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2017 International Conference on Alternative Energy in Developing Countries and Emerging

Economies 2017 AEDCEE, 25‐26 May 2017, Bangkok, Thailand

BiologicalTheHydrogen Sulfide
15th International and Sulfate
Symposium on DistrictRemoval
Heating and from
CoolingRubber
Smoked Sheet Wastewater for Enhanced Biogas Production
Assessing the feasibility of using the heat demand-outdoor
temperature function for a long-term district heat demand forecast
a a,b *
Kanathip Promnuan and Sompong O-Thong 0F

a
Biotechnology Program, Faculty of Science, Thaksin University, Phatthalung 93210, Thailand.
a,b,c a a b c
I. Research
AndrićCenter
a
*, A. Pina
in Energy , P. Ferrão
and Environment, , J.
Faculty Fournier
of Science, Thaksin.,University,
B. Lacarrière
Phatthalung,93210,
O. Le Correc
Thailand.

a
IN+ Center for Innovation, Technology and Policy Research - Instituto Superior Técnico, Av. Rovisco Pais 1, 1049-001 Lisbon, Portugal
b
Abstract Veolia Recherche & Innovation, 291 Avenue Dreyfous Daniel, 78520 Limay, France
c
Département Systèmes Énergétiques et Environnement - IMT Atlantique, 4 rue Alfred Kastler, 44300 Nantes, France
The sulfate-rich wastewater from rubber smoked sheet industry could generate hydrogen sulfide (H2S) under anaerobic
condition, which created bad smell to the community and might cause toxicity and damage to the environment. The H2S can be
removed from the biogas by sulfur-oxidizing bacteria (SOB) with the ability to converted H2S to sulfate. Sulfate-reducing
Abstract
bacteria (SRB) could remove sulfate in wastewater before anaerobic treatment for biogas production. The microbial sludge from
wastewater and anaerobic digestion system was collected and test for sulfate and H2S removal efficiency. Anaerobic microbial
District heating networks are commonly addressed in the literature as one of the most effective solutions for decreasing the
sludge has a high ability to produced methane from gelatin with a specific methane production rate of 92.4 ml CH4 gVSS-1 day-1.
greenhouse gas emissions from the building sector. These systems require high investments which are returned through the heat
Anaerobic microbial sludge has lower methane production when gelatin and sulfate used as a substrate with a specific methane
sales. Due to the changed climate conditions and building renovation policies, heat demand in the future could decrease,
production rate of 81.4 ml CH4 gVSS-1 day-1. The biomethane potential, hydrogen sulfide removal and sulfate removal in
prolonging the investment return period.
anaerobic digestion system by addition of enriched cultures of SOB and SRB were investigated. The methane yield of SRB
The main scope of this paper is to assess the feasibility of using the heat demand – outdoor temperature function for heat demand
consortium was 60.1 ml CH4/gCOD with 20% sulfate reduction from wastewater and no sulfide reduction. The methane yield of
forecast. The district of Alvalade, located in Lisbon (Portugal), was used as a case study. The district is consisted of 665
SOB consortium was 41.9 ml CH4/gCOD with no sulfate and sulfide reduction from wastewater. The addition of SRB
buildings that vary in both construction period and typology. Three weather scenarios (low, medium, high) and three district
consortium could increase methane production by reducing sulfate concentration in wastewater consequently to a reduced
renovation scenarios were developed (shallow, intermediate, deep). To estimate the error, obtained heat demand values were
concentration of H2S in biogas.
compared with results from a dynamic heat demand model, previously developed and validated by the authors.
©The results
2017 showed that
The Authors. when only
Published weatherLtd.
by Elsevier change is considered, the margin of error could be acceptable for some applications
© 2017 The Authors. Published by Elsevier Ltd.
Peer-review
(the error inunder responsibility
annual demand was of lower
the scientific
than 20% committee of the 2017
for all weather International
scenarios Conference
considered). on Alternative
However, Energy renovation
after introducing in
Peer-review
­D under responsibility
eveloping the
Countries of the Organizing Committee of 2017 AEDCEE.
and Emerging
scenarios, error value increased Economies.
up to 59.5% (depending on the weather and renovation scenarios combination considered).
The value of slope coefficient increased on average within the range of 3.8% up to 8% per decade, that corresponds to the
Keywords: Biogas; Hydrogen sulfide; Sulfur-oxidizing bacteria; Sulfate-reducing bacteria
decrease in the number of heating hours of 22-139h during the heating season (depending on the combination of weather and
renovation scenarios considered). On the other hand, function intercept increased for 7.8-12.7% per decade (depending on the
coupled scenarios). The values suggested could be used to modify the function parameters for the scenarios considered, and
improve the accuracy of heat demand estimations.

© 2017 The Authors. Published by Elsevier Ltd.

Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and
* Corresponding author. Tel.: +66-74-693-992; fax: +66-74-693-992.
Cooling.
E-mail address: sompong.o@gmail.com
Keywords: Heat demand; Forecast; Climate change
1876-6102 © 2017 The Authors. Published by Elsevier Ltd.
Peer-review under responsibility of the Organizing Committee of 2017 AEDCEE.

1876-6102 © 2017 The Authors. Published by Elsevier Ltd.

Peer-review under responsibility of the Scientific Committee of The 15th International Symposium on District Heating and Cooling.
1876-6102 © 2017 The Authors. Published by Elsevier Ltd.
Peer-review under responsibility of the scientific committee of the 2017 International Conference on Alternative Energy in
­Developing Countries and Emerging Economies.
10.1016/j.egypro.2017.10.161
570 Kanathip Promnuan et al. / Energy Procedia 138 (2017) 569–574
2 Kanathip Promnuan and Sompong O-Thong./ Energy Procedia 00 (2017) 000–000

1. Introduction

Rubber smoked sheet wastewater containing high sulfate and the sulfate could converse to hydrogen sulfide
(H2S) under anaerobic condition by sulfate-reducing bacteria. Hydrogen sulfide created a bad smell (rotten egg
odor) to public health. If people received a high concentration of H2S, it affects the respiratory system. Biogas
production from sulfate-rich wastewaters resulted in high hydrogen sulfide concentration in biogas. It may cause
serious problems in the application of biogas because the H2S concentration in the gas phase higher than 1000 ppmv
could not direct combustion. The limitation of H2S for internal combustion engine fuel is 100 ppmv and much lower
16 ppmv is set for compressed natural gas for transportation fuel [1]. Moreover, sulfide in the liquid phase
(dissolved sulfide) generated during the anaerobic treatment of sulfate-rich wastewater also imposes toxicity to
methanogens [2]. Treatment technologies to remove the high concentrations of H2S in the gas phase are physical
(adsorption, absorption, and dilution), chemical (chemical absorption, neutralization, and combustion) and
biological (activated sludge and biofilter) methods [3]. H2S in Biogas is difficult to remove because of
methanogenic archaea and sulfate-reducing bacteria growth in the same condition. Under anaerobic environment
with the presence of chemical oxygen demand (COD), the sulfate-reducing bacteria (SRB) can convert sulfate in
wastewaters to sulfide. Biological conversion of sulfide to elemental sulfur or sulfate using sulfide-oxidizing
bacteria (SOB) is a capable process to remove H2S from biogas [4, 5]. In natural, the SRB and SOB can coexist in
hydrothermal vents, microbial mats, marine sediments and wastewater biofilms as a response of high organic input
and low dissolved oxygen (DO) concentration [6]. Therefore, the combination of the SRB and SOB for remove
sulfide and sulfate in biogas reactor is of practical interest.
The aim of this study was to gain more insight into the syntrophic interactions between the different bacterial
trophic groups in biogas process. For this purpose, activity tests in the presence of various substrates were carried
out to fully elucidate the outcome of competition between the sulfate reducers and the other anaerobic consortia in
high sulfate wastewater. The biomethane production, hydrogen sulfide removal and sulfate removal in anaerobic
digestion of rubber smoked sheet wastewater by adding enriched cultures of SRB and SOB were investigated.

2. Methodology

2.1. Sampling and Inoculum

The rubber smoked sheet wastewater was collected from Yangkaw cooperative rubber sheet plant. The chemical
and physical compositions of wastewater were analyzed according to standard water and wastewater examination
methods [7]. The sludge inoculum was collected from biogas plant of the Yangkaw cooperative rubber sheet plant.
Wastewater adjusting the initial pH in the range of 7-7.5 by ash and NaHCO3 was used as the substrate for seed
sludge. Prior to use, the sludge inoculums were incubated for 1-2 weeks to activate microorganism activities. The
sulfate-reducing bacteria also enriched from anaerobic sludge collected from the Yangkaw cooperative rubber sheet
plant. It was kept at a temperature of 4°C during transport to the laboratory. Postgate medium C was used for
enrichment and cultured sulfate-reducing bacteria [8]. Postgate medium C 1 L contained of 6.0 ml Lactic acid, 4.5 g
Na2SO4, 1.0 g NH4Cl, 1.0 g Yeast extract, 0.5 g KH2PO4, 0.3 g Sodium citrate·2H2O, 0.06 g CaCl2·6H2O, 0.06 g
MgSO4·7H2O, 0.004 g FeSO4·7H2O and pH adjusted at 7.0±2. The sulfide-oxidizing bacteria were enriched from
biofilter sludge collecting from Pitak palm oil CO., LTD. Thiosulfate mineral medium was used for enrichment and
culturing sulfide-oxidizing bacteria [9]. Thiosulfate mineral medium 1 L contained of 5.1 g Na2S2O3, 2.0 g K2HPO4,
2.0 g KH2PO4, 0.4 g NH4Cl, 0.2 g MgCl2·7H2O, 0.01 g FeSO4·7H2O and pH adjusted at 7.0±2.

2.2. Specific methanogenic activity

A specific methanogenic activity (SMA) test was used to determine for microbial activity in anaerobic digestion
[10]. SMA tests were carried out in the presence and absence of sulfate (NaSO4 30 mM). The substrates tested
including acetic, avicel, glucose and gelatin and combinations at the concentrations of 4 g/L COD. SMA was carried
out in test vials (60 ml) with anaerobic sludge and incubated under mesophilic condition (35 °C) for a period of 7
days. All tests were performed in the presence and absence the addition of sulfate into the medium. The biogas
 Kanathip Promnuan et al. / Energy Procedia 138 (2017) 569–574 571
Kanathip Promnuan and Sompong O-thong./ Energy Procedia 00 (2017) 000–000 3

production was collected by water displacement (gas counter) and biogas composition analysis by GC-TCD every
day.

2.3. Batch assay for sulfide and sulfate removal

Batch assay was carried out in a 120-ml vial with working volume of 50 ml. Rubber sheet wastewater, anaerobic
sludge (SL), enriched SRB and enriched SOB were mixed as described previously by Angelidaki et al. [11]. The
initial pH was adjusted 7.0-8.0 and incubated under mesophilic condition (35 °C) for a period of 20 days by variated
proportions of inoculum (%SL: %SOB: %SRB) as follows: 60:20:0, 60:0:20, 60:10:10, 60:15:5 and 60:5:15. The
biogas production was collected by water displacement (gas counter) and biogas composition analysis by GC-TCD
every day. All tests were run in triplicate for at each condition with avicel as a positive control.

2.4. Analytical methods

Biogas composition was daily monitored by gas chromatography. The TS, VS, alkalinity, and pH of the materials
were determined using the standard water and wastewater examination methods [7]. Elemental composition (C, H,
O, N, and S) of the materials was performed in a CHNS-O Analyzer, CE Instruments Flash EA 1112 Series with
dynamic flash combustion at 900 ºC, for C, H, N and S element, at 1060 ºC for O element [12]. Sulfide, Sulfate, and
chemical oxygen demand (COD) were measured by Merck Cell test. The pH was measured with Adwa waterproof
pH-Temp Pocket tester.

3. Results and Discussion

3.1. Substrate characterization

The characteristics of rubber smoked sheet wastewater were shown in Table 1. The wastewater was high sulfate
and protein concentrations that can converse to ammonia and hydrogen sulfide. Ammonia is toxic to aquatic animals
and methanogens. In addition, ammonia also inhibited anaerobic digestion process. Methanogenesis was completely
inhibited at total ammonia nitrogen of higher than 9 g N L-1 [7]. Ammonia nitrogen is less inhibitory in its ionic
form (NH4+) than as free ammonia (NH3), but the partitioning between these forms is dependent on temperature and
pH. For this reason, ammonia inhibition has been reported in a wide range of total ammonia nitrogen concentrations
between 1500 and 7000 mg N L-1 [13]. Wastewater also contains a high amount of sulfate. The sulfate is reduced to
sulfide by the sulfate-reducing bacteria (SRB). SRB inhibition is due to competition for common organic and
inorganic substrates that suppresses methane production. SRB inhibition also results from the toxicity of sulfide
[13]. Therefore, the addition of SOB and/or SRB can be removal sulfate and sulfide by syntrophic between SOB that
convert sulfide to element sulfur and SRB that converts sulfate to sulfide.

3.2. Specific methanogenic activity

Table 2 summarizes the specific methanogenic activity (SMA) for anaerobic sludge samples from biogas plant of
rubber smoked sheet wastewater. The SMA in all substrates demonstrated that methanogens and SRB were
competed for consuming of substrates. The SMA of gelatin gave the high specific methane production of 92.41 ml
CH4 gVSS-1 day-1. The SMA values from gelatin as a substrate indicated that high growth proteolytic bacteria in the
systems [11]. Proteolytic bacteria are a hydrolysis bacterium that converts the insoluble complex organic matter,
such as cellulose, into soluble molecules such as sugars, amino acids, and fatty acids. Protease was secreted by
proteolytic bacteria and convert proteins into amino acids. The SMA of acetic, avicel and glucose as a substrate was
7.37, 13.96, 32.41 ml CH4 gVSS-1 day-1 respectively. There were very low when compared with the values obtained
for gelatin. High acetate degradation in vials containing sulfate was suggesting that acetate-utilizing SRB is a
significant role in acetate conversion. The SRB may compete with methanogens, acetogens, or fermentative
microorganisms for available acetate, H2, propionate, and butyrate in anaerobic systems [14, 15]. Anaerobic sludge
572 Kanathip Promnuan et al. / Energy Procedia 138 (2017) 569–574
4 Kanathip Promnuan and Sompong O-Thong./ Energy Procedia 00 (2017) 000–000

from Yangkaw cooperative rubber sheet plant can be degraded gelatin better than another substrate. Furthermore,
the presence of sulfate in acetic as a substrate shown inhibited methanogens in anaerobic digestion.

Table 1. Chemical and physiological characteristics of rubber smoked sheet wastewater

Parameter Influent wastewater
COD (mg/l) 6,500
pH 4
Total solid (mg/L) 6,115
Volatile solid (mg/L) 4,367
Sulfate (mg/L SO42-) 5,101
2-
Sulfide (mg/l S ) 0.25
Total alkalinity (mg/L. CaCO3) 3,000
Total nitrogen (mg/L) 1,360
a
Protein (mg/L) 8,500
a
total nitrogen multiplies factor of protein 6.25

Table 2. Specific methanogenic activity and sulfate reduction of sludge from rubber smoked sheet biogas plant
Treatment SMA
(ml CH4 gVSS-1 day-1)
Acetic 7.37
2-
Acetic+ SO4 27.03
Avicel 13.96
2-
Acetic+ SO4 7.58
Glucose 32.41
Glucose+ SO42- 27.32
Gelatin 92.41
2-
Gelatin+ SO4 81.41

3.3. Sulfide and sulfate removal for enhanced methane production

The results show that biogas increased was improved with sulfate removal from the wastewater (Table 3).
Methane production rate in all experiment was high on Day 2 and gradually decreased until the end of the
experiment (Fig 1a). Addition of enriched SRB at 20% into the batch fermentation system can remove sulfate (20%)
and increase methane yields (60.07 ml CH4/COD). Methanogens and SRB did not compete for common substrates.
Syntrophic propionate and ethanol conversion bacteria were likely performed primarily by SRB, while H2, formate,
and acetate were consumed primarily by methanogens. Some cases of sulfate, certain SRB such as Desulfovibrio
spp. may grow together with H2-utilizing methanogens to convert ethanol or lactate to acetate syntrophically [16].
Addition of enriched SRB and SOB was shown high methane accumulation more than the control (Fig 1b). Sulfide
in all treatments could not be removed by the addition of SOB. It is possible that SOB cannot grow in anaerobic
fermentation. Normally, SOB use the energy of oxidized inorganic sulfur compounds (hydrogen sulfide,
thiosulfates, sulfites, elemental sulfur) in aerobic condition [17], but some SOB can grow in micro-aeration [18].
Therefore, the conditions of this experiment may not be suitable for growth of SOB.

4. Conclusion

The specific methanogenic activity of anaerobic sludge from Yangkaw cooperative rubber sheet biogas plant has
a high capacity to degradation of the protein. Addition of enriched SRB could improve biogas volume and biogas
quality. Addition of enriched SRB 20%v/v has the capacity to remove sulfate and increase methane production.
 Kanathip Promnuan et al. / Energy Procedia 138 (2017) 569–574 573
Kanathip Promnuan and Sompong O-thong./ Energy Procedia 00 (2017) 000–000 5

Table 3. Effect of sulfide sulfate removal and methane yield after added proportions of SRB and SOB
No. Treatment Methane yield Sulfide Sulfide Sulfate Sulfate
(ml CH4 g-1 COD) (mg/l S2-) Removal (g/l SO42-) Removal
before after (%) before after (%)
1 SL 52.58 50 62 0 4.6 5.3 0
2 SOB 0 0.5 0.6 0 6.6 8.6 0
3 SRB 0 61 280 0 5.3 4.4 17
4 SL + SOB 41.93 54 250 0 4.3 4.7 0
5 SL + SRB 60.07 50 200 0 4.5 3.6 20
6 SL+ 1SOB + 1SRB 51.72 60 230 0 3.7 3.5 5
7 SL + 2SOB + 1SRB 45.75 51 230 0 4.7 4.2 11
8 SL + 1SOB + 2SRB 56.75 68 230 0 3.7 3.4 8

a b

Fig. 1 Effect of methane production rate (a) and cumulative methane yield (b) after added proportions of SRB and SOB

Acknowledgements

The authors would like to thank the Research and Development Institute, Thaksin University, Research Center in
Energy and Environment, Faculty of Science, Thaksin University, Research Group for Development of Microbial
Hydrogen Production Process, Khon Kaen University, and Thailand Research Fund through grant Research and
Researchers for Industries (RRI) (PHD59I0069) for the financial support.

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