8/11/2019 Firth99p353_k2opt

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of

cultural

conditions

n denitrification by

stutzeri

by Membrane Inlet

Spectrometry

Firth* and C. Edwards

o f B iological

S ciences,

U niversity o f L iverpool,

U K 7056/02/99: received 1 February

1

999 and accepted 27

1999

RTH

AN

D C.E DW ARDS.1 999.

Denitrification

is a

important

process

leading to loss

of fertiliser

and the

production

of the greenhouse gas

oxide

and

nitric

oxide, an ozone depleter.

inlet mass

spectrometry

MIMS)

was

to study the effect of

different

variables on

process

of

denitrification by P seudomonas

stutzeri

a

defined salts

medium. MIMS

was

used for

measurements

of

nitrous oxide, nitrogen

8/11/2019 Firth99p353_k2opt

2/23

oxygen and showed that denitrification

occurred

the presence of

dissolved oxygen.

A

nitrate

of

15

mmol l1 and a

nitrite

of 5 mmol l1 were found to be

for

complete

denitrification

of

nitrate

or

to nitrogen and varying these concentrations

a

marked effect

on the ratio

of

gaseous

products

Denitrification products

were also dependant

pH with

neutral or alkaline

conditions

being

best

r

production

of gaseous

end

products.

Our

results

that under nutrient

rich conditions the

most

factor

in the regulation of denitrification b

stutzeri is

the amount

of

nitrite

generated

at

the

enzymatic

stage

of

the

process.

This

appears

to

inhibition of the

denitrification

pathway

above 5

l

1

and

at

high

enough

concentrations

(15

mmol l

1)

restricts

growth.

UCTION

is

an

important

step

in the nitrogen cycle

nitrate NO3), is converted via nitrite NO2),

an

oxide

(NO),

to nitrous

oxide (N2O),

or dinitrogen g

It

is

the

only

biological mechanism

by

which N2

to the

atmosphere

(Ferguson 1994). The process

8/11/2019 Firth99p353_k2opt

3/23

out by a variety of b acterial species, which are foun

oughout natural environments. NO3 is used in place

(O2), as a terminal

electron

acceptor

during

respiratio

1991).

Because

this is an energetically unfavour

option, it has often been suggested that denitrification

anaerobic

process

(Tiedje

etal.

1982).

The beneficia

of denitrification include control or bioremediation

contaminated waters, which can cause eutrophicatio

cera et

al. 1986).

However, it

has

also

been

linked

cancer

(O%Donnell and Edwards 1992), and

b lu

syndrome

Mirvish

1985).

Denitrifiers

may

play

a

part in the b reakdown

of various

hydrocarbo

to : J .R. Firth,

School

of Biological Sciences,

University of

Life Sciences

Building,

Crown

Street,

Liverpool, L69

7ZB

jfirth@liverpool.ac.uk).

(Evans et al. 1991; Altenschmidt and Fuch

In

agricultural areas

denitrification leads to

a

loss

efficiency (Goulding et

al.

1993),

and incomplet

can lead to the release of NO and/or N2O

of

which

are

known

to

be

involved

in

the

formation

rain,

ozone depletion and

global warming

(Knowles

198

et al. 1991; Culotta and

Koshland

1992).

The method used here to

study

denitrification was

mem

inlet

mass

spectrometry

MIMS).

This technique

ha

used

to

study

various

microbial

processes,

such

8/11/2019 Firth99p353_k2opt

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nitrification,

and

the rumen ecosystem,

as denitrification (Lloyd and Scott 1983). The principle

MIMS have been

described

elsewhere

(Lloyd

et

a

and it provides continuous,

sensitive

and virtuall

measurements

of

gases

and volatiles

in the

gas

phase.

MIMS was employed here to

assess

the effect

of

differen

on denitrification by Ps. s tutzeri, a known den

which is

widely disseminated

in

many

environment

van Rijn et

al.

1996). It possesses the

full

complemen

nitrifying

enzymes

and

is therefore

capable

of producin

nitrogen

as

the

end product.

R IALS AN D

M

ETHO DS

and

growth conditions

stutzeri NCIMB 11056 was maintained

on

nutr

t agar

(Amersham, Bury,

UK)

at

4 C and

subculture

three weeks.

Seed

cultures

were

grown from

individua

inoculated into 50

ml of

minimal salts medium

250

ml

conical

flasks at 30 C. The

salts

medium con

(g per

litre): sodium

succinate, 27; KNO3,

1;

K2HPO4

(NH4)2SO4,

1;

Na2SO4, 0054; CaCl2, 005; MgCl2, 0025

d 1ml

trace element

solution containing (g

per

litre

1;

FeSO4,

1; ZnSO4, 1

BDH).

The final pH was

7

for inocula were

collected by

centrifugation

and

washe

times

in

equal

volumes

of

25

w/v) Ringer

s

solutio

8/11/2019 Firth99p353_k2opt

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prevent carry over

of nutrients.

medium experiments

stutzeri was grown in

a

2-L fermentor L

Products,

Reading,

UK),

with

temperature,

p

d stirrer control. The salts medium

was

amended wi

nitrate

or

nitrite concentrations, and

pH

varied

Washed

cell suspensions

were

inoculated to give a

OD550 of

0 1.

Temperature was

maintained at

30

a

stirring

rate

of

200

r.p.m. to

keep

the

cells

in

suspen

A

reservoir

of

5 mol1 HCl was used to control p

necessary. No oxygen

other than that

diffusing fro

atmosphere was added.

methods

was monitored by measuring OD550

of culture

sam

and cell

free

supernatant

fluids were

used

to analys

concentration by

the hydrazine

reduction

metho

1984).

Gas

analysis

was carried out using

a

quadrupol

inlet

mass

spectrometer

(Hiden

Analytical,

Wa

England), that had

a 4-probe

manifold

capable

sampling. The

probes

were covered with

a

silico

membrane and had an external diameter

of

07 mm

were

drawn into

the probe b y a vacuum and ionize

an

electron

beam.

The

ions

produced

were

then

sorte

8/11/2019 Firth99p353_k2opt

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to their mass/charge ratio m/z). Gases producin

ions of

the

same

m/z

ratio (e.g. CO2 and N2

e ions which

both

have an

m/z-value

of

44),

were

the

by

studying additional

m/z channels

whic

minor

ion peaks produced from these

gases.

T

between CO2

and

N2O gases, CO2 was

measure

m/z channel 12

where

it

causes a peak

corresponding

of its contribution

at

channel

44.

By multiplying up

th

to

give

a

100

value and

subtracting

this amount

fro

44,

the

contribution

made

by

N2O could

then

b

These

data

were

obtained

from

a

mass

spectr

pattern (Lloyd and Scott 1983). The data we

stored on

computer disk and analysed in

a spr

at

the

end of

the

experiment

using

Excel 40

(Micro

LTS

during growth in

the

defined medium.

was followed

during

batch growth of Ps. s tu

with 10 micromoles

l1

nitrate and

pH controlled

to

of 7

(Fig.

1). Under these growth conditions

the oxyge

in the

media remained

at

a level of approx

25

50 mmol l1

throughout the

experiment.

Den

occurred during

the

exponential

growth

phase an

s

monitored

as

nitrogen

production.

As

the culture

entere

8/11/2019 Firth99p353_k2opt

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phase, nitrite

levels

rose

to a peak

concentratio

7 mmol l1 after

9

h;

subsequently,

they fe

undetectable

levels

by

13 h.Nitrogen production continue

8/11/2019 Firth99p353_k2opt

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1 Denitrification by

a

14h-batch

culture

of

P seudomonas

in a defined salts medium

measured

using Membrane

Inle

Spectrometry.

The

gas

traces

illustrated

are

nitrogen

oxide

carbon dioxide and

oxygen

- - . Als

are nitrite concentrations detected in

the

medium

and growth as measured

as OD550

R

.

rise

during

this period, along

with nitrous

oxide and carbo

but no

nitric

oxide

could

b e measured.

When

nitrit

fell,

nitrogen

production

continued

to

a

peak value

7

mmol

l

%

1

at 11h. At the end of exponentia

maximum levels

of carbon

dioxide and nitrous oxid

re detected.

Nitrous oxide could still

be

measured

eve

net

nitrogen production had

finished.

When the exper

was

repeated

without

pH

control

so

that the

pH ros

8/11/2019 Firth99p353_k2opt

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growth occurred, less

nitrous

oxide

was released and highe

of nitrogen were detected (results

not

shown

of

nitrate

and nitrite

concentration

for denitrification

were

optimized

by

varyin

of

the parameters for the experiment

described

in

Fi

Peak

nitrogen

and nitrous

oxide

concentrations, used as

of efficiency of nitrogen

production,

revealed that

concentration of approximately

15

mmol l1

was opt

l

for

maximum nitrogen

production;

above

this

valu

of nitrogen produced fell

(Fig.

2a). Nitrous

oxid

8/11/2019 Firth99p353_k2opt

10/23

. 2 The effect of nitrite and

nitrate concentrations

on the

of nitrogen

E,

and nitrous

oxide

by

stutzeri

during

denitrification

in

a

defined

salts

Data are

representative

of

typical

experiments.

was also maximal at 15 mmol l

%

1 nitrate; howeve

nitrogen, the

amount of nitrous

oxide

measured

on

slightly to a concentration of approximately

420 micro

l

%

1 even when the nitrate supplied was at

100

mm

In contrast,

an

optimum

of

5 mmol

l%1

nitrite was

foun

maximum nitrogen

and

nitrous

oxide

production.

Th

corresponded

well

with the peak nitrite concentratio

mmol l

%

1)

measured

in

the

medium

during denitrificatio

the

optimum nitrate concentration of

15

mmol l%1 (Fi

Nitrite

accumulation in the medium over the range

8/11/2019 Firth99p353_k2opt

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concentrations examined increased to a maximum

20 mmol l1 in cultures

grown

with 30 mmol

l

1

nitrat

3).

Above

30

mmol

l

1

nitrate,

the

levels

ofnitrite

whic

be detected in the medium fell steadily to a value of

l

1

when the nitrate

concentration

supplied was 25

l

1.

Although nitrogen production was

inhibited

at nitrate con

above 15 mmol l1 this was not

true

for growth

final

stationary

phase biomass,

measured

as OD550,

culture

after

24 h incubation was highest at 30

mmols l

and

only

above

this

concentration

was

growth

yie

Reduced biomass values

after

24

h incubatio

in higher

residual

oxygen concentrations (Fig.

4 a

denitrification using nitrite,

final

OD550

value

fairly

constant up

to 15

millimoles

l

1 and then

fe

general,

cultures

grown

on

nitrite

as

terminal

electro

produced lower cell

concentrations

compared wit

grown

on nitrate.

Residual

oxygen

concentrations

re

during

the experiments remained

at

approximately 5

8/11/2019 Firth99p353_k2opt

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3

Maximum nitrite

concentrations detected during

by

P seudomonas

stutzeri in a defined salts

containing different initial nitrate concentrations.

Data

taken

from

representative experiments.

8/11/2019 Firth99p353_k2opt

13/23

8/11/2019 Firth99p353_k2opt

14/23

4

The effect of nitrite and nitrate concentration on

of

residual oxygen

and

growth

as

as OD550 R of

Pseudomonas

stutzeri

during

in

a

defined salts medium. Data are taken

from

experiments.

l

%

1

until

growth

yield

became

reduced,

at

whic

its concentration rose noticeably

Fig.

4b).

of

pH

optimum pH of 7 was

found for

both

nitrogen and nitrou

production

Fig.

5a),

although the

process

proceede

8/11/2019 Firth99p353_k2opt

15/23

efficiently when no

pH

constraint was applied

and

th

allowed

to b ecome more

alkaline

throughout

th

as a consequence

of denitrification.

Co

growth values measured as OD550

also

increase

to pH 7 at

which point

they remained fairly constant Fi

A more acidic

medium caused a more severe reductio

nitrogen production than nitrous oxide production.

production

and

accumulation

in

the

medium

togethe

nitrous

oxide

and nitrogen

were

always

detected

8/11/2019 Firth99p353_k2opt

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. 5 The effect of pH on (a) the production of nitrogen

and nitrous

oxide

?

b y

and

(b) growth of

stutzeri during denitrification

in

a

defined

salts medium

are

taken

from

representative experiments.

cultures

of

Ps.

s tutzeri.

Nitrite rose durin

growth in

the

presence ofnitrate, but disappeare

stationary phase was reached.

Other work

has show

Ps. stutzeri

released nitrogen

only, from nitrate withou

detectable nitrous oxide,

and

that its production occurre

any

nitrite could

b e

detected (Carlson and Ingraham

Xu and

Enfors

(1996)

found

that nitrate availabilit

the

24 h

prior

to inoculation

had

a

marked effect

on

th

accumulation

of

nitrite

due

to

a

need

to

synthesiz

8/11/2019 Firth99p353_k2opt

17/23

nitrite reductase. Different volatile

fatty

acids used

sources

have also been

shown

to influence nitrit

in Ps. stutzeri (van

Rijn

etal. 1996),

and p

the

process

in Paracoccus denitrificans Ku cer

K orner and Zumft (1989) found that nitrate inhibitio

nitrite

reductase

could

lead

to

a

nitrite

build

up

in

P

Nitrate reduction occurred more rapidly than nitrit

and

this

did seem

to

have an inhibitory effect o

oxide

reductase.

This probably

explains

the shoulde

in the

nitrogen

production curve in Fig. 1at around 4

where

the

rate

of

nitrogen

production

was

temporaril

until

the

nitrite levels

in the medium began

to

fa

z alez et al. (1994) have previously

demonstrated

the ina

of nitrate reductase by

nitrite

in

the

yeast Hansenul

Although the

starter cultures used

in

this

study

wer

grown

aerobically

overnight,

denitrification

commence

immediately

indicating that

the

enzymes required fo

were already

present

or synthesized very rap

as

oxygen levels

fell. This

is contrary

to the

work

(1992), who showed that nitrite was utilized b efor

production of

nitrogen

and

without any

nitrous oxid

detected. In Ps. s tutzeri, nitrous

oxide

and nitroge

occurred simultaneously with concentrations

peaking around 2 h

before

nitrous

oxide

reached

The nitrogen peak corresponded

to

the

maximum

of carbon

dioxide

detected,

but began

to

fall at th

d

of

exponential

growth.

If

complete

conversion

of

each

8/11/2019 Firth99p353_k2opt

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intermediates of the denitrification pathway had occurre

utilization of the next, peak nitrogen

levels would

hav

expected after that of its

precursor

nitrous oxide. Th

ofmaximum

amounts

ofnitrous oxide

could

be expla

d by the observation that all nitrite had disappeared fro

medium

at

this point.

Nitrate concentration affected denitrification, both directl

d indirectly. Although denitrification was inhibited

concentrations higher

than

15

mmol

l

1, higher

ce

were measured above this concentration

denitrification

being

a

growth-related

process.

T h

be explained

by different rates

of

nitrogen

productio

the

range of nitrate concentrations.

Nitrite

accumulatio

y

be the most critical step

during

denitrification in th

cterium. Reduction in growth

yield

at nitrate

con

in

excess

of

30

mmol

l

1

is

probably

due

to

cel

nitrate to nitrite concentrations sufficient to inhib

and thus reducing denitrification further.

Nitrite

is

bacterial

inhibitor

and is even used as

The optimum level of nitrate in this study (15 mmol l

s

similar to

the

lower

optimum

of approximately

20 mm

found

by

Thomas et

al.

(1994) for denitrification

by P

and Ps. f luorescens, but

m uch

lower than the valu

100

mmol l

1

found

for

denitrifying

Ps. aeruginosa reporte

Williams et

al.

(1978). Our failure

to

detect nitric oxide

medium

is

not

a

limitation

of

MIMS

as

a

technique

8/11/2019 Firth99p353_k2opt

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we have

been

able to

distinguish nitric

oxide

fro

oxide and

nitrogen

during

denitrification by a

denitrifier

isolated

from aquatic environment

and Edwards,

Unpublished Data). As

no

nitric oxid

detected it appears

that the

nitric

oxide

reductase

efficient in

Ps.

s tutzeri,

as reported by

Goretsk

al. (1990), producing

nitrous

oxide

at

approximately

th

time as

nitrite

is produced. Similarly, there

was

n

of

nitrite

reductase

which

would

have led to

in

nitrous

oxide concentrations. One possibility

inhibition

of

the nitrous

oxide

reductase

leading

to

of nitrous

oxide.

At elevated concentrations

th

rous oxide may then exert a

negative

feedback effect upo

nitrite or nitrate reductases, switching off

the

pathway

etal. (1995) found

that high nitrite concentration

all

denitrification

enzymes

leading

to

a

build

up

and

nitrous oxides

in a

denitrifying

activated sludge.

The most preferred situation, where

the

highest possibl

of nitrate

is

removed

from the

medium

wit

least amount

of nitrous

oxide

released

to the atmosphere

one in

which

the

nitrate

concentration

is at 10

mmol

l

nitrous

oxide

concentrations

produced

did

not

fall

as nitrogen concentrations at nitrate concentration

optimum, a higher percentage of

the

nitrate

wa

into the

atmosphere as

this

harmful gas. Nitrit

was not quite as

critical

in these terms, sinc

difference

was

seen

in

the ratio

of nitrogen

to nitrou

8/11/2019 Firth99p353_k2opt

20/23

production when the culture was grown in the presenc

between

5 and 15

mmol l1 nitrite.

Although

it

has

been

suggested that

denitrification

is

anaerobic process,

this

work

clearly showed that P

is capable of carrying out

the process in the

presenc

oxygen.

The

use of

a

stirred

fermentor

vessel reduce

possibility

of

anaerobic

microniches

occurring.

Oxyge

remained approximately

constant

throughou

r experiments as an equilibrium was

reached

between di

into solution from the headspace and consumption b

respiring

cells.

The

culture

as

a

whole,

and perhaps eve

cells,

appeared

capable

of utilizing oxygen

an

as electron acceptors

simultaneously, despite

the latte

less

energetically favourable.

Membrane

inlet

mass

spectrometry is

proving

to b e

tool

for

studying

elemental

cycling.

The

ability

real time data provides new evidence in support

ideas on how denitrification is regulated

within th

cell and

these studies

show the complexity

of

inte

between

the denitrification enzymes, their product

d the

surrounding environment.

DG

EM

ENTS

work was supported by

a studentship

from

the Natura

Research

Council,

UK.

8/11/2019 Firth99p353_k2opt

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EFER ENCES

U.

and

Fuchs, G. (1992) Anaerobic toluene

oxidatio

to benzyl

alcohol and

benzaldehyde in a denitrifying

P seudomona

strain. Journal of Bacteriology

174, 4860

4862.

M. (1992)

Comparison

of

denitrification

by

P aracoccu

denitrificans,

P seudomonas stutzeri and Pseudomonas

aeruginosa

Acta

M icrobiological

Pologica 41, 203

210.

C.A. and Ingraham, J .M.

(1983)

Comparison

of den

trification

b y Pseudomonas

s tutzeri, Pseudomonas

aeruginosa

an

Paracoccus

denitrificans. Applied Environmental M icrobiology 4

1247

1253.

E. and Koshland,

D.E. (1992) NO news

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P.J., Mang, D.T. and Young, L.Y. (1991) Degradation

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S.J. (1994) Denitrification and its control. Antonie va

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Edwards Unpublished data.

C., Gonz alez, G.,

Avila,

J . et al. (1994) Nitrite cause

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inactivation

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P.R. (1993) Denitrification losses of nitrogen fertiliser applied

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(1982)

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Boddy, L. (1986) Mass spectrometry

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Direct measurement of dissolve

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