Radiorespirometric assays for the detection of biogenic sulfides from sulfate-reducing bacteria

ORIGINAL ARTICLE

Radiorespirometric assays for the detection of biogenicsulfides from sulfate-reducing bacteriaJ.C. de Queiroz1, A.C. de Melo Ferreira2 and A.C.A. da Costa1

1 Instituto de Qu�ımica, Programa de P�os-Graduac�~ao em Qu�ımica, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brasil

2 Servic�o de An�alises Ambientais, Instituto de Radioprotec�~ao e Dosimetria, Rio de Janeiro, Brasil

Keywords

culture medium, most probable number,

radiorespirometry, sulfate-reducing bacteria,

sulphide.

Correspondence

Antonio Carlos Augusto da Costa, Instituto

de Qu�ımica, Programa de P�os-Graduac�~ao em

Qu�ımica, Universidade do Estado do Rio de

Janeiro, Pavilh~ao Haroldo Lisboa da

Cunha, R. S~ao Francisco Xavier 524,

Maracan~a, Rio de Janeiro, Brasil.

E-mail: [email protected]

2013/1804: received 8 October 2012, revised

22 December 2012 and accepted 31

December 2012

doi:10.1111/jam.12122

Abstract

Aims: The detection of trace concentrations of biogenic sulfides can be carried

out through radiorespirometric assays. The objective of this work was to

improve the methodology for detection of H2S in trace concentrations, to

correlate with sulfate-reducing bacterial activity.

Methods and Results: Serial dilutions of synthetic sea water with a pure

culture of Desulfovibrio alaskensis, a mixed anaerobic microbial culture and a

natural saline sample from a petroleum offshore platform indicated that

dilutions were followed, accordingly, by sulfate reduction.

Conclusions: Tests performed indicated that increasing the time of incubation

of a mixed anaerobic microbial culture contributed to an increase in the

sulfate reduction rates, as well as the amount of carbon source and inoculum.

Significance and Impact of the Study: The technique here developed proved

to be a rapid test for the detection of biogenic sulfides, particularly those

associated with corrosion products, being an useful tool for monitoring and

controlling oil/water storage tanks, petroleum continental platforms and several

types of reservoirs.

Introduction

One of the first attempts to use radiorespirometric tests

for the detection of microbial activity was performed by

Levin and Straat (1976), studying the labelled release of

carbon for extraterrestrial life detection onboard the Vik-

ing spacecraft on the surface of Mars. At that time, the

authors described the intention of detecting heterotrophic

life by supplying a dilute solution of radioactive organic

substrates to a sample of Martian soil and monitoring

the evolution of gas.

Later, Hardy and Syrett (1983) used the same tech-

nique to evaluate inhibitors of sulfate-reducing bacteria

(SRB), concluding for its effectiveness and short-time

duration of the technique.

After that, a few papers were published on the sub-

ject, all of them emphasizing the classical monitoring

methods may give misleading results and that radiore-

spirometric techniques may give a better understanding

of the processes taking place within the systems

(Maxwell and Hamilton 1986; Hamilton et al., 1989;

NACE 2004)

Sulfate-reducing bacteria constitute a common, wide-

spread and harmful microbial group of great environ-

mental and economic impact for the petroleum industry,

commonly found in injection waters, corrosion products

and usually constituting a biofilm matrix scraped from

the surface of corrosion coupons (Sanders 1988). They

have the ability of using sulfate as final electron acceptor

in the respiration, with H2S as the final metabolic prod-

uct (Barton 1995; Roychoudhury et al. 2003). Problems

associated with the anaerobic corrosion due to the activ-

ity of SRB cells become even worse due to the use of sea

water for secondary oil recovery in offshore platforms

(Gaylarde 1990; Edyvean 1991). The resulting environ-

ment favours bacterial growth, offering anaerobic condi-

tions with sulfate and nutrients. In this particular case,

during the production and storage of petroleum, there

are many points at which biocorrosion is a problem

(Ara�ujo-Jorge et al. 1992). In the case of growth of a

1008 Journal of Applied Microbiology 114, 1008--1019 © 2013 The Society for Applied Microbiology

Journal of Applied Microbiology ISSN 1364-5072

consortium of microbes, SRB adhere to inert surfaces

with subsequent development of biofilms, which mediate

the interaction between metal surfaces and the environ-

ment (Videla 1989). Routine methods to prevent and

treat microbial contamination and biodeterioration

involve the use of biocides, which are toxic and with

some degree of environmental impact. Then, an accurate

diagnosis of biocorrosion is always required prior to a

treatment decision (Gaylarde and Johnston 1984; Mcken-

zie and Hamilton 1992). According to Maxwell et al.

(2002), it is extremely difficult to estimate the costs

related to corrosive processes attributed to the activity of

micro-organisms (SRB and other bacteria) in the oil

industry. However, in the last few years, the costs for the

control of their activity are significant, with annual

income values ordering about US$150 000 per continen-

tal platform, only considering the use of biocides.

Recently, considerable efforts have been carried out for

the development of new methods for SRB enumeration.

In general, the most usual methods to enumerate SRB

can be divided in two categories: direct detection meth-

ods and culture methods (Flemming and Ingvorsen

1998). The last category includes methods based on the

most probable number (MPN) technique, a worldwide

used method. However, this technique does not provide

an accurate determination of the number of SRB cells

within a natural ecosystem or sample. According to

Oliveira (2005), the uncertainty in this method can reach

68%, and according to American FDA, several improve-

ments and statistical changes were introduced in the

MPN method to decrease bias and uncertainties. McCra-

dy (1915) published the first precise estimation of the

bacterial numbers by the MPN, followed by Halvorson

and Ziegler (1933), Eisenhar and Wilson (1943) and

Cochran (1950), who published articles on the statistical

foundations of the MPN technique. Woodward (1957)

recommended that MPN tables should omit those combi-

nations of positive tubes (high for low concentrations

and low for high concentrations) that are so improbable

that they raise concerns about laboratory errors or possi-

ble contaminations. de Man (1983) published a confi-

dence interval method that was modified to make the

tables for this procedure. In relation to direct detection

methods, like radiorespirometric assays, they constitute a

promising technique for SRB activity determination, but

still require some improvement for practical applications.

The radiotracer technique used for radiorespirometric

assays for sulfide determination was introduced by Ivanov

(1956). This method consists in adding trace amounts of35SO2�

4 to a natural sample of sea water, injection water

or any other process water. In the end of an incubation

period, the biologically reduced sulfate can be quantified.

Rosser and Hamilton (1983) developed a radiorespiro-

metric assay for the study of sea sediments and, later,

adopted it to monitor the microbial sulfate reduction

activity in biofilms formed on mild steel corrosion cou-

pons. According to them, the efficiency of 35S-sulfide

recovery under their experimental conditions (16–20 h

equilibrium in a shaking water bath at 100 rev min�1 at

35°C) was about 96%.

Fossing and Jorgensen (1989) developed a more

advanced technique, which separates the reduced 35S-sulfur

by reflux distillation. The disadvantage of this method is

the time-consuming procedures for the test and the more

expensive equipment required. As stated by Ulrich et al.

(1997), the method developed for quantifying reduced

inorganic sulfur from sediments and water may also be

used for estimation of sulfate reduction rates. In this case,

the methodology based on the use of radiotracers was

adapted to a simple and less time-consuming procedure.

The great advantage of these assays is the use of 35SO2�4 by

SRB and, consequently, the detection of the H235S pro-

duced, through liquid phase scintillation, in shorter peri-

ods, and with greater specificity. Hamilton et al. (1989)

concluded that sulfide levels may offer the most reliable

parameter for the detection of long-term ongoing corro-

sive processes in which SRB are implicated. Based on these

considerations, the main objective of this work was to

improve the methodology for the detection of H2S in low

concentrations, to correlate sulfate reduction rates with

SRB activity and enumeration by the MPN method.

To reach this goal, a series of experiments were con-

ducted aiming at determining ideal conditions for the

production of biogenic sulfides under conservative condi-

tions of nutritional substances in the medium. This

seemed to be a suitable simulation of environments with

low availability of nutrients, as expected in the natural

environments where SRB cells are present in sea water.

At last, this work attempted to correlate the number of

SRB cells present in distinct laboratory-made and natural

samples and the corresponding production of biogenic

sulfides. All the experiments were performed in a reaction

flask specially designed for this purpose and patented by

the authors and others at Brazilian INPI under number

PI 0904216-4 A2 (da Costa et al. 2012).

Materials and methods

Strains used in the study

For the development of the present tests, distinct strains/

samples were tested, as follows: (i) A pure culture of the

sulfate-reducing bacteria Desulfovibrio alaskensis (NCIMB

13491T or DSM 16109T) was used in radiorespirometric

test 1. This bacterium was recovered from a soured oil

well in Alaska. These are Gram-negative, vibrio-shaped

Journal of Applied Microbiology 114, 1008--1019 © 2013 The Society for Applied Microbiology 1009

J.C. de Queiroz et al. Biogenic sulfides and bacteria

and motile by means of a single polar flagellum. The car-

bon and energy sources used by the isolate, and the salin-

ity, temperature and pH ranges facilitating its growth,

proved to be typical of a partial lactate-oxidizing, moder-

ately halophilic, mesophilic and sulfate-reducing bacte-

rium. It clustered closely with Desulfovibrio vietnamensis

DSM 10520T, from a similar habitat. However, there is

sufficient dissimilarity at the DNA sequence level between

D. vietnamensis DSM 10520T and D. alaskensis strain

(10�2% similarity) to propose that it belongs to a separate

species within the genus Desulfovibrio. Based on the

results obtained, the name D. alaskensis sp. nov. is there-

fore proposed, with Al1T (or NCIMB 13491T or DSM

16109T) as the type strain; (ii) A mixed culture of anaer-

obic bacteria, obtained from the bottom of an oil/water

storage tank, without biological characterization, how-

ever, with a high sulfide production activity, was used in

radiorespirometric tests 2, 5 and 6; (iii) A natural saline

sample from an offshore platform was used in radio-

respirometric tests 3 and 4.

Culture conditions

The pure culture tested in the radiorespirometric assays

was the species D. alaskensis grown in Postgate C med-

ium in synthetic Tropic MarinTMsea water. The inocula-

tion of the cells (10% v/v), in 50-ml flasks, was carried

out to obtain a test culture aged 18 h, in the exponential

growth phase of the culture. Some variations in this

procedure were included in the present work: substitution

of the pure bacterial culture by a mixed culture of anaer-

obic bacteria and substitution of the pure bacterial cul-

ture by a natural saline sample from an offshore

platform, with a high microbiological activity. From test

to test, parameters such as volume of saline solution,

amount of Postgate C medium, number of dilutions and

time of incubation were changed. Specific objectives for

every radiorespirometric test performed are presented in

Table 1.

The steps of each radiorespirometric assay performed

are described as follows. To each test, initially, it is neces-

sary to add the culture medium (Postgate C medium) to

a glass tube, with the help of a syringe and needle; the

side and upper septa of the flask are closed; and the sys-

tem is sterilized by autoclaving at 120°C for 20 min. The

second step of the test is the addition of a 1 lCi 35SO�24

(IPEN/USP) solution to the sterilized flask, and the fur-

ther addition of a filter paper support to the flask, con-

taining 0�75 ml of a 2 mol l�1 zinc acetate solution,

through the upper septum of the flask. The filter paper

support must not touch the liquid in the flask. It is

essential to observe whether both septa of the flask are

locked, to start the introduction of nitrogen gas, to create

an anaerobic atmosphere inside the flask, suitable for

SRB cells. Immediately after, a known volume of micro-

bial culture or natural sample with biological activity is

transferred to the flask, through the side septum. The

septa of the flasks are closed with parafilm, to prevent

Table 1 Summary of the radiorespirometric tests performed

Test

Tropic marin

sea water or 3

�5% saline

solution (ml)

Postgate

medium

(ml)

Type of microbial culture or

sample

Number of

dilutions

Time of

incubation (h) Objective of the test

1 4�3 ml 0�2 Desulfovibrio alaskensis (0�5 ml) 3 4 To check the effect of dilutions in a pure

culture in the biogenic production of

sulfides

2 4�3 ml 0�2 Anaerobic mixed culture (0�5 ml) 1 4 To check the effect of dilutions in an

anaerobic mixed culture in the biogenic

production of sulfides

3 3�8 ml 0�2 Natural saline sample (1�0 ml) 2 4 To check the effect of dilutions in a natural

saline sample from an offshore petroleum

platform

4 3�8 ml 0�2 Natural saline sample (1�0 ml) 1 4 and 6 To test the effect on the incubation time

5 3�8 ml* 0�2/0�5 Anaerobic mixed culture

(1�0 and 0�7 ml)

0 6 To check more conservative conditions for

the production of biogenic sulfides. Also

to investigate the importance of Postgate

C medium. Simulation of deep-sea

conditions

6 3�8 ml 0�2 Anaerobic mixed culture (1�0 ml) 12 6 To check the reproducibility of the method

for decreasing most probable number

populations

*In this test, two sets of experiments were conducted: one using TM synthetic sea water and a second one using 3�5% saline solution.


Biogenic sulfides and bacteria J.C. de Queiroz et al.

the release of H235S, and the glass tubes are incubated at

30°C at predetermined periods. At the final incubation

period, 0�5 ml of a 6 mol l�1 hydrochloric acid solution

is introduced in the solution through the side septum of

the flask, stimulating the release of biogenic sulfides due

to the acid attack. To stimulate the release, this procedure

is conducted in a rotary shaker, at 35°C and 100 rpm,

during 16 h. At last, the filter paper supports are

removed from the tubes to have the biosulfides quantified

through liquid phase scintillation. The filter papers are

introduced in a glass flask containing 15 ml of scintilla-

tion solution. The time needed for the quantification of

biogenic sulfides was predetermined as 100 min for each

sample (counts of ß-emissions from 35S) and also for the

control flasks of standard solutions.

The results obtained can be expressed as% 35SO�24

reduced that represents a direct function of the biological

activity. Alternatively, as a mean of comparison, biologi-

cal reduction of sulfates can be correlated with MPN cells

quantification (McCrady 1915), to check the possible

correlation between the two techniques.

Each radiorespirometric test was followed by a quanti-

fication of MPN cells in the same sample or inoculum.

The tests also included radioactive and microbiological

controls. In the case of the radioactive control test, all the

reagents are added to the tubes, except the inoculum. In

the case of the microbiological control, all the reagents

are introduced, except the radiotracer.

Inoculum preparation

If necessary, when dilution of the test cultures and samples

was performed during the test, 1�0 ml of the culture was

transferred to a penicillin flask containing 9�0 ml of arti-

ficial sea water and homogenized. From that initial dilu-

tion, 1�0 ml was transferred to another flask in the same

conditions, until the final dilution needed. After each

dilution performed, the MPN of cells was performed.

Procedures for the preparation of the prototype flasks

used in the tests were performed inside a laminar flux

chamber and equipped with nitrogen gas fluxes. The

locker and rubber septum located at the top of the proto-

type flask were removed and the radiotracer was intro-

duced (35SO�24 in a known mass of a solution with a

fixed activity) directly in the solution containing the sea

water and Postgate C medium immediately, except in the

microbiological control tests. Following this procedure,

flasks were washed with 1�0 ml of sterile sea water. After

that, the filter paper support was introduced; the proto-

type was completely closed and purged with nitrogen gas

to initiate the tests. We can confirm that the total

amount of H2S produced during sulfate reduction,

released in the reaction flask, was recovered by the filter

paper. We tested more than one type of solvent solution

to wet the filter paper to recover H2S, and the shaking

conditions during H2S evolution were enough to ensure

total H2S recovery. To confirm this, we also made scin-

tillometric determinations in the remaining liquid solu-

tion to validate that all H2S produced was in the filter

paper. This information was incorporated in the present

version of the article.

Radiorespirometric assays performed

All the tests described below were conducted five times to

ensure reproducibility of the results. Results reported in

this work correspond to the average values obtained from

five equivalent experiments with standard deviations. Sul-

fide quantification by scintillometry presented a detection

limit of 4 9 10�3 cpm. These counts were converted to

nmol 35SO�24 reduced per day. Final results presented were

expressed as per cent 35SO�24 reduction. These radiorespi-

rometric tests for the detection of biogenic sulfides were

carried out with the addition of a radiotracer (35SO�24 ) to

the sample assayed at the trapping system as represented

below (Fig. 1). This prototype was protected for industrial

safety at Brazilian INPI, as previously informed.

Radiorespirometric test 1

The medium used was the synthetic sea water Tropic

MarinTM (Forsters & Smith, Rhinelander, WI, USA)

(4�3 ml) added to 0�2 ml of Postgate C medium and

0�5 ml of a pure culture of D. alaskensis. Three consecu-

tive tenfold dilutions of this solution were prepared. The

incubation period was equal to 4 h. This test was

performed according to the previous description.



MarinTM (4�3 ml) added to 0�2 ml of Postgate C medium

and 0�5 ml of a mixed anaerobic microbial culture. One

tenfold dilution of this solution was prepared. The incu-

bation period was equal to 4 h. This test was performed

according to the previous description.



MarinTM(3�8 ml) added to 0�2 ml of Postgate C medium

and 1�0 ml of a natural saline sample from an offshore

platform, with a high microbiological activity. Two ten-

fold dilutions of this solution were prepared. The incuba-

tion period tested was 4 h. This test was performed

according to the previous description.





MarinTM (3�8 ml) added to 0�2 ml of Postgate C medium

and 1�0 ml of a natural saline sample from an offshore

platform, with a high microbiological activity. One ten-

fold dilution of this solution was prepared. The incuba-

tion periods were 4 and 6 h.



MarinTM(3�8 ml) added to 0�2 or 0�5 ml of Postgate C

medium and 1�0 ml of a mixed anaerobic microbial cul-

ture. In substitution to the Tropic MarinTM, a saline solu-

tion (3�5%) was tested in the same previous conditions.

The incubation period was 6 h, for all the experiments

performed in this set.


An anaerobic culture containing SRB cells at

109 MPN ml�1 was used in this test, after serial dilutions

that produced distinct anaerobic cultures containing SRB

cells from 108 to 102 MPN ml�1. The medium used was

the synthetic sea water Tropic MarinTM(3�8 ml) added to

0�2 ml of Postgate C medium and 1�0 ml of each diluted

mixed anaerobic microbial culture containing SRB cells.

The incubation period was 6 h, for all the experiments

performed in this set.

A summary of all the experiments can be found in

Table 1.

Results

For the first set of radiorespirometric assays, we expected

to find a direct correlation between 35SO�24 reduction and

MPN quantification. However, from the results obtained,

we could observe that it was not easily observed due to

the uncertainty associated with cell quantification by

MPN technique.

Figure 2 presents the results obtained from test 1 in

terms of 35SO�24 reduction, against the corresponding

MPN results used in the same test.

The results indicate a decrease in the per cent values of

sulfate reduced ranging from 4�5 to 3�5 9 10�4%, for the

three dilutions made; however, it can be seen that these

results were statistically significant only from the nondi-

luted to the first dilution. For the remaining dilutions,

the results were statistically equivalent.

Rubber cover with aluminum seal

Line to fix the filter paper

Filter paper with H2Srecovery solution

Glass tube with upperand side openings

Rubber cover withaluminum seal

Palstic catheter for N2 injection

Side opening for introduction ofreagents and the plastic catheter

Sample and/or standards forthe metabolism of the radiotracer

H2S

Figure 1 Schematic representation of the trapping system used in

the development of the radiorespirometric assays of 35SO�24 reduction

by sulfate-reducing bacterial cells present in natural samples.

0

1

2

3

4

5

75000 47·5 55 5·5MPN/ml

% 3

5 SO

4–2 R

educ

ed ×

10–

4

Figure 2 Radiorespirometric test 1. Conditions: synthetic sea water

Tropic MarinTM solution, Postgate C medium, Desulfovibrio alaskensis

pure culture, incubation period of 4 h, three serial tenfold dilutions.



The results observed for the MPN of sulfate-reducing

bacteria showed a concentration of 7�5 9 104 MPN ml�1,

in the first sample. For the remaining dilutions, cell

concentrations ranged from 55 to 5�5 MPN ml�1, a fact

that was not expected, as serial dilutions were performed.

In the second radiorespirometric test, we expected to

observe 35SO�24 reduction by a mixed microbial anaerobic

culture in comparison with the previous experiment per-

formed with the use of a pure culture of Desulfovibrio

alaskensis. According to our expectation, 35SO�24 reduc-

tion was much more pronounced in comparison with the

previous experiment.

In test 2, however, just one dilution of the inoculum

was performed. The results from radiorespirometric test 2

are presented in Fig. 3. The results indicated a markedly

higher sulfate reduction by the mixed anaerobic bacterial

population: 18�5 9 10�4% for the original bacterial

culture and 9�0 9 10�4% for the diluted bacterial culture.

In the third radiorespirometric test, authors investi-

gated 35SO�24 reduction obtained by a natural saline sam-

ple from an offshore platform, probably associated with a

more specific and active microbial consortium, able to

reduce sulfate at rates considerably higher than previously

observed.

Analogously, results from test 3 are presented in Fig. 4.

The results obtained in this test indicated a much higher

microbial sulfate reduction, markedly greater than the

results obtained with pure and mixed microbial cultures.

The sample that was used as inoculum presented a high

population of SRB cells and a high sulfate reduction

activity, equal to 366 9 10�4% without dilution and 183

and 22�4 9 10�4% after the subsequent tenfold dilutions

and 4 h of incubation.

After concluding that the anaerobic consortium was

much more active for 35SO�24 reduction, in comparison

with the previous investigated conditions, we decided to

study the effect of more conservative conditions on35SO�2

4 reduction. This would bring valuable informa-

tion about a more realistic condition, probably found in

deep-ocean environments.

Again, during test 4, the effect of the dilution of the

sample was tested, to evaluate the performance of the

method under more conservative conditions (lack of

nutrients for microbial growth, simulating seawater

conditions). Results are presented in Fig. 5.

In order to try to optimize 35SO�24 reduction, based on

the previous results obtained, we decided to perform the

fifth radiorespirometric assay, combining optimized

25

20

15

10

5

0

1250 225MPN/ml

% 3

5 SO

4–2 R

educ

ed ×

10–

4


Tropic MarinTMsolution, Postgate C medium, mixed anaerobic culture,

incubation period of 4 h, one tenfold dilution.

% 3

5 SO

4–2 R

educ

ed ×

10–

4

500

450

400

350

300

250

200

150

100

50

0

12·5 1·5 2·25MPN × 103/ml


Tropic MarinTMsolution, Postgate C medium, natural saline sample

from an offshore platform with high microbiological activity, incuba-

tion period of 4 h with two tenfold consecutive dilutions.

% 3

5 SO

4–2 R

educ

ed ×

10–

4

MPN × 105/ml

40

35

30

25

20

15

10

5

0

3·7547·5125 1·25


Tropic MarinTMsolution, Postgate C medium, natural saline sample

from an offshore platform with high microbiological activity, incuba-

tion periods of 6 h (1st and 2nd Bars) and 4 h (3rd and 4th bars)

with one tenfold dilution for each incubation period.



factors, such as inoculum, time, carbon source and salts

concentration.

Results from test 5 are presented in Fig. 6. The results

presented in Fig. 6 indicated, for all the tests performed,

a high microbial reduction of sulfates, in comparison

with all the previous tests. This is probably due to a com-

bination of factors that were changed in test 5, based on

previous tests: (i) The amount of inoculum used in the

tests, equal to 0�7 or 1�0 ml, was higher than the previous

ones; (ii) Time of incubation was 6 h; (iii) The increase

in the amount of carbon source (Postgate C medium)

was associated with a decrease in the total volume of

reaction medium; and, (iv) Change in the medium was

due to the substitution of Tropic MarinTMsea water by

saline solution.

As it was previously observed, those sulfate reduction

rates are dependent on a series of parameters and envi-

ronmental conditions; thus, a new test was planned under

optimized conditions (radiorespirometric test 6).

In this test, we worked with a mixed anaerobic culture

with a highly active sulfate-reducing activity. This culture

presented a SRB population equal to 108 MPN ml�1;

after serial dilutions, subcultures were produced with

concentrations up to 102 MPN ml�1. After that, with

each dilution obtained, a new radiorespirometric test was

performed, including the optimized conditions obtained

in previous tests. The results obtained are presented in

Figs 7 and 8.

From Fig. 7, it is clearly observed that for cell concen-

trations ranging from 102 up to 105 MPN ml�1, accord-

ingly sulfate reduction took place, indicating that for this

population range, sulfate reduction can be correlated with

SRB cell numbers. In addition, from sulfate reduction

measurements, an approximate estimation of SRB cell

numbers can be achieved. Of course, the idea is to have a

fast procedure for the quantification of sulfide produc-

tion, irrespective of the cell number present in the sam-

ple. However, the opposite procedure can be performed:

from sulfate reduction rates, an estimation of the number

of active microbial cells can be achieved, to evaluate

possible biocide treatments.

From Fig. 8, the same observations from Fig. 7 apply,

however, with a different correlation than that previously

observed. Here, it can be seen that for SRB concentra-

tions higher than 105 MPN ml�1, the reduction in sulfate

does not fit the exact pattern, as previously observed.

% 3

5 SO

4–2 R

educ

ed ×

10–

4

MPN × 105/ml4·75 47·5 3·752·25

350

300

250

200

150

100

50

0

Figure 6 Radiorespirometric test 5. Conditions: 1st bar: saline solu-

tion (3�5%), 0�2 ml of Postgate C medium and 0�7 ml of mixed

anaerobic culture; 2nd bar: saline solution (3�5%), 0�5 ml of Postgate

C medium and 0�7 ml of mixed anaerobic culture; 3rd bar: saline

solution (3�5%), 0�2 ml of Postgate C medium and 1�0 ml of mixed

anaerobic culture; 4th bar: saline solution (3�5%), 0�5 ml of Postgate

C medium and 1�0 ml of mixed anaerobic culture.

y = 6·3827e1·4448x

R2 = 0·9543

y = 1·1575e0·7542x

R2 = 0·9596

1·0E + 00

1·0E + 01

1·0E + 02

1·0E + 03

1·0E + 04

1·0E + 05

1·0E + 06

0 1 2 3 4 5 6 7 8

Figure 7 Radiorespirometric test 6. Correlation between most proba-

ble number technique and per cent sulfate reduced, for microbial

concentrations found up to 102–105 MPN ml�1 (x axys). Dark circles

(MPN ml�1) and open circles (% sulfate reduction). MPN, most

probable number.

y = 760138e1·0424x

R2 = 0·9268.

y = 154·71e–0·371x

R2 = 0·7488

1·0E + 00

1·0E + 02

1·0E + 04

1·0E + 06

1·0E + 08

0 1 2 3 4 5 6

Figure 8 Radiorespirometric test 6. Correlation between most prob-

able number technique and per cent sulfate reduced, for microbial

concentrations found higher than 105 MPN ml�1 (x axys). Dark circles

(MPN ml�1) and open circles (% sulfate reduction). MPN, most

probable number.



Anyway, it can be seen that if we consider the errors

associated with the quantification of SRB cells, sulfate

reduction rates can be correlated with cell numbers, as

well.

Discussion

Before discussing the main results obtained, a few statisti-

cal concepts involved in the application of MPN tech-

nique will be presented, according to procedures adopted

by the United States Food and Drug Administration

(2010).

The 95% confidence intervals in the MPN tables indi-

cate that before tubes are inoculated, there is a chance

that at least 95% of the confidence interval associated

with any result obtained will enclose the actual concen-

tration of cells. Consequently, there are many possibilities

of intervals that meet this criterion. de Man (1983) sug-

gested to calculate confidence limits iteratively from the

smaller to the higher concentrations. However, there is

an increasing tendency to estimate cell concentrations

(when MPN technique is selected) based on slight shifts

in intervals by iterating from the greater to the smaller

concentrations.

It is widely known that a MPN can be obtained for

any number of tubes and dilutions, and it is also known

that MPN based on three dilutions corresponds to very

close approximations to the procedure based on four or

more dilutions. Several possibilities arise from this

assumption, such as one or more dilutions can show all

tubes positive, or no dilutions show all tubes positive.

Conventionally, the available methods require that no

excluded lower dilutions may have any negative tubes.

However, based on the procedure suggested by FDA,

when the highest dilution that makes all tubes positive

follows a lower dilution that has one or more negative

tubes, no tubes should be excluded. This is performed to

reduce underestimations in the detection of target groups

of microbes.

This procedure, however, must be used considering

low and high confidence values. So, in the present work,

although average values are reported, the confidence lim-

its were considered; in these cases, distinct results

reported are statistically different results.

The most common problem with SRB in offshore sys-

tems is sulfide corrosion. Monitoring of SRB has been

almost solely by counting of bacteria and chemical analy-

sis of the bulk phase. However, bacterial counts give little

information on the in situ bacterial activity, that is, the

sulfate reduction/sulfide production rates. This technique

that uses labelled sulfate has been implemented, to allow

for the determination of SRB activity, trying to correlate

activity with microbial numbers.

The data from Fig. 2 confirm the difficulty to correlate

the two distinct techniques used; the liquid phase scintil-

lation is a highly precise technique, while the most prob-

able number technique is associated with a high level of

uncertainty, particularly for small cell concentrations, as

those observed in this test.

These results indicated that it was not recommended

to work with such number of dilutions, under the condi-

tions here stated. Those conclusions were partially solved

in the radiorespirometric test 2, performed with the use

of a mixed culture of anaerobic bacteria, under the same

conditions used in the previous test 1.

One more time, from the results presented in Fig. 3, it

was not observed a direct and perfect quantitative corre-

lation between MPN of cells and sulfate reduction. How-

ever, a good correlation could be reached. This

conclusion could be seen because the bacterial population

decreased five times after dilution and the same level of

sulfate reduction was not observed. Again, it is important

to emphasize that these results were obtained in the pre-

viously described conditions of the test, and it must be

considered that not all the cells in the culture are present

in the same level of metabolic activity.

From test to test, the optimization of distinct parame-

ters was performed to reach a closer correlation between

sulfate reduction and microbial quantification.

If results from test 1 are compared with the results

obtained in test 2, it can be observed a higher microbial

sulfate reduction in test 2, indicating that the mixed cul-

ture is presently metabolically more active than the pure

culture of Desulfovibrio alaskensis, even though it is

known that the mixed culture is not exclusively consti-

tuted of sulfate-reducing species. These results, however,

indicate an expected microbial activity, although not

directly and quantitatively proved: serial dilutions are fol-

lowed by a decreasing sulfate-reducing activity. This is

the expected result that confirms that the use of the

MPN technique, without a combination with another

technique, is not recommended. As known, MPN tech-

nique is a time-consuming technique, beyond being

highly imprecise due to the high errors associated. Thus,

the use of an accessory chemical technique that could be

used to indicate the presence of metabolic activity associ-

ated with SRB cells can lead to a fast and time-saving

procedure.

Considering the results obtained from test 1 (pure cul-

ture of SRB) and test 2 (mixed culture of anaerobic bac-

teria), test 3 was planned. The objective of this test was

to observe whether it would be possible to quantify the

biogenic sulfides produced by a natural saline sample,

collected from an offshore petroleum platform, probably

with a high population of microbial cells, particularly

SRB cells. This would serve as a standard for natural



samples from several similar environments, to detect the

potential of those samples to produce biogenic sulfides,

irrespective of the number of cells present in the samples.

The decreasing microbial sulfate reduction was not

quantitatively followed by the SRB number detected,

although the qualitative behaviour followed the expected

profile. Based on these results, test 4 was performed with

a natural saline sample from an offshore platform, again

during 4 and 6 h of incubation. This was performed to

check the need for higher incubation periods and the cor-

responding sulfide production. Under the present condi-

tions of test, it could be confirmed that the best

incubation period for radiorespirometric assays of micro-

biological sulfate reduction is 6 h, both for pure and

mixed cultures, as well as for natural samples with a high

biological activity.

The natural sample used in test 4 presented a higher

microbiological sulfate-reducing activity after 6 h of

incubation in comparison with 4 h, both for the diluted

and nondiluted samples tested. Results obtained after

6 h of incubation presented markedly higher microbio-

logical activity, emphasizing the importance of the incu-

bation period of the samples. A comparison between

results from test 4 and test 3 confirmed that an increase

in the microbial population present in the sample not

necessarily produces a higher amount of biogenic

sulfides.

For instance, microbial populations in test 3 ranged

from 2�25 to 12�5 9 103 MPN ml�1, and the corre-

sponding biological reduction in sulfates ranged from

22�4 to 366 9 10�4%35SO4. On the other hand, microbial

populations in test 4 ranged from 1�25 to

125 9 105 MPN ml�1, and the corresponding biological

reduction in sulfate ranged from 1�9 to

28�1 9 10�4%35SO4. This clearly indicated that the

higher population from test 4 produced less sulfide than

the smaller population from test 3. These results corrobo-

rate the importance of the quantification of the metabolic

product and not necessarily the microbial population;

this can be high, but with a decreased biological activity.

After concluding that 6 h was a better incubation time,

the next test (test 5) aimed at introducing changes in the

composition of the medium, for a more conservative

environment. To reach this goal, Tropic MarinTMsea water

was substituted by a saline solution (3�5% w/v) and the

amount of Postgate C medium used was also changed,

consequently changing the amount of carbon source for

microbial reduction of sulfates. However, it is important

to emphasize that without a minimum amount of carbon

source, no microbiological sulfate reduction could be

possible. To reach this goal, the objective of this test was

to verify whether the water sample itself, when trans-

ferred to the reaction flask, could supply the reaction

with the adequate amount of carbon for bacterial growth.

The importance of this procedure is to simulate seawater

conditions, where the availability of carbon source for

SRB cells is limited. Beyond these conditions, this proce-

dure would facilitate the preferential use of 35SO�24 by

SRB cells, once the availability of 32SO�24 would also be

reduced.

The change in the composition of the medium from

Tropic MarinTMsea water to saline solution in the pres-

ence of 0�2 ml of Postgate C medium (Fig. 6, 1st and 3rd

bars) did not contribute to a higher microbial reduction

of sulfates, although a higher amount of mixed anaerobic

culture has been used. It can be concluded that sulfate

reduction was observed in various media, including a sal-

ine solution with a low availability of carbon. This med-

ium contributed to the preferential use of the radiotracer

by the bacterial cells.

On the other hand, the microbial reduction of sulfates

was markedly increased when the saline solution was

tested, in higher concentrations of Postgate C medium. A

possible explanation for this fact is that the mixed culture

used in these experiments was obtained from a natural

environment, where the availability of organic substances

is quite limited. It can be thus concluded that under

these conditions, we tried to simulate a conservative nat-

ural environment that contributes to the maintenance

and activity of this type of microbial population, where

the cells were already adapted. This is a possible explana-

tion for higher sulfide production under more conserva-

tive conditions if compared with previous experiments

performed. However, it was still necessary to find what

type of correlation could be envisaged between sulfate

reduction and the amount of microbial cells responsible

for this reduction.

From 102 to 105 MPN ml�1 of an anaerobic mixed

culture, sulfate reduction took place, following the same

behaviour as observed for cells serial dilutions. This is an

indication of the wide range of sample applications of the

technique. For higher SRB cell concentrations, this equiv-

alent production of sulfides was not directly observed,

only after considering the errors associated with cell

quantification. Thus, qualitatively it was possible to cor-

relate microbial reduction of sulfates (activity) and MPN

results for samples under 105 MPN ml�1.

Ulrich et al. (1997) compared a passive extraction

method and a distillation method described by Fossing

and Jorgensen (1989) and found virtually identical results

in highly and moderately active sediments, with some

advantages of passive extraction, as the capacity to

process a large number of samples in a short time.

In a comparison among radiotracer techniques, Meier

et al. (2000) discussed three different procedures to quan-

tify sulfate reduction rates – two passive extractions, as



described by Rosser and Hamilton (1983) and Ulrich

et al. (1997), and reflux distillation, as suggested by Fos-

sing and Jorgensen (1989). They concluded that sulfate

reduction rates are reproducible both for passive and

active extraction methods for recovery of reduced 35S-sul-

fur. They consider that the diffusion procedure of Ulrich

et al. (1997) is a quick and simple method for the extrac-

tion of total reduced inorganic sulfurs, with a good

efficiency, comparable with that of the distillation

procedure.

Maxwell and Hamilton (1986) used the method pro-

posed by Rosser and Hamilton (1983) with the addition

of a 3�0 9 1�0 9 0�5 cm 50D mild steel corrosive cou-

pon. They described that the applicability of this assay to

high sulfide production systems, as water injection sys-

tems and oil storage cells, must be carefully assessed. It is

because the filter paper strip can be saturated with sul-

fide. Furthermore, it is possible to face a decrease in sul-

fate reduction rates, due to a nutrient limitation in the

closed assay system. This is a possible explanation for the

results observed in Fig. 8.

Hardy and Syrett (1983) tested a rapid and sensitive

method for measuring respiration of SRB with a paper

wick containing zinc acetate to trap the labelled sulfur

produced by biological activity. Authors observed that

with a contact time of 2 h, it was possible to observe dis-

tinct inhibiting effects of quaternary ammonium com-

pound–based products. The technique could be used to

assess inhibitor efficiencies in <6 h.

Rosser and Hamilton (1983) detected labelled reduced

sulfur species in sediments contaminated with domestic

and industrial effluents, with an efficiency of 99�3%.

Maxwell (1986) used the radiorespirometric technique

to assess SRB-mediated corrosion concluding that it is

not possible to correlate microbial production of sulfide

and corrosion rates, due to the chemical and physical

nature of the materials tested, that play a more important

role. The author indicates that understanding of the pro-

cesses taking place within a particular system, associated

with the detection of microbial activity measured by radi-

orespirometric techniques, can help in the remedial mea-

sures to be carried out, with a greater assurance than the

conventional methods.

Maxwell and Hamilton (1986) proposed a modified

radiorespirometric assay for determining sulfate-reducing

activity on biofilms formed on metal surfaces. They

concluded that the great advantage of the proposed

technique is such that the biofilm can be studied with-

out removing it from the surface. The authors obtained

the recovery of practically all sulfide produced by a

mixed culture containing SRB cells. However, no

attempts to correlate their results with cell numbers were

reported.

Hamilton et al. (1988) studied the mechanism of

anaerobic microbial corrosion in the marine environment

using radiorespirometric techniques, concluding that only

a qualitative agreement is possible between sulfate reduc-

tion and corrosion rates, probably due to the cathodic

protection. The authors indicate that knowledge of the

microbial numbers is needed to confirm this, but empha-

sizes that measuring microbial activity is more important

than microbial numbers.

The work of Mckenzie and Hamilton (1992) described

the use of a mixed culture of SRB cells, containing

2�5 9 108 cells ml�1, obtained from sediments from the

vicinity of an oil production platform. Authors observed

a 65% sulfide recovery. If we compare their results with

the ones obtained in the present work, we can see that

the closest correlation can be found in data from Fig. 7.

However, attempts to correlate their results with the pres-

ent ones must consider not only the size of the inoculum

but also the initial concentration of labelled sulfate in the

medium and the chemical and physical nature of the

samples used.

Edenborn and Brickett (2001) investigated the activity

of immobilized SRB cells with the sulfide production

from microbial cultures in wetland sediments. The

sediment cores were obtained from an underground

mine complex with a SRB concentration of

4�5 9 106 cells cm�1 of gel probe. In that work, authors

observed a 98�5% sulfate reduction, equivalent to the

results obtained in the present work. Although the results

obtained by those authors are quite similar to the ones

obtained in the present work (Fig. 7), if we consider the

concentration of sulfate used by those authors (6 lCi oflabelled suldate), we can conclude that our results were

much better, considering per cent sulfate reduction, from

a more diluted solution, indication a much lower

detection of biogenic activity.

However, we did not find similar articles in the scien-

tific literature to compare the present tests performed.

Papers found based on the use of this methodology

included experiments under distinct conditions, with a

different prototype reaction flask, thus making any com-

parison unsuccessful. No correlation between MPN tech-

nique and radiorespirometric techniques were found.

That is why, in the present article, authors highlight the

results for field applications due to its precision, safety,

time-saving operation and novelty for the petroleum

industry.

The radiorespirometric assay proved to be a useful tool

for the quantification of the microbial sulfate-reducing

activity, both for synthetic and natural samples. The great

advantage of the present methodology is the possibility of

quantification of microbial activity in a maximum of

48 h, in comparison with the conventional 672 h



(28 days) of the MPN technique, conventionally used for

the determination of the sulfate-reducing activity.

The obtained results showed a good detection limit,

making it possible the evaluation of the presence of

SRB in concentrations not detected by conventional

methods.

The radiorespirometry proved to be an excellent and

rapid alternative to quantify microbial sulfate-reducing

activity, irrespective of the knowledge available about the

microbial population.

Acknowledgements

The authors would like to thank Comiss~ao Nacional de

Energia Nuclear for a scholarship, Instituto de Pesquisas

Energ�eticas e Nucleares/Universidade de S~ao Paulo for

providing the radiotracer, Petrobras and Financiadora de

Estudos e Projetos for previous financial support to work

in this field.

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Radiorespirometric assays for the detection of biogenic sulfides from sulfate-reducing bacteria

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