shown

in

Fig.

The

demographical

data

of

the

patients

are

shown

in

Table

With

regards

to

co-morbidities

one

patient

had

hypercholesterolemia

and

one

had

obesity

in

the

placebo

group

In

the

group

one

patient

also

had

obesity

and

two

had

a

history

of

hypertension

After

treatment

with

the

inhibitor

there

was

no

change

in

mean

PASI

in

the

group

-LRB-

n

-RRB-

-LSB-

at

baseline

at

day

-LRB-

Fig.

-RRB-

-RSB-

Three

of

the

patients

treated

with

-LRB-

-RRB-

attained

an

improvement

of

or

more

compared

to

baseline

-LRB-

PASI

responders

-RRB-

showing

improvements

of

and

In

the

placebo

group

-LRB-

n

-RRB-

the

mean

PASI

slightly

decreased

-LRB-

at

baseline

at

day

-RRB-

of

the

patients

treated

with

placebo

showed

an

improvement

of

more

than

All

changes

observed

were

not

statistically

significant

In

the

follow-up

period

no

changes

in

mean

PASI

were

seen

in

either

treatment

groups

Fig.

chart

of

randomized

placebo

controlled

clinical

trial

ligand

inhibitor

SAE

serious

adverse

event

AE

adverse

eventTable

data

patientsRandomized

clinical

Age

-LRB-

years

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Duration

of

skin

disease

-LRB-

years

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Baseline

-LRB-

-RRB-

-LRB-

-RRB-

aMean

-LRB-

SD

-RRB-

ligand

inhibitor

PASI

psoriasis

area

and

severity

index

BSA

body

surface

areaFig

and

immunohistochemical

respons

after

treatment

with

a

receptor

inhibitor

In

a

randomized

placebo

controlled

clinical

trial

psoriasis

patients

were

randomized

for

treatment

with

a

receptor

inhibitor

-LRB-

-RRB-

or

placebo

for

days

Clinical

efficacy

was

measured

by

psoriasis

area

and

severity

index

-LRB-

PASI

-RRB-

-LRB-

a

-RRB-

Lesional

skin

biopsies

were

taken

from

all

patients

at

baseline

and

day

to

evaluate

immunohistochemical

effect

Immunohistochemical

single

staining

of

of

a

patient

treated

with

at

baseline

-LRB-

b

-RRB-

and

day

-LRB-

c

-RRB-

Immunohistochemical

markers

in

relation

to

clinical

response

-LRB-

d

-RRB-

PASI

non-responder

improvement

of

PASI

of

less

than

PASI

responder

improvement

of

PASI

of

or

more

percent

data

are

shown

as

mean

SD

ns

non-significant

In

the

treatment

group

four

patients

discontinued

One

patient

developed

an

erythrodermic

eruption

after

days

of

treatment

which

was

considered

by

the

site

physician

as

a

serious

adverse

event

-LRB-

SAE

-RRB-

Two

patients

discontinued

due

to

adverse

events

-LRB-

AEs

-RRB-

one

developed

shingles

in

the

n.trigeminus

area

of

the

right

side

of

his

face

after

days

of

treatment

and

one

patient

discontinued

due

to

hair

loss

after

days

of

treatment

One

patient

discontinued

due

to

non-compliance

In

the

placebo

group

two

patients

discontinued

due

to

AEs

both

exacerbation

of

their

psoriasis

after

weeks

of

treatment

expression

before

and

after

treatment

with

Immunohistochemical

analysis

of

lesional

tissue

samples

from

the

group

and

the

placebo

group

revealed

no

statistically

different

expression

of

between

baseline

and

day

in

both

treatment

groups

as

illustrated

by

Fig.

c

When

focusing

on

the

markers

elastase

and

in

relation

to

the

clinical

response

no

statistically

significant

difference

after

days

of

treatment

with

either

or

placebo

was

found

-LRB-

Fig.

-RRB-

except

for

elastase

and

dermal

cells

which

were

statistically

significantly

lowered

in

the

three

PASI

responders

treated

with

Additional

data

obtained

by

confocal

scanning

microscopy

corresponded

with

the

digital

image

and

SQA

-LRB-

data

not

shown

-RRB-

Discussion

The

primary

objective

of

this

study

was

to

explore

the

possibility

of

involvement

of

in

the

pathogenesis

of

chronic

plaque

psoriasis

Therefore

we

determined

the

expression

of

in

situ

at

the

protein

and

mRNA

level

by

immunohistochemical

analysis

and

quantitative

RT-PCR

respectively

The

total

number

of

single

positive

-LRB-

-RRB-

and

double

positive

-LRB-

and

-RRB-

cells

in

lesional

psoriatic

skin

significantly

outnumbered

those

in

non-lesional

skin

However

when

expressed

as

percentage

of

or

cells

the

difference

between

lesional

versus

non-lesional

expression

was

less

clear

With

the

latter

approach

we

found

that

the

proportion

of

expression

was

significantly

higher

in

the

epidermal

cells

and

dermal

cells

only

when

comparing

lesional

skin

to

non-lesional

skin

Analysis

of

mRNA

expression

demonstrated

a

slight

though

not

significant

increased

expression

of

in

lesional

psoriatic

skin

perhaps

due

to

the

small

numbers

of

patients

In

line

with

earlier

observations

we

found

that

the

mRNA

expression

for

RANTES

-LSB-

-RSB-

and

-LSB-

-RSB-

was

significantly

higher

expressed

in

the

lesional

samples

However

in

contrast

to

previous

research

-LSB-

-RSB-

the

expression

of

was

not

increased

in

lesional

psoriatic

skin

In

summary

our

results

do

not

provide

a

clear

answer

to

our

objective

of

determining

whether

the

percentage

of

expressing

cells

is

similar

in

lesional

and

non-lesional

skin

or

if

this

percentage

is

increased

in

lesional

skin

To

assess

any

possible

functional

participation

of

in

the

development

or

maintenance

of

psoriatic

plaques

we

investigated

the

clinical

response

to

treatment

with

a

inhibitor

as

well

as

the

effect

of

this

drug

on

the

inflamed

skin

in

situ

The

randomized

placebo-controlled

clinical

trial

revealed

no

significant

clinical

effect

and

changes

at

the

immunohistochemical

level

between

patients

treated

with

placebo

or

the

inhibitor

The

specific

type

and

dose

of

the

inhibitor

used

in

this

clinical

trial

has

proven

its

efficacy

previously

in

vitro

in

vivo

and

clinical

studies

with

other

diseases

in

which

is

known

to

play

a

pivotal

or

significant

role

-LSB-

-RSB-

However

only

three

patients

treated

with

the

inhibitor

demonstrated

a

clinical

improvement

of

or

more

It

can

not

be

excluded

that

this

low

and

not

statistically

significant

number

of

patients

is

due

to

a

spontaneous

improvement

reflecting

the

unpredictability

of

psoriasis

Surprisingly

the

immunohistochemical

markers

analyzed

in

the

skin

biopsies

of

the

so-called

PASI

responders

did

only

partially

correspond

with

the

clinical

response

Of

all

immunohistochemical

markers

only

elastase

and

dermal

showed

a

significant

decline

after

days

of

treatment

Notably

the

expression

of

a

marker

known

to

correspond

well

with

the

clinical

severity

as

measured

by

PASI

was

increased

after

treatment

with

in

two

out

of

the

three

responding

patients

-LRB-

Fig.

-RRB-

In

addition

their

baseline

expression

of

was

lower

in

comparison

with

the

other

patients

while

their

PASI

was

similar

This

inconsistency

could

be

due

to

the

low

number

of

patients

or

suggests

an

individual

difference

in

kinetics

The

increased

expression

of

observed

by

immunohistochemistry

and

the

increased

mRNA

expression

of

RANTES

in

lesional

psoriatic

skin

may

suggest

an

involvement

of

this

receptor

and

ligand

in

psoriasis

However

our

clinical

trial

with

an

effective

inhibitor

unequivocally

demonstrated

that

this

is

not

the

case

Previous

research

has

shown

increased

expression

of

several

chemokines

and

chemokine

receptors

in

psoriasis

-LSB-

-RSB-

indicating

that

multiple

receptors

may

participate

in

regulating

T

cell

recruitment

to

the

inflamed

skin

Furthermore

RANTES

is

known

to

also

bind

with

and

whereas

is

solely

connected

to

Given

this

complexity

of

interactions

between

chemokines

and

chemokine

receptors

it

is

not

unlikely

that

blocking

a

single

chemokine

receptor

-LRB-

i.e.

-RRB-

would

have

been

insufficient

in

diminishing

the

inflammatory

process

According

to

Homey

-LSB-

-RSB-

chemokine

antagonistic

approaches

to

impede

with

the

inflammatory

process

may

perhaps

be

preventive

rather

than

therapeutic

Chemokines

and

their

receptors

play

an

essential

role

in

the

trafficking

of

T

cells

to

all

kinds

of

tissue

including

the

skin

Yet

once

leucocytes

have

entered

the

target

organ

and

underwent

activation

processes

impairment

of

recruitment

of

pathogenic

T

cells

is

likely

to

be

less

effective

in

reducing

the

clinical

symptoms

When

combined

with

a

successful

eradicative

treatment

of

the

T

cells

chemokine

antagonists

could

perhaps

be

promising

candidates

for

prevention

of

acute

flares

prolongation

of

lesion-free

interval

and

therefore

provide

optimized

long-term

management

of

patients

suffering

from

chronically

relapsing

inflammatory

skin

disease

such

as

psoriasis

So

although

does

not

seem

to

be

a

key

chemokine

receptor

in

the

pathogenesis

of

psoriasis

further

efforts

are

needed

to

unravel

the

complete

set

of

chemokines

and

chemokine

receptors

significant

in

the

recruitment

of

inflammatory

cells

in

psoriasis

and

may

help

to

identify

crucial

molecules

as

demonstrated

previously

by

TNF

in

various

immune

mediated

inflammatory

diseases

O O O O O B O B O O O O O O O O O O B 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 O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O B 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 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 B 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 B 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 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 O O O O O O O O O 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 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 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 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 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 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 B 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 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 B 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 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 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 O O O O O 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 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 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 B 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 B 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B 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 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 B O O O O O O O O B O O O O B 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B 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 B O O O O O O O B 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 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 O O O O O O O O O O O O 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 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 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 B O O O O O O B 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 B O O O O O O B 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 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 B 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 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 B 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 B 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 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 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 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 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 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 O O O O O O O O O O O O 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Competition and coexistence of sulfate-reducing bacteria , acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio The microbial population structure and function of natural anaerobic communities maintained in lab-scale continuously stirred tank reactors at different lactate to sulfate ratios and in the absence of sulfate were analyzed using an integrated approach of molecular techniques and chemical analysis . The population structure , determined by denaturing gradient gel electrophoresis and by the use of oligonucleotide probes , was linked to the functional changes in the reactors . At the influent lactate to sulfate molar ratio of 0.35 mol mol − 1 , i.e. , electron donor limitation , lactate oxidation was mainly carried out by incompletely oxidizing sulfate-reducing bacteria , which formed 80 -- 85 % of the total bacterial population . Desulfomicrobium - and Desulfovibrio-like species were the most abundant sulfate-reducing bacteria . Acetogens and methanogenic Archaea were mostly outcompeted , although less than 2 % of an acetogenic population could still be observed at this limiting concentration of lactate . In the near absence of sulfate -LRB- i.e. , at very high lactate/sulfate ratio -RRB- , acetogens and methanogenic Archaea were the dominant microbial communities . Acetogenic bacteria represented by Dendrosporobacter quercicolus-like species formed more than 70 % of the population , while methanogenic bacteria related to uncultured Archaea comprising about 10 -- 15 % of the microbial community . At an influent lactate to sulfate molar ratio of 2 mol mol − 1 , i.e. , under sulfate-limiting conditions , a different metabolic route was followed by the mixed anaerobic community . Apparently , lactate was fermented to acetate and propionate , while the majority of sulfidogenesis and methanogenesis were dependent on these fermentation products . This was consistent with the presence of significant levels -LRB- 40 -- 45 % of total bacteria -RRB- of D. quercicolus-like heteroacetogens and a corresponding increase of propionate-oxidizing Desulfobulbus-like sulfate-reducing bacteria -LRB- 20 % of the total bacteria -RRB- . Methanogenic Archaea accounted for 10 % of the total microbial community . Introduction The anaerobic digestion of organic material is accomplished by the concerted action of various trophic groups of bacteria -LRB- Schink et al. 1992 -RRB- . The soluble organic compounds are degraded to CH4 , CO2 , and H2S via a syntropic interaction of fermentative and acetogenic bacteria with methanogens or sulfate-reducing bacteria -LRB- SRB -RRB- . The electrons for CO2 reduction to methane or acetate , or sulfate reduction to sulfide are derived from the fermentative metabolism . The fermentative metabolism may include homoacetogenic or heteroacetogenic microorganisms depending upon whether acetic acid is the only major metabolic product -LRB- Drake 1994 ; Thauer et al. 1977 -RRB- or other organic acids , such as propionic acid , in addition to acetic acid are produced -LRB- Seeliger et al. 2002 -RRB- . The products of fermentative acetogenesis and CO2-dependent homoacetogenesis can serve as substrates for acetate - and propionate-oxidizing SRB or acetoclastic methanogens depending upon the presence or absence of sulfate as electron acceptor . SRB are believed to outcompete methanogens in the presence of nonlimiting sulfate concentrations because they compete better for common substrates . This is explained on the basis of their kinetic properties -LRB- Ks and μmax -RRB- and given the favourable thermodynamic conditions -LRB- Ward and Winfrey 1985 ; Widdel 1988 -RRB- . In contrast , methanogens tend to dominate in low-sulfate environments -LRB- Stams 1994 -RRB- . In most anaerobic environments , hydrogen is present as an intermediate for which SRB , hydrogenotrophic methanogens and homoacetogens will compete . Under standard conditions , sulfate reduction and methanogenesis are thermodynamically more favourable than homoacetogenesis . However , due to the interspecies transfer of fermentation products , such as hydrogen , the real thermodynamic values may be far from standard conditions and even less to reverse reactions . Studies with the sludge from the bioreactors have indicated that hydrogen is mainly consumed by SRB when H2 is limiting and sufficient sulfate is present -LRB- Isa et al. 1986 -RRB- . However , when H2/CO2 was added as the sole substrate , a coexistence of heterotrophic SRB and homoacetogens was reported by Weijma et al. -LRB- 2002 -RRB- , suggesting that when SRB are dependent for growth on homoacetogens , it is likely that the growth kinetics of homoacetogens determines the competition -LRB- Stams et al. 2005 -RRB- . The composition of the influent chemical oxygen demand -LRB- COD ; electron donor -RRB- to ratio tends to have a significant effect on the competition for electron flow between different microbial communities -LRB- McCartney and Oleszkiewics 1993 -RRB- . The theoretical mass ratio needed to achieve complete removal of organic matter is 0.67 , assuming that eight electrons are transferred per molecule of sulfate -LRB- Lens et al. 1998 -RRB- . In general , low and high mass ratios should favor sulfidogenesis and methanogenesis , respectively . However , values reported in the literature vary considerably -LRB- Annachhatre and Suktrakoolvait 2001 ; Choi and Rim 1991 ; Mizuno et al. 1994 -RRB- . Although the effect on the major processes like sulfidogenesis and methanogenesis on the whole has been well studied -LRB- McCartney and Oleszkiewicz 1993 -RRB- , our understanding of its influence on the diversity and dynamics of the microbial communities is still limited . This is partially due to the limitations of the traditional microbiological techniques , including pure culture isolations , most probable number estimation , selective enrichments , etc. . Fortunately , molecular techniques , like denaturing gradient gel electrophoresis -LRB- DGGE ; Muyzer et al. 1993 -RRB- and fluorescence in situ hybridization -LRB- FISH ; Amann et al. 1995 -RRB- , have provided alternative approaches to overcome the limitations associated with culture-based analyses . DGGE profiling of 16S rRNA gene fragments amplified from both genomic DNA and reverse-transcribed RNA , i.e. , cDNA not only determines the presence and distribution but also the actively growing members in the mixed microbial communities -LRB- Dar et al. 2007a -RRB- . FISH , on the other hand , allows for the identification and quantification at the level of populations and even single cells -LRB- Amann et al. 1995 -RRB- . In the present study , the structure and function of anaerobic communities maintained in lab-scale continuously stirred tank reactors -LRB- CSTR -RRB- under sulfidogenic and nonsulfidogenic conditions was investigated . An integrated approach of molecular techniques including DGGE and FISH together with chemical analysis was used to link the microbial population dynamics with changes in the lactate -LRB- electron donor -RRB- concentration and sulfate availability . The focus of our study was the competition and interaction between the dominant microbial communities of SRB , acetogens and methane-producing Archaea . Materials and methods Reactor operation The schematic representation of the experimental design is given in Fig. 1 . The start-up reactor -LRB- R-0 -RRB- was inoculated with sludge from a methanogenic wastewater treatment plant treating lactate-containing wastewater . The reactor was operated in a continuous mode for about five to six volume changes at a dilution rate of 0.02 h − 1 with lactate as the only electron donor and maintained under excess of sulfate . The lactate -LRB- 12 electron -RRB- to sulfate -LRB- 8 electron -RRB- molar ratio -LRB- r -RRB- was 0.65 mol mol − 1 . The culture in the reactor R-0 was used as an inoculum for the reactors , R1 and R2 . The reactors R1 and R2 were operated at 30 °C with a working volume of 2 l and at two different concentrations of lactate , 3.5 and 20 mM , respectively . Sulfate was fed to the reactors at a concentration of 10.32 mM , i.e. , at ratios far below and far above the theoretical ratio of 0.67 . Nonsulfidogenic conditions were applied to reactor R2 by further reducing influent sulfate feed and reducing the lactate concentration to 6.7 mM -LRB- r = 20.9 -RRB- . The composition of influent mineral medium was as follows -LRB- concentration in mM -RRB- : KH2PO4 , 3.6 ; Na2SO4 , 10 or 0 ; NH4Cl , 6 ; MgSO4 · 7H2O , 0.3 ; CaCl2 · 2H2O , 0.4 ; FeSO4 · 7H2O , 0.02 ; a stock trace element solution , 1 ml/l ; a stock selenite -- tungstate solution , 1 ml/l ; and a stock vitamin solution , 1 ml/l . The stock trace element solution contained -LRB- mg/l -RRB- : H3BO3 , 30 ; MnCl2 · 4H2O , 100 ; CoCl2 · 6H2O , 190 ; NiCl2 · 6H2O , 24 ; CuCl2 · 2H2O , 2 ; ZnCl2 , 140 ; Na2MoO4 · 2H2O , 36 . The stock selenite -- tungstate solution contained -LRB- mg/l -RRB- : Na2SeO3 · 5H2O , 6 and Na2WO4 · 2H2O , 8 . The stock vitamin solution was composed of -LRB- mg/l -RRB- : biotin , 10 ; vitamin B12 , p-aminobenzoic acid , and calcium D -LRB- + -RRB- pantothenate , 50 ; thiamine , pyridoxine -- HCl , and nicotinic acid , 100 . The medium was autoclaved , and the dissolved oxygen removed from the hot media by sparging it with oxygen-free nitrogen . The medium was maintained in an oxygen-free atmosphere by continuously flushing it with nitrogen . The pH in the reactors was maintained at 7.5 by automatic titration with 1 M NaOH and 1 M HCl . An automatic level controller maintained the working volume of 2 l inside the reactor vessels . The cultures were continuously sparged with oxygen-free nitrogen to flush the gaseous H2S . Sulfide precipitated as ZnS after letting the gaseous H2S through a zinc acetate trap connected to the gas exhaust of the reactor . Cultures were run at D = 0.02 h − 1 for at least five to six volume changes -LRB- one volume change is 50 h -RRB- and until chemical analysis of effluent showed constancy over at least two consecutive days . Fig. 1Schematic representation of the experiment . Reactor R-0 was the start-up reactor . The culture in R-0 was used as an inoculum for the reactors R1 and R2a . The cultures were run at a dilution rate of 0.02 h − 1 for five to six volume changes with each volume change of 50 h Chemical analysis Acetate and other volatile fatty acids were analyzed either by gas chromatography -LRB- GC -RRB- or by high-performance liquid chromatography -LRB- HPLC -RRB- . Sulfate was analyzed by ion chromatography as described previously -LRB- Scholten and Stams 1995 -RRB- . Sulfide was measured quantitatively by a colorimetric assay -LRB- Cline 1969 -RRB- . As sulfide was stripped from the culture liquid with nitrogen to reduce sulfide toxicity , the sulfide produced was calculated from the sulfate consumed . Hence , the given sulfide values indicate minimum values . Nucleic acid extraction , reverse transcription of RNA , and PCR amplification Nucleic acid extraction was carried out as described previously -LRB- Dar et al. 2007b -RRB- . One microliter -LRB- 80 -- 100 ng -RRB- of RNA template was used to carry out the reverse transcription -LRB- RT -RRB- of the isolated RNA into cDNA . Reverse transcription was performed using the iScript cDNA Synthesis Kit -LRB- BioRad , California , USA -RRB- according to the manufacturer 's protocol . Amplification of the 16S rRNA gene fragment was performed using the primer pair 341F-GC and 907R and the protocol as described previously -LRB- Muyzer et al. 1995 -RRB- . Nested amplification with group-specific primers for SRB was also performed as described previously -LRB- Dar et al. 2005 -RRB- . We used 1 μl of genomic DNA and 2 μl of cDNA as template for the amplification reactions . The quality of the PCR products was examined on 1 % -LRB- w/v -RRB- agarose gel , and the yield was quantified by absorption spectrophotometry using the Nanodrop ND-1000 TM -LRB- NanoDrop Technologies , Delaware , USA -RRB- . DGGE of 16S rRNA gene fragments and phylogenetic analysis DGGE was performed as described by Schäfer and Muyzer -LRB- 2001 -RRB- using the D-Code system -LRB- BioRad Laboratories , California , USA -RRB- . After electrophoresis , the gels were incubated for 30 min in a solution containing ethidium bromide -LRB- 0.5 μg/ml -RRB- , rinsed for 20 min in Milli-Q water , and photographed using a BioRad GelDoc station -LRB- BioRad , California , USA -RRB- . Individual bands were excised , resuspended in 20 μl of Milli-Q water , and stored overnight at 4 °C . A volume of 3 to 5 μl of the supernatant was used for reamplification with the original primer sets . The reamplified PCR products were run again on a denaturing gradient gel to check their purity . Before sequencing , the PCR products were purified using the Qiaquick PCR purification kit -LRB- QIAGEN , Hilden , Germany -RRB- . The obtained 16S rRNA gene sequences were first compared to the sequences stored in publicly accessible database using the NCBI BLAST search tool -LRB- http://www.ncbi.nlm.nih.gov/BLAST ; McGinnis and Madden 2004 -RRB- . Subsequently , the sequences were imported into the ARB software program -LRB- Ludwig et al. 2004 -RRB- and aligned using the automatic aligner function . The alignment was further corrected manually , and an optimized tree was calculated using the neighbor-joining algorithm with Felsenstein correction . Design of oligonucleotide probes Specific oligonucleotide probes for the 16S rRNA of the dominant anaerobic communities in the reactors were designed using the probe design tool of the ARB software package -LRB- Ludwig et al. 2004 -RRB- as described previously -LRB- Dar et al. 2007a -RRB- . The oligonucleotides used for in situ hybridisation are given in Table 1 . Table 1Oligonucleotides used in this studyProbe nameTarget organismProbe sequence -LRB- 5 ′ -- 3 ′ -RRB- ReferenceEUB338IMost bacteriaGCT GCC TCC CGT AGG AGTAmann et al. 1990EUB338IIPhylum PlanctomycetesGCA GCC ACC CGT AGG TGTDaims et al. 1999EUB338IIIPhylum VerrucomicrobiaGCT GCC ACC CGT AGG TGTDaims et al. 1999ARCH915ArchaeaGTG CTC CCC CGC CAA TTCStahl and Amann 1991SPS770Genus SporomusaATC CCG TTC ACT CCC CTGThis studySRB385Most DeltaproteobacteriaCGC GTC GCT GCG TCA GGAmann et al. 1990SRB385DbSome DeltaproteobacteriaCGG CGT TGC TGC GTC AGGRabus et al. 1996DSR660Genus DesulfobulbusGAA TTC CAC TTT CCC CTC TGDevereux et al. 1992DSM1265Genus DesulfomicrobiumAGA TTC GCT CGA CCT CGCThis studyDSV139Desulfovibrio sp. strain L7CGC TGT TAT CCC GAT CACDar et al. 2007aDSCOC814Desulfococcus groupACC TAG TGA TCA ACG TTTDevereux et al. 1992 Fluorescence in situ hybridization Fixed cell samples from the reactors were immobilized on Teflon-coated multiwell microscopic slides as described previously -LRB- Dar et al. 2007b -RRB- . Hybridization was carried out according to the protocol as described previously -LRB- Manz et al. 1992 -RRB- using a formamide concentration of 35 % -LRB- v/v -RRB- . Quantification of the hybridized cells was performed as described previously -LRB- Neef et al. 1996 -RRB- . The hybridized cells were analyzed by two independent observers for determining the fraction of positive signal from each probe relative to the signal visualized with general probes for bacteria -LRB- EUB338 I , II , and III -RRB- , sulfate-reducing bacteria -LRB- SRB385 and SRB385Db -RRB- , or with the general DNA stain DAPI -LRB- 4 ′ ,6 ′ - diamidino-2-phenylindole -RRB- . In addition , a general probe specific for members of the domain Archaea -LRB- ARC915 -RRB- was used . The hybridization experiments were done in duplicate , using different fluorochromes for each probe . Different microscopic fields on each slide were analyzed to confirm the results . Hybridization stringencies of the newly designed probes were determined by performing hybridizations with increasing formamide concentrations as described previously -LRB- Manz et al. 1992 -RRB- using target organism -LRB- s -RRB- and nontarget organism displaying three mismatches within the target region . Sequence accession numbers The sequences determined in this study were submitted to GenBank under accession numbers EU276620 -- EU276626 . Results Microbial community dynamics at different lactate/sulfate ratio Figure 2 summarizes the effluent composition in the bioreactors , after a minimum selection period of five to six volume changes , as a function of the different lactate to sulfate ratios imposed . The start-up reactor R-0 had a lactate/sulfate ratio -LRB- r = 0.65 mol mol − 1 -RRB- theoretically sufficient to allow full lactate oxidation to CO2 with sulfate -LRB- also see Table 2 -RRB- . The results for R1 with excess sulfate -LRB- r = 0.34 mo/mol -RRB- indicate that after five to six volume changes only sulfate , sulfide , and acetate , and no other organic acids were detected in the effluent of the bioreactor -LRB- Fig. 2 -RRB- . Nearly all lactate -LRB- 3.5 mM -RRB- had been converted to acetate -LRB- 3.2 mM -RRB- with the equivalent conversion of sulfate to sulfide . These measurements indicate partial oxidation of lactate to acetate , coupled to sulfate reduction was an important reaction in R1 -LRB- reaction 2 , Table 2 -RRB- . At a high lactate to sulfate ratio -LRB- r = 1.94 mol mol − 1 -RRB- in the reactor R2a , propionate was detected in the effluent besides acetate -LRB- Fig. 2 -RRB- , demonstrating that lactate was partially fermented to a propionate and acetate mixture -LRB- reaction 3 , Table 2 -RRB- . In this sulfate-limited reactor , nearly all sulfate had been converted to sulfide . At a very low sulfate concentration -LRB- r = 20.9 -RRB- , lactate fermentation to acetate and propionate was observed as a dominant reaction -LRB- reactor R2b -RRB- . The shift in the microbial population structure due to different lactate to sulfate ratios after five to six volume changes was recorded by DGGE profiling of 16S rRNA gene fragments , which were amplified either from genomic DNA or from reverse-transcribed RNA , i.e. , cDNA . Fig. 2Effluent concentrations of sulfide , sulfate , acetate , and propionate in reactors R1 , R2a , and R2b . The sulfide data are minimum numbers due to N2 strippingTable 2Stoichiometry of anaerobic degradation reactions relevant to this studyNo.ReactionΔG01 kJ reaction − 11 − 128.52 − 80.83 − 55.74 − 37.75 − 85.46 − 31.1 The comparative analysis of the profiles based on the DNA and RNA of the same sample reflect not only the presence but also the actively growing populations . Figure 3 illustrates the results of the bacterial 16S rRNA -- DGGE reflecting the shift in the predominant populations with the changing lactate to sulfate ratios . For the three lactate to sulfate ratios used -LRB- i.e. , r = 0.34 , 1.94 , and 20.9 -RRB- , the DGGE profiles based on 16S rRNA were not identical to the 16S rDNA-based profiles of the same sample . The phylogenetic affiliation of 16S rRNA gene sequences is presented in Fig. 4 . A neighbor-joining tree was generated using the sequences of the DNA fragments excised from the denaturing gel . Band B3 , representing the most persistent species of all the microbial communities in the DNA based profiles , showed high sequence similarity to the Sporomusa -- Pectinatus -- Selenomonas group with the closest relative being Dendrosporobacter quercicolus . The DGGE profile of the samples taken from R2a to R2b showed an increase in the intensity of fragment B3 . Fragment B5 was abundant in reactors R1 and R2a but with varying degrees of intensity . The fragment was not only persistently present but also belonged to an actively growing population as suggested by its simultaneous presence in the RNA-based profiles . However , the fragment was not present in the DGGE profile when the sulfate feed to the reactor R2b was brought down -LRB- r = 20.9 -RRB- . Comparative sequence analysis showed that band B5 clustered with the genus Desulfovibrio with Desulfovibrio mexicoense as the closest relative among the cultured representatives . Fragment B6 , which was also significantly present , especially in the RNA-based profiles of R1 and R2a , was related to the members of genus Desulfobulbus with the closest relative being Desulfobulbus rhabdoformis . Band B4 was observed only in R1 maintained at a limiting lactate concentration -LRB- r = 0.34 mol mol − 1 -RRB- . The fragment was found closely related to Desulfomicrobium apsheronum . Two other fragments excised , band B1 and band B2 , although present , did not form a significant proportion of the active microbial communities in R1 and R2 as indicated by the RNA-based profiles . The two fragments were affiliated to an uncultured group of Cytophaga and Spirochetes , respectively . Nested amplification with group-specific primers -LRB- results not shown -RRB- resulted in the identification of an acetate-utilizing SRB represented by band B7 . The sequence of the fragment was found closely related to Desulfococcus biacutus . Fig. 3DGGE analysis of 16S rRNA gene fragments using DNA and RNA samples from reactors R-0 , R1 , R2a , and R2b . Lanes 1 -LRB- DNA -RRB- and 2 -LRB- RNA -RRB- sample from reactor R-0 -LRB- influent lactate 6.7 mM -RRB- ; lanes 3 -LRB- DNA -RRB- and 4 -LRB- RNA -RRB- sample from reactor R1 -LRB- influent lactate 3.5 mM -RRB- ; lanes 5 -LRB- DNA -RRB- and 6 -LRB- RNA -RRB- from reactor R2a -LRB- influent lactate 20 mM -RRB- ; lanes 7 -LRB- DNA -RRB- and 8 -LRB- RNA -RRB- from reactor R2b -LRB- in the near absence of sulfate -RRB- . Bands indicated with a dot were excised and sequencedFig . 4Phylogenetic tree based on 16S rRNA gene sequences obtained from the DGGE bands -LRB- see Fig. 3 -RRB- . Sequences determined in this study are in boldface . Black dots on the nodes indicate bootstrap values of 90 % and higher -LRB- 1,000 replicates -RRB- . The scale bar indicates 10 % sequence difference The archaeal 16S rRNA gene-targeted DGGE indicated that the diversity was limited to a single species -LRB- results not shown -RRB- and it was found at all lactate to sulfate ratios except the lowest ratio -LRB- r = 0.34 -RRB- with 3.5 mM lactate . Sequence analysis of the excised fragment indicated its close relatedness to a group of uncultured Archaea distantly related to Methanosaeta sp. . Microbial community dynamics using FISH The FISH results were obtained using oligonucleotide probes that targeted dominant microbial communities present under different substrate concentrations . The results are presented as relative abundances of populations targeted by specific probes and are expressed as the percentage of the total positive signal obtained by general probes for bacteria -LRB- EUB338 I , II , and III -RRB- or with the general DNA stain DAPI -LRB- 4 ′ ,6 ′ - diamidino-2-phenylindole -RRB- . In addition , the general probe specific to Archaea -LRB- ARC915 -RRB- gave a measure of Archaea . The percentage abundance of the specific probes relative to the general probes is summarized in Fig. 5a and b . In general , probe EUB338 stained more than 90 % of all the cells in the samples . Probe SPS770 specifically targeted the species that was closely related to D. quercicolus . At the influent lactate concentration of 3.5 mM -LRB- reactor R1 -RRB- , the relative abundance of this organism was < 2 % of the total bacterial population -LRB- Fig. 5a and 6b -RRB- . When compared to the start-up reactor R-0 , the relative abundance of the cells targeted by probe SPS770 was 8 -- 10 % of the total bacterial cells -LRB- Fig. 5a and 6a -RRB- . The relative percentage of cells that hybridized with this probe was significantly higher -LRB- 40 -- 45 % of the total bacterial cells -RRB- , in samples taken from reactor R2a -LRB- lactate excess , 20 mM ; Fig. 5a and 6c -RRB- and increasing even higher to more than 70 % -LRB- Fig. 5a and 6d -RRB- , when the sulfate feed to reactor R2b was minimized . Probes DSR660 , DSV139 , and DSM1265 were used to target different sulfate-reducing communities -LRB- Table 1 -RRB- that were identified in the reactor samples using DGGE . Desulfomicrobium-like SRB targeted by probe DSM1265 were present in high proportions at excess sulfate -LRB- reactor R1 , r = 0.34 mol mol − 1 -RRB- . The percentage abundance relative to the total bacteria was more than 65 % compared to 15 -- 20 % in the inoculum -LRB- R-0 ; Fig. 5b -RRB- . The numbers were about 5 -- 8 % at limiting sulfate concentrations in reactor R2a -LRB- r = 1.94 -RRB- and decreased further to undetectable levels in the virtual absence of sulfate in reactor R2b -LRB- r = 20.9 -RRB- . Desulfovibrio-like SRB targeted by probe DSV139 were the most resilient of all the SRB . Their numbers did not change significantly , fluctuating between 10 % and 7 % of the total bacteria in the two reactors R1 and R2-a , respectively -LRB- Fig. 5b -RRB- . They were reduced to undetectable levels when the sulfate supply to reactor R2-b was virtually stopped . Fig. 5a Relative abundance of Archaea , sulfate-reducing bacteria , and acetogenic bacteria . b Relative abundance of Desulfomicrobium - , Desulfovibrio - , Desulfobulbus - , and Dendrosporobacter-like bacteria . The percentage abundance is relative to the signal obtained with probe EUBmixFig . 6Whole-cell hybridization of samples from reactors R-0 , R1 , R2a , and R2b probe EUBmix labeled with Cy5 -LRB- blue -RRB- , probe SRB385 and 385Db labeled with Fluos -LRB- green -RRB- , and probe SPS770 labeled with Cy3 -LRB- red -RRB- . a Reactor R-0 -LRB- influent lactate 6.7 mM -RRB- ; b reactor R1 -LRB- influent lactate 3.5 mM -RRB- ; c reactor R2a -LRB- influent lactate 20 mM -RRB- ; d reactor R2b -LRB- in the near absence of sulfate -RRB- The relative abundance of Desulfobulbus-like SRB targeted by probe DSR660 showed the most significant increase among all SRB detected , responding to an increase in lactate concentration combined with sulfate limitation . The percentage abundance increased from about 10 -- 12 % in the inoculum -LRB- reactor R-0 ; Fig. 5b -RRB- to about 20 % of the total bacteria in reactor R2a maintained at limiting sulfate concentration -LRB- r = 1.94 -RRB- . In reactor R1 -LRB- i.e. , at sulfate excess conditions -RRB- , the Desulfobulbus-like SRB were < 5 % of the total bacteria -LRB- Fig. 5b -RRB- . The change to a low sulfate concentration in the reactor resulted in a considerable increase in the methanogenic population . FISH with probe ARCH915 indicated an increase of the methanogenic population from 5 % in the inoculum -LRB- R-0 -RRB- to more than 10 -- 15 % under limiting sulfate -LRB- R2-a -RRB- and nonsulfidogenic -LRB- R2-b -RRB- conditions -LRB- Fig. 5a -RRB- . The relative abundance of the methanogenic population decreased to undetectable levels after the shift from R-0 to the lactate-limited reactor R1 . Discussion This study evaluates the shift in microbial structure in lab-scale CSTR reactors as affected by changing lactate to sulfate ratios using a combined approach of molecular methods -LRB- PCR -- DGGE and FISH -RRB- and chemical analysis . The combination of different methods allowed the establishment of a link between the population structure and function of the anaerobic communities in the reactors under sulfidogenic and nonsulfidogenic conditions . PCR -- DGGE profiles produced using DNA templates may not reflect the dynamic changes in community structure . Metabolically active cells have a much higher ratio of rRNA to DNA than dormant cells -LRB- Weller and Ward 1989 -RRB- . Therefore , the use of rRNA templates may emphasize the actively growing populations in the community as opposed to those that are relatively inactive but persistent in the community . Because PCR-based approaches for the analysis of microbial diversity in mixed populations can be influenced by several constraints -LRB- von Wintzingerode et al. 1997 -RRB- , our results based on PCR -- DGGE do not necessarily reflect the abundance of target sequences in the reactor samples . We , therefore , tried to confirm the relevance of the sequence data by whole-cell hybridization using fluorescently labeled oligonucleotide probes . Depending on different lactate to sulfate ratios , theoretically , various scenarios involving SRB in the degradation of lactate are possible as shown in Table 2 . Complete oxidation of lactate to CO2 and sulfide by SRB -LRB- reaction 1 , Table 2 -RRB- . Incomplete oxidation of lactate to acetate by SRB -LRB- reaction 2 , Table 2 -RRB- . Fermentative degradation of lactate to propionate and acetate and the utilization of fermentation products by SRB and acetoclastic methanogens -LRB- reactions 3 , 4 , 5 , and 6 , Table 2 -RRB- . In the absence of sulfate the fermentative degradation of lactate coupled to acetoclastic methanogens -LRB- reactions 3 and 6 , Table 2 -RRB- . The net metabolic pathway of the degradation of lactate by a mixed culture inoculum used in this study suggests its dependence upon the relative quantities of lactate and sulfate . Theoretically , the complete oxidation of lactate to inorganic carbon requires a minimum lactate to sulfate ratio of 0.67 mol mol − 1 -LRB- reaction 1 , Table 2 -RRB- and if the ratio is below 0.67 , additional electron donor is required for complete sulfate reduction . At lower molar ratio of lactate to sulfate -LRB- 0.34 mol mol − 1 , reactor R1 -RRB- , only sulfate , sulfide , and acetate were detected in the effluent . These results directly show the quantitative oxidation of lactate to acetate coupled to sulfate reduction as a dominant reaction -LRB- reaction 2 , Table 2 -RRB- . This is consistent with high percentage abundance of Desulfomicrobium - and Desulfovibrio-like SRB -LRB- known , incompletely oxidizing , SRB capable of performing reaction 2 , Table 2 ; Figs. 5a and 6b ; Widdel 1988 -RRB- . Complete lactate oxidation to carbon dioxide with sulfate as electron acceptor would be energetically favorable -LRB- reaction 1 , Table 2 -RRB- . However , the kinetics of this complete oxidation may not enable efficient competition with the incomplete lactate oxidizers . This is evident from the observation that in batch culture in the presence of excess sulfate , incompletely oxidizing SRB outcompete completely oxidizing species due to their superior maximum specific growth rate on fermentation intermediates like lactate , ethanol , propionate , etc. -LRB- McCartney and Oleszkiewicz 1993 ; Widdel 1988 -RRB- . The actual competition between partial and complete lactate oxidizing SRBs will be determined by the affinity for lactate -LRB- μmax/Ks -RRB- . Given the fact that the nested amplification suggested that the numbers of the complete oxidizers were very low in the inoculum , a long period of time would have been required before such SRB would have grown to a significant number . Therefore , we can not rule out that eventually the complete oxidizers might have become dominant under lactate-limiting conditions in R1 . In the near absence of sulfate , lactate fermentation to acetate and propionate -LRB- reaction 3 , Table 2 -RRB- was observed as a dominant reaction in the reactor R2b . This was reflected by a significant increase in the relative abundance of Dendrosporobacter-like species and a near washout of SRB species -LRB- Fig. 5a and 6d -RRB- . D. quercicolus is an obligate anaerobe that clusters with the Sporomusa -- Pectinatus -- Selenomonas group -LRB- Strompl et al. 2000 -RRB- . The members of this group are known to perform acetogenic and propionic acid fermentation -LRB- reaction 3 , Table 2 ; Biebl et al. 2000 -RRB- . Major fermentation products of D. quercicolus and other closely-related members of the group are acetate and propionate . Theoretically -LRB- reaction 3 , Table 2 -RRB- , the fermentation of 6.7 mM of lactate should yield approximately 4 mM of propionate and 2 mM of acetate assuming that 0.5 mM is assimilated into the biomass . However , the acetate concentration in the effluent of the reactor R2b -LRB- Fig. 2 -RRB- indicated a net loss of 15 % of the electron donor . The plausible explanation for this loss can be acetoclastic methanogenesis -LRB- reaction 6 , Table 2 -RRB- that can be assumed from an increase in the methanogenic population to 15 % under nonsulfidogenic conditions -LRB- Fig. 5a -RRB- . The lactate to sulfate molar ratio in reactor R2a -LRB- 1.94 mol mol − 1 -RRB- was increased by a factor of three compared to the inoculum , reactor R-0 -LRB- 0.67 mol mol − 1 -RRB- and a factor of about six compared to reactor R1 -LRB- 0.34 mol mol − 1 -RRB- . The conditions in reactor R2a were changed to sulfate limitation with a substantial increase in influent lactate concentrations . Indeed , the sulfate concentration had dropped to < 0.2 mM , showing that the SRBs could no longer compete for the remainder of the substrates . The products in the effluent included propionate and acetate besides sulfide , suggesting that lactate was partially fermented to a propionate and acetate mixture -LRB- reaction 3 , Table 2 -RRB- . This indicates a change in the net metabolic pathway for the degradation of lactate compared to the reactor R1 . This change may be attributed to the lactate fermenting bacteria catalyzing reaction 3 -LRB- Table 2 -RRB- . The same is suggested by a significant increase in the population of D. quercicolus-like species , 5 times increase relative to reactor R-0 and even 20 -- 25 times relative to reactor R1 -LRB- Fig. 5a and 6c -RRB- . This is in agreement with the previous studies performed -LRB- McCartney and Oleszkiewicz 1993 ; Mendez et al. 1989 ; Pichon et al. 1988 ; Ueki et al. 1986 -RRB- . In all these studies , an increase in the buildup of propionate was observed with increasing organic substrate to sulfate ratio . McCartney and Oleszkiewicz -LRB- 1993 -RRB- attributed this change to the net metabolism of lactate , the combined influence of the increased lactate to sulfate ratio , and the time for which the culture was exposed to excess sulfate . This is clearly also the case in our experiments : if a steady state has been reached , the DNA and RNA bands in the DGGE are expected to be similar , if not identical . As demonstrated in Fig. 3 , this was not the case . Again , if growth rates would have been only marginally different , a true steady state might have taken several weeks to become established , and hence , we can not rule out that we have looked at a snapshot during a shift in the population . In this study , the exposure of the original inoculum from the anaerobic wastewater plant to excess sulfate was approximately 13 days during start-up reactor , R-0 , before an increase in the lactate to sulfate ratio was imposed in reactor R2a -LRB- 1.94 mol mol − 1 -RRB- . This exposure time was enough to enrich SRB in sufficiently high numbers for the development of substantial sulfidogenesis leading to sulfate depletion . As pointed out above , the consequence was that the culture runs into sulfate limitation and , under this condition , acetogens and methanogens experience no competition for the remaining substrate . It is interesting to note that the buildup of propionate in reactor R2a was concurrent to the increase in D. rhabdoformis-like SRB -LRB- Fig. 5b -RRB- , which are known to degrade propionate -LRB- reaction 4 , Table 2 -RRB- . This increase in the relative abundance of D. rhabdoformis-like SRB , suggesting a synergistic association between heteroacetogenic bacteria and SRB , is a feasible scenario in the presence of limiting sulfate . An overall electron balance over the system was established , assuming a biomass yield of 0.07 °C - mol C-mol − 1 . In the experiment , at a lactate to sulfate ratio of 0.34 mol mol − 1 -LRB- reactor R1 -RRB- , the electron balance was nearly 95 % . However , in the other experiments with R2 , a net loss of reduction equivalents between 10 % and 50 % is observed . This may be attributed to methane or H2 formations , which were stripped out with the sulfide . Assuming the dominance of partial lactate oxidation coupled to sulfate reduction in reactor R1 -LRB- 0.35 mol mol − 1 -RRB- and a partial lactate fermentation to propionate and acetate in reactor R2-a -LRB- 1.94 mol mol − 1 -RRB- , both H2 and acetate are the most plausible precursor for methane formation . However , the observation that relatively high concentrations of acetate were found in the reactor effluent -LRB- 1 -- 4 mM -RRB- and the detection of methanogenic Archaea which clustered with Methanoseata sp. suggests that , given the dilution rate of 0.02 h − 1 , only a low affinity acetoclastic methanogen catalyzing reaction -LRB- reaction 6 , Table 2 -RRB- could be maintained in the system . These results were consistent with the increase in the percentage abundance of methanogenic Archaea to more than 10 -- 15 % of the total DAPI-stained cells . Because the conditions in the reactors did not favor the growth of nonmethanogenic Archaea -LRB- extreme halophiles , thermoacidophiles , the Archaeoglobales , and thermophiles placed in Crenarchaeota kingdom ; Woese 1987 -RRB- , cells targeted by probe ARCH915 can be assumed to be the total methanogenic representation in this environment . In conclusion , this study points to different metabolic routes being followed by a mixed anaerobic community , even in the mineralization of simple substrates like lactate . We could establish a link in microbial population dynamics to major perturbations caused by changing influent lactate to sulfate ratios . This study demonstrated that at low lactate to sulfate molar ratios in the influent , SRB had a competitive advantage over acetogens and methanogens and that , in the near absence of sulfate , heteroacetogens formed a syntropic association with methanogens . Higher lactate to sulfate ratio resulted in a pathway that had propionate and acetate as products , and the majority of sulfidogenesis and methanogenesis was dependent on the fermentation products . All these results were substantiated by corresponding shifts in relative abundance of the microbial communities present as analyzed by DGGE and FISH .

O O O O O O O B 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B 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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B 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 B O B O

anaerobic consortia
sulfate-reducing bacteria