Desalination 248 (2009) 377–383

Sulphate reduction and biomass growth rates

for Desulfobacterium autotrophicum in yeast
extract – Supplemented media at 38C

C. Sáez- Navarretea , A. Zamoranob, C. Ferradab, L. Rodrı́guezb

a,b
Department of Chemical and Bioprocess Engineering, College of Engineering, Pontificia Universidad
Católica de Chile, Santiago 7820436, Chile
Tel. þ56 2 354 4927; Fax þ56 2 354 5803; email: C.Saez-Navarrete@ed.ac.uk
Received 31 January 2008; revised accepted 15 May 2008

Abstract

Water pollution by heavy metals and sulphates is common in areas with mineral deposits. Sulphate-reducing bacteria (SRB)
are microorganisms widely distributed, and can be used as heavy metals removal agents. Furthermore, the use of organic substrates
or wastes in SRB systems could reduce the cost and increase the treatment efficiency. The aim of the study was to assess the sul-
phate reduction capacity and growth rates of the bacterium Desulfobacterium autotrophicum in batch culture media supplemented
with different concentrations of yeast extract. At 38 C, in the growth phase, there was a direct relationship between the concen-
tration of yeast extract employed and the biomass growth and sulphate reduction rates. In the presence of low sulphide concentra-
tions, supplementary 0.5 g/L of yeast extract maximized sulphate reduction on a per-cell basis, but at high concentrations of H2S it
would be advisable to use 2 g/L to reduce the product inhibition.
Keywords: Sulphate-reducing bacteria; Sulphide production; Microbial growth rates; Hydrogen; Inhibition; Temperature

1. Introduction electrons from thiosulphate, sulphite, dithionate,

trithionate or elemental sulphur [2]. In nature, SRB are
Sulphate-reducing bacteria (SRB) are anaerobic
commonly found in marine sediments, in volcanic eco-
microorganisms that employ sulphate as an electron
systems, in the rumen of a number of mammalian spe-
acceptor terminal to produce hydrogen sulphide. SRB
cies as well as in some geothermal waters. Some
can grow heterotrophically or in an autotrophic med-
groups are also found in sulphur-contaminated waste-
ium using H2 as electron donor and CO2 (exclusively
water. Nevertheless, in terms of sheer volumes, mining
or mixed with CO) as the carbon source [1]. In addition
is the principal source of water and soil pollution invol-
to reducing sulphate, most SRB can also accept
ving oxidised sulphur species and heavy metals [3–5].
Biological reduction of sulphate produces hydro-
 gen sulphide (H2S), which is used for separating heavy
Corresponding author.

Presented at the Water and Sanitation in International Development and Disaster Relief (WSIDDR) International
Workshop Edinburgh, Scotland, UK, 28–30 May 2008.

0011-9164/09/$– See front matter © 2009 Published by Elsevier B.V.

doi:10.1016/j.desal.2008.05.078
378 C. Sa´ez-Navarrete et al. / Desalination 248 (2009) 377–383
metals in liquid residues, precipitated as sulphides
[6–8]. Other applications include the production of
commercially viable molecules and the recovery of
many metals [9–11]. SRB metabolism results in alkali-
nity (because of H2S production consumes protons in
the media) that can be used to neutralize acidic treat-
ment flows [6,12].
Anaerobic processes and SRB activity are strongly
temperature dependent [13]. For the strain Desulfobac-
terium autotrophicum, optimal autotrophic growth is
reputedly attained between 25 C and 28 C (at pH
6.7) [14]. In studies of these bacteria in heterotrophic
media, the optimal temperature for growth is 28 C,
although the highest sulphate reduction rates occur
between 36 C and 40 C [15]. The effect on per-cell
sulphate reduction rates is not clear, however, nor is the
combined effect of adding yeast extract supplements to
the culture medium at the higher temperature range
mentioned.
For continuous SRB systems, using autotrophic
media sulphate reduction rates of 30,000 mg/L/d have
been obtained at 30 C [16] and of 7500 mg/L/d at 55 C
[17]. From heterotrophic media, on the other hand,
reported sulphate reduction rates are 475 mg/L/d at
25 C [7], and 6330 [18] and 3700 mg/L/d [19] both
at 30 C. Using H2 as the energy source and sodium
acetate as the carbon source, a sulphate reduction rate
of 4800 mg/L/d at 30 C [9] was obtained in a mixo-
trophic medium. In studies with the strain D. autotro-
phicum, sulphate reduction rate of 109 mg/L/d at
30 C, was achieved in a batch bioreactor using an auto-
trophic medium [20]. Sulphate reduction rates are gen-
erally reported in terms of total cellular density of the
given system. Per-cell reduction rates will vary in sus-
pended or stationary cultures and, consequently pro-
vide a less ambiguous way of comparing system
behaviour under different conditions.
Sulphides, the metabolic products of sulphate
reduction, inhibit SRB growth and activity [21]. It has
been reported hydrogen sulphide to be the most toxic
form of sulphur for SRB, but also has been found the
effects to be reversible [22]. Other author observed
an inverse relationship in which as sulphide concentra-
tions increased (up to a limit of 280 mg/L) cell size
diminished. Beyond this concentration the size of the
cell remained constant, but yields fell sharply [23].
In regard to how supplementary yeast extract
functions in the media, differences in the way the
culture medium’s inorganic carbon source was used
at different supplement concentrations and preferred
carbon uptake from the yeast extract have been
found [24]. There have been no reports, however,
of how supplementary yeast extracts affect sulphate
reduction. It is important to optimize reduction rates
when developing technologies for bioproducing H2S
using SRB. The aim, therefore, of the present study is
to assess sulphate reduction and cellular growth rates
of the bacterial strain D. autotrophicum in yeast
extract-supplemented autotrophic culture media at
38 C. The temperature was selected based on the
research developed for Rabus et al. [15].
2. Materials and methods
2.1. Culture medium
The autotrophic culture medium composition, used
in both the preparation of the inoculum and the subse-
quent experiments, was as follows (per liter of distilled
water): 21 g of NaCl; 3 g of MgCl2  6H2O; 3 g of
Na2SO4; 0.5 g of KCl; 0.3 g of NH4Cl; 0.2 g of
KH2PO4; 0.15 g of CaCl2  2H2O; 1 mg of resarzurine;
2.5 g of NaHCO3; 0.4 g of Na2S  9H2O; 1 mL of trace
element solution; 10 mL of Wolfe vitamin solution;
and 1 mL of a solution comprising 3 mg Na2SeO3 
5H2O in a liter of NaOH 0.01 M. The trace element
solution contained the following (per liter of distilled
water): 1.5 g/L FeCl2  4H2O; 0.19 g/L CoCl2 
6H2O; 0.1 g/L MnCl2  4H2O; 0.07 g/L ZnCl2;
0.036 g/L Na2MoO4  2H2O; 0.024 g/L NiCl2 
6H2O; 6 mg/L H3BO3; 2 mg/L CuCl2  2H2O; 10
mL/L HCl (solution at 25%). The ferrous chloride was
dissolved in 10 mL of hydrochloric acid, and then
diluted with water whereupon the remaining com-
pounds were added. The solution was mixed with a
magnetic stirrer, and then adjusted to pH 6.0 with
NaOH. Finally, the Wolfe vitamin solution contained:
0.01 g/L pyridoxine  HCl; 5 mg/L thiamine  HCl; 5
mg/L riboflavin; 5 mg/L nicotinic acid; 5 mg/L
calcium pantotenate; 5 mg/L p-aminobenzoic
acid; 2 mg/L biotin; 2 mg/L folic acid and 0.1 mg/L
of cyanocobalamine. The yeast extract-supplemented
 C. Sáez-Navarrete et al. / Desalination 248 (2009) 377–383
autotrophic culture solutions are the result of adding
specific amounts of yeast extract to the autotrophic
solution described above.
2.2. Experimental setup
Samples of the bacterium D. autotrophicum HRM2
(DSM 3382) were obtained from the American Type
Culture Collection. The bacteria were cultivated in a
1.5 L glass batch bioreactor in a liter of lactateless
ATCC 1875 culture medium. Experiments were con-
ducted in 250 mL glass batch bioreactors containing
190 mL of previously sterilized autotrophic or yeast
extract-supplemented media and 10mL of inoculum
at a concentration of 5  107 cells/mL. Experiments
were performed in duplicate with yeast extract concen-
trations of 0, 0.1, 0.5, 1 and 2 g/L. Each bioreactor had
two glass tubes inserted into a rubber stopper for the
hydrogen feed and for purging hydrogen sulphide.
After sampling, the bioreactors were cleared using
nitrogen for two minutes to displace the H2S produced
and to reduce inhibitory effects. Once the hydrogen
sulphide was liberated, the bioreactors were filled with
hydrogen slightly above atmospheric pressure (from 1
to 1.5 psig). Immersed in a thermo-regulated Memmert
bath, model WB45, the bioreactors were held at a con-
stant 38 C. Each time a sample was taken bioreactor
pH was adjusted, by adding sufficient HCl 1 N, to
pH 7.0. Experiments were run for 8 days to ensure each
biomass completed the growth phase.
2.3. Analytical methods
Biomass concentration was measured periodically
through the direct count of bacteria in each bioreactor,
using a Neubauer camera linked to a Leite Biomed
optical microscope.
Sulphate and sulphide concentrations were deter-
mined in a HACH, model DR2010, spectrophot-
ometer. Sulphate concentration was measured
periodically using the manufacturer method 680
(equivalent to USEPA 375.4 for wastewater), which
is precise to +0.9 mg/L for sulphate yet shows inter-
ference in the presence of calcium, chlorine, magne-
sium and silica (quantified as CaCO3) at
concentrations above 8000, 14,000, 2400 and 500
mg/L, respectively. Sulphide concentration, S2, was
379
Fig. 1. Biomass growth.
measured at the beginning and end of each run using
manufacturer method 690 (equivalent to methylene
blue and USEPA 376.2 for wastewater). Method 690
is precise to +0.003 mg/L for sulphur and is subject
to interference from strong reducing anions, such as
sulphites, thiosulphates and hydrosulphites.
The concentration of aqueous hydrogen sulphide
(H2S) in the system was estimated with a non-
stationary, phenomenological model that considered
bioproduced H2S, ionic equilibria, ionic forces in the
media and the gas–liquid mass transfer. As bioreactor
pH varied from pH 7 (following post-sampling adjust-
ment) to pH 8.5 (prior to sampling), only the first ioni-
zation of H2S was considered when determining the
H2S/HS relationship. Equilibrium constant adjust-
ments for the ionic forces in each medium were derived
from the classical theory of Debye–Hückel. The com-
bined gas–liquid mass transfer coefficient for H2S, kLa,
was determined experimentally, obtaining a value of
7.14  104s1 [25].
3. Results
3.1. Cellular growth rates
Temporal cellular concentrations are presented in
Fig. 1 for each bioreactor over the duration of the
experiments, all of which were conducted at 38 C.
Each data point is the average of the results from dupli-
cated bioreactor experiments and dual-counted
380 C. Sa´ez-Navarrete et al. / Desalination 248 (2009) 377–383
Fig. 2. Maximum microbial growth rates for the growth
phase.
samples, for the various concentrations of yeast
extract-supplemented media studied, with their respec-
tive standard deviations. Increased cellular density was
found at higher concentrations of supplementary yeast
extract in the culture media, particularly in the first 3
days, although by the end of the experiment for the
bioreactors containing 1 and 2 g/L, cell densities were
similar. Plots reveal similar behaviour of weak cellular
growth for the bioreactors containing 0 and 0.1 g/L of
yeast extract. The 0.5 g/L yeast extract-supplemented
bioreactors produced a similar profile to the experi-
ments conducted with concentrations of 0 and 0.1 g/L.
It seems most of the cultures needed a day to acclima-
tize to the higher temperature although the bioreactor
containing 2 g/L adjusted quicker.
Peak biomass growth rates, and when they occurred
during the experiments in the growth phase, are pre-
sented in Fig. 2. Microbial growth rates increased in
stronger concentrations of yeast extract-
supplemented culture. The relationship was not linear,
however.
Taking the bioreactor containing 1 g/L yeast extract
as a reference (since this is the commonly used concen-
tration in heterotrophic media), doubling the yeast
extract (2 g/L) only produced a 10% rise in the biomass
growth rate, whereas halving the concentration of yeast
extract (0.5 g/L), the cellular growth rate was 60%
lower. Growth rates declined by 77% and 87% at yeast
Fig. 3. Sulphate concentration.
extract concentrations of 10% (0.1 g/L) and in the auto-
trophic media (0 g/L), respectively.
3.2. Sulphate reduction
All cultures started with a sulphate concentration of
approximately 2200 mg/L. Although none of the media
reduced sulphate the first day, by day 8, concentrations
ranged from 975 to 2180 mg/L, see Fig. 3. The pure,
autotrophic media (0 g/L) reduced very little sulphate.
A similar trend was exhibited for the culture medium
containing 0.1 g/L yeast extract, which also revealed
negligible sulphate-reducing properties.
The cultures containing 0.5 and 1 g/L yeast extract
exhibited similar sulphate reduction profiles, reducing
675 and 750 mg/L of sulphate to sulphur, respectively,
by the end of the experiment. Over the full 8 days, the
batch system containing 2 g/L yeast extract achieved
the largest total reduction in sulphate (1250 mg/L)
from an initial 2225 to 975 mg/L. Peak sulphate reduc-
tion rates (Fig. 4) were practically negligible for the
media containing 0 and 0.1 g/L yeast extract. In the
0.5 and 1 g/L bioreactors sulphate reduction rates
were over 200 mg/L/day and the bioreactors containing
2 g/L yeast extract reduced sulphate at a rate of
300 mg/L/day. Maximum sulphate reduction occurred
from days 2 to 3 in every medium containing yeast
extract, and although the differences are not easily
appreciable early on, the effects are clear by the end
of the experiments.
 C. Sáez-Navarrete et al. / Desalination 248 (2009) 377–383
Fig. 4. Maximum sulphate reduction rates.
All of the maximum per-cell sulphate reduction
rates in the yeast extract-supplemented media occurred
on the third day of the experiments. The highest per-
cell sulphate-reducing rates were attained in the culture
medium containing 0.5 g/L yeast extract that rose stea-
dily to a peak of 7.6  106 mg/cell/day (Fig. 5), but
then tailed off. Per-cell performance for the 2 g/L cul-
ture was also high (6.6  106mg/cell/day) and also
diminished, but not as quickly.
For comparative purposes, published reduction rate
figures have been converted into micrograms per liter
considering 1 gm of protein equivalent to two grams
of biomass [18], and that a cell has a mass of 1012
gm [26]. Higher per-cell sulphate reduction was
obtained for the culture medium containing 0.1 g/L
yeast extract at 38 C than Londry and Des Marais
Fig. 5. Maximum sulphate reduction per cell and biomass
growth.
381
[20] found at 30 C for D. autotrophicum (1.24 
108 mg/d/cell). Results here for the 0.5 g/L culture
at 38 C were far higher van Houten et al. [17] reported
in autotrophic conditions at 55 C from mixed strains a
values of 0.93  106 to 1.03  106 mg/d/cell. At this
supplement concentration, the rates were higher than
Sáez-Navarrete [25] who found, at 30 C 2.42  106
mg/d/cell, but lower than 9.8  106 mg/d/cell [27] at
the same temperature, and 1.39  105 mg/d/cell
[28], achieved at 20 C. In 1 and 2 g/L yeast extract cul-
tures at 38 C the sulphate reduction rates were similar
to those Sáez-Navarrete [25] encountered with a mix-
otrophic medium at 30 C.
4. Discussion
Little biomass growth (Fig. 1) or sulphate reduction
(Fig. 3) was observed in the bioreactors containing 0
and 0.1 g/L yeast extract. Our results for cellular
growth corroborated earlier findings for the auto-
trophic culture of D. autotrophicum at 38 C, but the
lack of sulphate reduction at this temperature was
unexpected. DNA profiling was performed to confirm
that the culture was indeed pure, and it did contain the
single strain D. autotrophicum. Adding 0.1 g/L yeast
extract had scant impact on either performance curve,
so neither candidate would prove useful other than for
cell maintenance. At higher concentrations, neverthe-
less, yeast extract did exert an effect upon biomass
growth and sulphate reduction rates at 38 C when
cultivating the bacterium D. autotrophicum. While the
bioreactors containing 2 g/L yeast extract achieved
the highest biomass growth rate (Fig. 2) on day 1 and
the highest sulphate-reducing rate on day 3 (Fig. 4), the
highest sulphate reduction per cell was from the media
containing 0.5 g/L (Fig. 5). Aside from the bioreactor
containing 1 g/L, optimizing on biomass growth that
occurred on different days for the distinct batch yeast
extract supplement concentrations (Fig. 2) would not
provide optimal sulphate reduction (that occurred on
day 3 for every yeast extract enriched media). Indepen-
dent of the issue of biomass sustainability, in terms of
costs and in light of the per-cell sulphate-reducing rates
encountered, 0.5 g/L would be more cost effective than
2 g/L for cultivating D. autotrophicum. In more general
terms, the optimization of the rate of overall sulphate
removal in a continuous system and high scale will
382 C. Sa´ez-Navarrete et al. / Desalination 248 (2009) 377–383
Fig. 6. Accumulated H2S during the batch experiments.
depend on the maintenance of high concentrations of
biomass to high rates of reduction of sulphate per cell.
In batch systems, this operational point matches with
the exponential cell growing. In this study, the results
show that at a yeast extract concentration media of
0.5 g/L it is produced a very satisfactory outcome with
respect to the results obtained to lower concentrations
(0 and 0.1 g/L), and the similar it was observed that
at higher concentrations of yeast extract (1 and 2 g/L).
Moreover, in a high scale system, if the organic sub-
strate is inexpensive and effluents generated can be
managed properly, the operation should be carried out
at high concentrations, which in the case studied it is
up to 2 g/L.
H2S, or the species in equilibrium (HS), at high
concentrations constrain cell size and biomass growth.
Concentrations of H2S are shown in Fig. 6 for the three
bioreactor sets containing the highest amounts of yeast
extract. Despite the evident discontinuities, all three
bioreactor systems produced a sustained increase in
H2S concentration, which demonstrates need to
improve liquid-mass transfer for clearing the system
of H2S. Studying the strain Desulfovibrio desulfuricans
in heterotrophic media and at a similar temperature
(35 C), Okabe et al. [23] found that cell production fell
slightly at a total sulphide concentration of 150 mg/L
(equivalent to 100 mg H2S/L), and that at 280 mg/L
(equivalent to 187 mg H2S/L) biomass growth declined
sharply. In the bioreactors containing 0.5, 1 and 2 g/L,
the fall in biomass growth from day 3 (Fig. 1) may be
due to the effect accumulated sulphide concentrations
had on D. autotrophicum. The minimum H2S concen-
tration from day 3 of 81 mg/L coincides with the
reports of Okabe et al. [21] as the starting point for
an impact on cellular growth (108 mg S/L, equivalent
to 72 mg H2S/L), and is just below the value the same
author found in 1992 at which yields started to drop
(150 mg S/L, equivalent to 100 mg H2S/L).
5. Conclusions
We were able to determine design parameters from
the study to guide the future development of a good,
continuous sulphate-reducing system.
Use of per-cell sulphate reduction rates should
make comparing different systems much easier with
a view to paving a way to understanding the mechan-
isms involved.
Autotrophic cultures promoted little growth and
surprisingly poor sulphate reduction. At 0.5 g/L and
above of yeast extract supplement, biomass was rea-
sonable and high sulphate reduction rates were
achieved.
Which concentration of yeast extract supplement to
use in a continuous system will depend on how accu-
mulated H2S is dealt with. In the presence of low sul-
phide concentrations, 0.5 g/L would maximize
sulphate reduction on a per-cell basis, but at high con-
centrations of H2S it would be advisable to use 2 g/L
supplementary yeast extract.
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