Professional Documents
Culture Documents
PCBR Volume 4 Issue 2 Pages 191-206
PCBR Volume 4 Issue 2 Pages 191-206
Due to the presence of sulfur bacteria in natural sulfur sources, the sulfur sources of sewage
Thiobacillus thioparus – one of the significant bacteria consuming sulfur compounds – was evaluated as
a control sample.
The performance of bacteria in different culture conditions (carbon source and aeration conditions) was
evaluated, and suitable.
Conditions for their growth were determined.
Sodium sulfide was used to create the sulfide medium.
The production of sulfur during the process was evaluated using the ANOVA data analysis method.
The optimal points for sulfur production were predicted using the Response Surface Methodology
(RSM).
GRAPHICAL ABSTRACT
192 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
sulfur granules formed by microorganisms are oxidizing ability has made it possible to use them
non-toxic and non-edible and have a high sulfur in various industries to remove sulfide in various
content, which can be widely used in the processes. All thiobacillus bacteria can oxidize
manufacture of raw materials. It is also used in sulfide to sulfate, producing elemental sulfur as
bio-leaching processes, agricultural products, an intermediate[18, 19]. The most important
and as an adsorbent for the removal of heavy factors influencing biological treatment
metals from waste [9-11]. Also, in 1990, processes are the aeration rate, the number of
environmentally friendly and cost-effective nutrients required by the bacterial culture
desulfurization processes based on medium, temperature, and pH. Among these, the
biotechnological knowledge were developed to aeration rate is one of the vital factors because
substitute conventional chemical and physical oxygen plays the primary role as the electron
methods such as the Amine Clous process[12]. receptor for the metabolism of sulfur-oxidizing
Another biological process used is the SHELL- bacteria (SOB)[20].
PAQUES Process, in which sulfide is rapidly Since many countries have to import large
absorbed from a gas into an alkaline liquid in an amounts of sulfur to meet industrial needs, which
absorption tower. The alkaline liquid is in turn increases environmental pollution from
automatically collected at the end of the tower sulfur, researchers are not only concerned about
and directly enters the second stage of the removing sulfide from pollutants in waste to
process (bioreactor). Inside the bioreactor, the protect the environment but are also concerned
adsorbed sulfide is converted to sulfur and about recovering the sulfur resources in the
sulfate. Biologically produced sulfur particles are wastewater. Many researchers have studied the
used after dewatering (sulfur sludge). The removal of sulfide in waste. Nevertheless, given
biological process of removing sulfur compounds the little research that has been done on the
is based on the biological cycle of sulfur process of biological removal of sulfide and
production[13, 14]. elemental sulfur recovery, in this study, the
The potential and ability of the hydrogen sulfide performance of different bacteria for sulfide
biological removal process are enhanced by the removal during various processes is measured to
presence of microorganisms, including a wide achieve bacteria with high biological oxidation
range of bacteria, which are classified according ability of sulfide, after cultivation and
to different criteria, and they are called sulfur- proliferation of used bacteria. After selecting the
oxidizing bacteria (SOB). Categorical criteria appropriate bacterium, different operating
include the source of carbon used, how oxygen is conditions for the selected bacterium are
consumed, the source of hydrogen consumed, as evaluated. Then, the process of removing
well as the color of bacteria, the shape, and hydrogen sulfide was performed on a laboratory
temperature of the growing medium[15, 16]. scale, and its results were analyzed using Design
Besides the fact that SOB plays an essential role Expert 10.1.1 through Central Compound Design
in H2S removal, adjusting the pH to about 8 can (CDD) method.
prevent the growth of sulfate-reducing bacteria,
which plays an essential role in H2S Materials and methods
production[17]. One of the most critical bacteria Materials
used for sulfur oxidation is thiobacillus. Standard samples were used to evaluate natural
Thiobacillus is acid-friendly bacteria that oxidize samples. The standard sample used in this study
iron or sulfur. This type of bacterium is the most was the Thiobacillusthioparus bacterium. It is the
crucial sulfur oxidizer in the soil, and their best species used in industrial applications in
193 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
194 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
195 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
196 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
197 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
Table.4 Test design for two factors using a central combination design
198 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
Fig.6. Investigating the growth of T.R.w & M.H.S Fig.7. Investigating the growth of T.R.w& M.H.S
bacteria in aerobic and anaerobic conditions bacteria in autotrophic and heterotrophic conditions
199 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
However, according to the results and At this pH, the performance of bacteria has
consumption of both carbon sources, samples changed slightly compared with the lower pH,
can be considered as mixotrophic (optional) but it can still be claimed that they have a high
bacteria. ability to remove sulfide. The performance of
these bacteria at this pH is desirable and can be
Investigation of the performance of bacteria in used on a larger scale. In a similar experiment,
alkaline PHs the results of thiobacillusthioparusare are shown
At neutral and weak acidity pH, sulfide species in Figure 9.
are often soluble in hydrogen sulfide (H2Saq, HS-). The results show that the thiobacillusthioparus
Because the species are volatile and enter the air species show good activity and growth only at
due to aeration and contact with the air, the and pH = 7 and close to it, and with increasing
performance of the process at this pH repels the pH, the yield and activity of this bacterium
sulfur gas entering the air, and the bacteria do decrease sharply. In other words, at higher pH,
not have access to the sulfide. High pH the bacterium is inactive and cannot remove the
performance not only causes the generation of sulfide in the solution. This result is especially
dominant species as S2- but also increases the noticeable at higher pH such as 10. This pH does
rate of uptake in the adsorption tower. not correctly remove sulfide and spends more
Therefore, performance at high pH is desirable. time annihilating it. Increasing the time will
On the other hand, due to the alkalinity of the drastically reduce the removal rate and
medium and poor performance or the inability of significantly increases operating and process
many species to operate in it, the possibility of costs, so it is important to use species that have a
contamination is reduced[27, 28]. The results of high sulfide removal rate.
these experiments for refinery wastewater For these reasons, as well as the poor
bacteria are shown in Figure 8. performance of the thiobacillusthioparus
As can be seen, refinery wastewater bacteria at bacterium at high sulfide concentrations, the
pH = 9 perform well, showing that sulfide bacterium has been removed from tests, and
removal rates at this pH such as pH = 7as the pH further testing will be performed for bacteria
increases to 10, and there is a difference in the isolated from the refinery's wastewater.
function of the bacteria.
200 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
201 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
the process of sulfide removal. The central the data obtained from the experiments, the
compound multi-level test design method was value of R2wascalculated for different models,
used to begin the work and determine the test and the appropriate model was selected and
levels. In this design, 9 experiments were suggested using the maximum value for it. In
performed as the main experiment of the these experiments, the proposed model was a
process, and 4 experiments were performed second-order model. The results are shown in
repeatedly to control the usefulness of the model Table.6. In the next step, using analysis of the
for the central point. Subsequently, experiments data variance, the importance of the parameters
were performed, and samples were measured to was examined. The results are shown in Table.7.
produce sulfur and sulfate. In this section, using
In this section, various parameters were using a loading rate and the amount of dissolved
examined. The higher the F-Value and the lower oxygen with good approximation.
the Prob> F values, the more critical the
parameter is, and this parameter can be called Sulfur Produce =-31.95143+0.67280* Sulfide Loading
the process-affecting parameter. From Table.6, it Rate -124.54906* O2 -0.52381* Sulfide Loading Rate *
can be concluded that the loading rate is an O2 +8.59722E-005* Sulfide Loading Rate2
influential parameter in this process (sulfur +221.93878* O2 2 (1)
production). Other parameters, such as the
amount of oxygen and the interaction of the two In the next step, the results of the prediction and
factors of loading rate and oxygen, are also useful the actual values obtained are evaluated. These
parameters. However, their importance is not results are shown in Fig.11. In Figure 11, the
equal to the mentioned parameter. actual values obtained and the predicted values
In the next step, the software provides a second- are shown as horizontal and vertical axes.
order equation Eq.1 for analyzing the data, which
can predict the amount of sulfur production
202 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
The midline indicates the complete conformity Finally, the optimal conditions for sulfur
of these two values, and the predicted results can production were examined. For maximum sulfur
be used with high accuracy to continue production in this range, optimal conditions
experiments. were analyzed, at the end of which the sulfide
The results of data analysis are shown as the loading rate was 3700 mg/L, and the oxygen
effect of these two parameters on the amount of content was0.3 mg/L. The amount of sulfur
sulfur production as a three-dimensional obtained from these conditions was equal to
procedure in Figure12. As shown in this Fig.12, 3035.55mg/L. The optimization results are
this procedure covers all points in the range shown in Figure13.
shown. Red dots are the points where the
maximum sulfur is produced. This part of the Conclusion
surface occurs at points with high sulfide loading According to preliminary experiments, some
rate and low oxygen content, which go to blue bacteria consume sulfur compounds in both
dots with decreasing loading rate and increasing samples from hot springs and sewage of Shahid
oxygen rate, which shows sulfide reduction. Tondgooyan Refinery Co. in Tehran. A
Design points above predicted value
comparison of the results of bacterial culture
Design points below predicted value
experiments in a culture medium containing
3200
thiosulfate suggests that the refinery's sewage
2400 bacteria performed well in consuming and
Sulfur Produce
1600
removing sulfide. Measurement of solution
turbidity, as well as microscopic observations,
800
1.00 3800.00
amount of sulfide emerged, the results of
0.82
0.65 2300.00
3050.00
measuring optical density and microscopic
B: O2 0.48
0.30 800.00
1550.00
A: Sulfide Loading Rate observations showed the growth and activity of
this sample.
Fig.12. 3D procedure resulting from the effect of Since bacteria are isolated from different sources,
parameters they are likely to be of different species and have
different nutritional needs. Examining these
203 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
conditions can be useful in improving the refinery's wastewater were used in subsequent
function of bacteria and identifying their needs. experiments.
One of the conditions studied is the aerobic or In the study of the concentration of sulfide on the
anaerobic mechanisms of bacteria. Some species performance of bacteria, they were exposed to
oxidize sulfur compounds in aerobic conditions, high levels of sulfide concentration. According to
in which oxygen is considered as an electron the results obtained, bacteria from the refinery’s
donor, and some species grow better in wastewater removed high sulfide concentration
anaerobic conditions, in which other compounds well and at the rate of 40 mg/h.L, and the
act as electron donors. Based on the results of the thiobacillusthioparus yielded acceptable rates.
experiments, it was observed that both samples Results in amounts were less than 1000 mg/L,
have the ability to grow in both cases, but their whereby the elimination rates were 30 mg/h.L.
growth is higher in aerobic conditions. Then a In lower amounts of sulfide, such as (250-3000
test was designed to examine autotrophy and mg), the bacteria isolated from the refinery's
heterotrophy. According to the results of the wastewater outperformed and had a higher
sample test, the used bacteria are considered to removal rate. In smaller amounts, it could be a
be among the mixotroph (optional) bacteria, good option for sulfide removal operations. At
after examining the growth conditions of bacteria the end of the experiment, sulfur production was
in the culture medium containing thiosulfate and analyzed. At this stage, with the experiments and
increasing the population of bacteria, obtaining with the help of Design Expert, sulfur production
the desired results. Because the goal was to was analyzed. The results showed that the rate of
remove sulfide, to create a sulfide medium, the sulfide loading, the amount of oxygen, and the
amount of thiosulfate was reduced, and sodium interaction of these two parameters were more
sulfide was added to the culture medium as a active in the movement of the reaction towards
sulfur source. The sulfide consumption by sulfur production. In limited amounts of oxygen,
bacteria was then evaluated, and only the such as 0.3 mg/L, and high sulfide loading rate
Thiobacillusthioparusbacteria and the bacteria such as 3700 mg/h, we obtained the optimal
from the refinery's wastewater were able to amount of sulfur, which was 3035/5 mg/L.
remove the sulfide in the aquatic medium. Due to Besides, according to the results obtained in this
the need for process performance in alkaline pH paper and investigating the effectiveness of two
to prevent hydrogen sulfide and sulfide removal factors, aeration rate, and sulfide loading rate, it
from the aquatic environment, as well as the is necessary to provide samples from various
benefits of performance in alkaline pH, attempts other sulfur sources and evaluate their
were made to evaluate performance in this pH. performance in sulfide removal. Among these
Bacteria from the wastewater of refineries at sources, we can mention the sewage of metal
higher pH did not show a functional difference. industries and other hot springs with high sulfide
This process continued until pH = 10, but in this content. It can also be suggested that the bacteria
pH, the activity of bacteria decreased but did not obtained from the T.R.W be subjected to genetic
indicate that these bacteria did not work. engineering operations and that these bacteria
However, in the thiobacillusthioparus, the be more accurately evaluated. Other operating
increase in pH severely impaired their function conditions using these bacteria were evaluated to
so that in pH 9 and 10, it can be argued that these achieve high yield and removal rates, such as
bacteria are incapable of activity. Therefore, process temperature, the effect of agitation
according to the results, the bacteria from the intensity, by these bacteria.
204 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
Given that bacteria grew well in organic matter, [6] J. Kanjanarong, B.S. Giri, D.P. Jaisi, F.R. Oliveira, P.
and functional results were obtained from it, Boonsawang, S. Chaiprapat, R. Singh, A.
Balakrishna and S.K. Khanal, Removal of
other organic carbon sources can be evaluated to
hydrogen sulfide generated during anaerobic
achieve affordable carbon resources ultimately. treatment of sulfate-laden wastewater using
On the other hand, since in industries, the biochar: evaluation of efficiency and
exhaust gases from the units that use natural gas mechanisms. Bioresource Technology, 234
as fuel contain a high concentration of carbon (2017) 115-121.
[7] S. Hall, Rules of thumb for chemical engineers.
dioxide, this type of carbon source can also be
(2017): Butterworth-Heinemann.
used. Therefore, the performance of bacteria can [8] S. Mokhatab and W.A. Poe, Handbook of natural gas
be evaluated by performing experiments in the transmission and processing. (2012): Gulf
laboratory by combining carbon dioxide in CO2 professional publishing.
and air capsules. [9] K. Rabbani, W. Charles, R. Cord-Ruwisch and G. Ho,
Recovery of sulfur from contaminated air in
wastewater treatment plants by biofiltration: a
Acknowledgments critical review. Reviews in Environmental
This work was a part of the M.Sc. thesis carried Science and Bio/Technology, 14 (2015) 523-
out in Sheikh Bahaei's department research 534.
laboratory in Science and Research Branch, [10] J. He and J.P. Chen, A comprehensive review on
biosorption of heavy metals by algal biomass:
Islamic Azad University, Tehran, Iran.
materials, performances, chemistry, and
modeling simulation tools. Bioresource
Conflict of interest Technology, 160 (2014) 67-78.
The authors confirm that this article's content [11] J. Klok, G. van Heeringen, R. De Rink, and H.
has no conflict of interest. Wijnbelt, Introducing the next generation of
the THIOPAQ O&G process for biotechnological
gas desulphurization: THIOPAQ-SQ. Sulphur,
References 18260 (2017) 1-2.
[1] R.-C. Jin, G.-F. Yang, Q.-Q. Zhang, C. Ma, J.-J. Yu and [12] A. ter Heijne, R. de Rink, D. Liu, J.B. Klok and C.J.
B.-S. Xing, The effect of sulfide inhibition on Buisman, Bacteria as an electron shuttle for
the ANAMMOX process. water research, 47 sulfide oxidation. Environmental science &
(2013) 1459-1469. technology letters, 5 (2018) 495-499.
[2] B.R. Gurjar, L.T. Molina and C.S.P. Ojha, Air pollution: [13] C. Cline, A. Hoksberg, R. Abry and A. Janssen.
health and environmental impacts. (2010): CRC Biological Process for H~ 2S Removal from Gas
Press. Streams: The Shell-Paques/THIOPAQ™ Gas
[3] J. Sun, X. Dai, Y. Liu, L. Peng and B.-J. Ni, Sulfide Desulfurization Process. in Proceedings of the
removal and sulfur production in a membrane Laurance Reid gas conditioning conference.
aerated biofilm reactor: model evaluation. 2003.
Chemical Engineering Journal, 309 (2017) [14] M. O Brien, C. Wentworth, A. Lanning, and T.
454-462. Engert. Shell-Paques® Bio-Desulfurization
[4] Q. Mahmood, P. Zheng, J. Cai, Y. Hayat, M.J. Hassan, Process Directly and Selectively Removes H~ 2S
D.-l. Wu and B.-l. Hu, Sources of sulfide in from High-Pressure Natural Gas-Start-Up
waste streams and current biotechnologies for Report. in PROCEEDINGS OF THE LAURANCE
its removal. Journal of Zhejiang University- REID GAS CONDITIONING CONFERENCE. 2007.
Science A, 8 (2007) 1126-1140. [15] N. Hou, Y. Xia, X. Wang, H. Liu, H. Liu, and L. Xun, H
[5] B. Khoshnevisan, P. Tsapekos, N. Alfaro, I. Díaz, M. 2 S biotreatment with sulfide-oxidizing
Fdz-Polanco, S. Rafiee and I. Angelidaki, A heterotrophic bacteria. Biodegradation, 29
review on prospects and challenges of (2018) 511-524.
biological H2S removal from biogas with focus [16] H.-K. Namgung and J. Song, The effect of oxygen
on biotrickling filtration and microaerobic supply on the dual growth kinetics of
desulfurization. Biofuel Research Journal, 4 Acidithiobacillus thiooxidans under acidic
(2017) 741-750. conditions for biogas desulfurization.
205 | P a g e
Mirnezami S.M. et. al./ Prog. Chem. Biochem. Res. 2021, 4(2), 191-206
International journal of environmental research method in river water and fish muscles:
and public health, 12 (2015) 1368-1386. Optimization by experimental design. J Hazard
[17] L. Yan, J. Ye, P. Zhang, D. Xu, Y. Wu, J. Liu, H. Zhang, Mater, 403 (2021) 123716.
W. Fang, B. Wang, and G. Zeng, Hydrogen [24] Z. Rezayati zad, B. Moosavi, and A. Taheri,
sulfide formation control and microbial Synthesis of monodisperse magnetic
competition in batch anaerobic digestion of hydroxyapatite/Fe3O4 nanospheres for
slaughterhouse wastewater sludge: Effect of removal of Brilliant Green (BG) and Coomassie
initial sludge pH. Bioresource Technology, 259 Brilliant Blue (CBB) in the single and binary
(2018) 67-74. systems. Advanced Journal of Chemistry-
[18] M. Monachon, M. Albelda-Berenguer, T. Section B, 2 (2020) 159-171 .
Lombardo, E. Cornet, F. Moll-Dau, J. Schramm, [25] Y. Cheng, T. Yuan, Y. Deng, C. Lin, J. Zhou, Z. Lei, K.
K. Schmidt-Ott and E. Joseph, Evaluation of Shimizu and Z. Zhang, Use of sulfur-oxidizing
Bio-Based Extraction Methods by bacteria enriched from sewage sludge to
Spectroscopic Methods. Minerals, 10 (2020) biologically remove H2S from biogas at an
203. industrial-scale biogas plant. Bioresource
[19] T. Satyanarayana, B.N. Johri and A. Prakash, Technology Reports, 3 (2018) 43-50.
Microorganisms in environmental [26] J. Cai, P. Zheng, M. Qaisar and J. Zhang, Elemental
management: microbes and environment. sulfur recovery of the biological sulfide
(2012): Springer Science & Business Media. removal process from wastewater: a review.
[20] J.-H. Kang, H.-G. Namgung, J.-I. Cho, S.S. Yoo, B.-J. Critical reviews in environmental science and
Lee and H.W. Ji, Removal of Hydrogen Sulfide technology, 47 (2017) 2079-2099.
in Septic Tanks for Treating Black Water via an [27] R. de Rink, J.B. Klok, G.J. van Heeringen, K.J.
Immobilized Media of Sulfur-Oxidizing Keesman, A.J. Janssen, A. ter Heijne and C.J.
Bacteria. International Journal of Buisman, Biologically enhanced hydrogen
Environmental Research and Public Health, 17 sulfide absorption from sour gas under
(2020) 684. haloalkaline conditions. Journal of hazardous
[21] B. Sörbo, A colorimetric method for the materials, 383 (2020) 121104.
determination of thiosulfate. Biochimica et [28] J. Mormontoy and J.E. Hurtado. Hydrogen sulfide
Biophysica acta, 23 (1957) 412-416. production at alkaline, neutral, and acid pH by
[22] D.P. Kelly, L. Chambers and P. Trudinger, Cyanolysis a bacterial consortium isolated from Peruvian
and spectrophotometric estimation of mine tailing and wetland. in Advanced
trithionate in mixture with thiosulfate and Materials Research. 2013. Trans Tech Publ.
tetrathionate. Analytical Chemistry, 41 (1969) [29] B. Huber, B. Herzog, J.E. Drewes, K. Koch, and E.
898-901. Müller, Characterization of sulfur oxidizing
[23] M. Fattahi, E. Ezzatzadeh, R. Jalilian, and A. Taheri, bacteria related to biogenic sulfuric acid
Micro solid-phase extraction of cadmium and corrosion in sludge digesters. BMC
lead on a new ion-imprinted hierarchical microbiology, 16 (2016) 1-11.
mesoporous polymer via dual-template
206 | P a g e