Elsevier

Bioresource Technology

Volume 272, January 2019, Pages 40-47
Bioresource Technology

Comparison of sulphide and nitrate removal from synthetic wastewater by pure and mixed cultures of nitrate-reducing, sulphide-oxidizing bacteria

https://doi.org/10.1016/j.biortech.2018.09.125Get rights and content

Highlights

  • New strains of nitrate-reducing, sulphide-oxidizing bacteria (NR-SOB) were isolated.

  • Mixture of NR-SOB strains showed the highest N-NO3 removal rate (5.5 mg N-NO3/L h)

  • The NR-SOB strains were closely related to Paracoccus spp. (99% similarity)

  • The pure and mixed strains of NR-SOB removed 100% gas-phase H2S.

Abstract

In this study, the activities of hydrogen sulphide (H2S) oxidation and nitrate (N-NO3) reduction by three pure and mixed strains of nitrate-reducing, sulphide oxidizing bacteria (NR-SOB) were determined. Batch experiments were performed at 35 °C and pH 7.0–8.0 with initial H2S concentrations of 650–900 ppmv and N-NO3 concentrations of ∼120 mg/L. The strains MAL 1HM19, TPN 1HM1 and TPN 3HM1 were capable of removing 100% gas-phase H2S. The co-cultures showed better performance for H2S and N-NO3 removal. The mixed NR-SOB strains showed a higher H2S oxidation rate (143 ± 18 ppmv/h), while the highest N-NO3 removal rate (5.5 ± 0 and 5.1 ± 0.6 N-NO3 mg/L·h) was obtained by a mixture of two NR-SOB strains. The 16S rDNA sequence analysis revealed that all strains belonged to the sub-class Alphaproteobacteria and are closely related to Paracoccus sp. (>99%).

Introduction

Hydrogen sulphide (H2S) is one of the sulphur compounds that is of major environmental concern because it is flammable, toxic and corrosive and has an unpleasant odor (smells like rotten eggs) (Kanjanarong et al., 2017). Increasing amounts of H2S are released into the atmosphere as a by-product of several industries, including petroleum refining (10–887 mg/L), biogas production (2–6 g/L), and the pulp and paper (35–55 mg/L) industry (Sposob et al., 2017). In addition, H2S leads to the corrosion of machine structures, internal combustion engines and similar equipment (Namini et al., 2012).

The human olfactory system can detect H2S at concentrations less than 50 parts per million (ppmv). However, at 100–150 ppmv, the symptoms include loss of smell or olfactory fatigue, and concentrations above 500 ppmv can cause rapid unconsciousness and death (Namini et al., 2012). Nevertheless, the long-term, chronic exposure of living organisms to low H2S levels is not well inferred in the literature. Therefore, the permissible exposure level of H2S in ambient air has been set at 10 ppmv for a 8 h work shift according to the U.S. Occupational Safety and Health Administration (OSHA) (da Silveira Petruci et al., 2014). In the liquid phase, where it occurs in both ionic (HS or S2−) and undissociated (H2Saq) forms, high H2S concentrations are toxic to microorganisms (Kimura, 2014). For example, depending on the bacterial species, the 50% maximal inhibition concentration (IC50) of sulphide (H2S, HS, S2−) has been reported to be in the range of 30–250 mg/L (Pokorna and Zabranska, 2015).

Another compound of environmental concern in drinking water and wastewater is nitrate (N-NO3), which is the stable form of nitrogen in oxygenated systems. The contamination of water with N-NO3 is mainly due to agricultural fertilizers and manure, animal feedlots, horse corrals, septic tanks, geologic sources and soil organic matter (Rezvani et al., 2017, Chen et al., 2018). Although N-NO3 does not directly affect the health of an organism itself, it can be converted to highly carcinogenic nitrosamine and nitrosamide compounds that are usually chemically reactive and metabolically unstable (Fernández et al., 2014). Moreover, the ingestion of high NO3 levels causes methemoglobinemia (blue baby syndrome) in the mother and child, which decreases the transport of oxygen in the blood.

Nitrate-reducing, sulphide oxidizing bacteria (NR-SOB) are important biocatalysts for the removal of both sulphide and N-NO3 in different water environments (Lee and Wong, 2014), where the NR-SOB utilize sulphide as an electron donor coupled with N-NO3 reduction via the autotrophic denitrification pathway to form sulphate (SO42−) or elemental sulphur (S0) and nitrogen (N2) gas. Recent studies have reported the application of NR-SOB, including autotrophic, heterotrophic and phototrophic bacteria for H2S or N-NO3 removal (Pokorna and Zabranska, 2015). From these studies, the most suitable bacteria for H2S removal are the chemolithotrophic bacteria, such as Thiobacillus sp., Sulfurimonas denitrificans (formerly known as Thiomicrospira denitrificans), Beggiatoa sp. and Thiothrix sp. (Pokorna and Zabranska, 2015).

The discovery of new NR-SOB from non-conventional sources, for example continental hydrothermal environments, is interesting in order to improve sulphide and N-NO3 removal from water under varying environmental conditions. The screening of such new microorganisms is a promising approach because it has been estimated that 99% of the existing bacteria in the world are still undiscovered or uncultivated in the laboratory (Panda et al., 2013). Hot springs are a potential natural source for NR-SOB, since they are often rich in H2S or sulphide and therefore, they are potential sources of mesophilic or thermophilic sulphide oxidizing bacteria (Tamazawa et al., 2012).

Isolates of bacteria for H2S and N-NO3 removal can be considered as an alternative inoculum because pure cultures have a short lag-phase and require short culture times to deplete the sulphide and N-NO3 in synthetic wastewater (Watsuntorn et al., 2017). In addition, the pure culture can improve the removal capacities and efficiencies of bioreactor systems by its incorporation in biofilms (Aroca et al., 2007). In a recent study, Watsuntorn et al. (2017) reported simultaneous H2S and N-NO3 removal by a pure strain of NR-SOB (MAL 1HM19) that was isolated from the Mae Um Long Luang hot spring (Thailand). However, use of this isolate still requires sterile conditions to maintain the purity of the microorganism. Recently, the concept of using a combination of different pure cultures has emerged as a potential means to enhance the robustness of pure strain systems in ex-situ biodegradation studies (Masset et al., 2012). These could be applied to treat industrial waste streams, e.g. wastewater from the Kraft’s process of the pulp and paper industry containing high N-NO3 levels (∼350 mg/L). Besides, the gaseous emissions from this industry contain high H2S concentrations (8.8 mg/L) (Kamali et al., 2016).

There are a few studies that have applied both pure and mixed NR-SOB cultures for bioleaching of chalcopyrite (Zhu et al., 2011), but there are to the best of our knowledge no reports on the simultaneous removal of H2S and N-NO3 from wastewater by mixtures of different pure NR-SOB strains. Therefore, the objectives of this study were: (i) to investigate the role of sulphide oxidation in both the gas and liquid phases and N-NO3 reduction by the pure NR-SOB strains, namely MAL 1HM19, TPN 1HM1 and TPN 3HM1 isolated from different hot springs in Thailand, and (ii) to enhance the capacity of sulphide and N-NO3 removal by using different combinations of these NR-SOB strains.

Section snippets

Sources and enrichment of NR-SOB strains

Four strains of NR-SOB were tested for sulphide and N-NO3 removal from synthetically prepared wastewater. Strain MAL 1HM19 was isolated from the Mae Um Long Luang hot spring (Mae Hong Son province, Thailand) (Watsuntorn et al., 2017), while the strains TPN 1HM1 and TPN 3HM1 were isolated from the Thep Pha Nom hot spring (Chiang Mai province, Thailand). For the isolation step, mixed sediment and water samples were collected from the Mae Um Long Luang hot spring (Mae Hong Son province) and Thep

H2S removal rate and SO42−production by pure and mixed NR-SOB cultures

The H2S in the gas-phase of the incubations of the pure and mixed NR-SOB strains was investigated at 35 °C which is the temperature of both the gas and liquid phases during biogas production (N/S molar ratio = 3.3). Moreover, Fernández et al. (2014) reported the inhibition of NR-SOB at temperatures below 30 °C. The H2S in the gas phase was completely removed within 6–10 h by all the cultures, except by P. pantotrophus. The H2S removal rate by the pure culture of strain MAL 1HM19 was the highest

Conclusions

In this study, pure and co-cultures of the NR-SOB Paracoccus sp. namely MAL 1HM19, TPN 1HM1 and TPN 3HM1 showed good removal of both H2S and NO3 in batch tests within a short time (24–48 h). The novelty of this study was to create co-cutures of NR-SOB for simultaneous H2S and N-NO3 removal, which gave better removal rates of both H2S and N-NO3 than the single-strain incubations. The highest H2S removal rate was obtained when all the three NR-SOB strains were used.

Acknowledgement

This work was supported by the 100th Anniversary Chulalongkorn University Fund for Doctoral Scholarship and the Graduate School of Chulalongkorn University, Thailand.

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