Isolation of Sulfide Remover Strain Thermithiobacillus Tepidarius JNU-2, and Scale-Up Bioreaction for Sulfur Regeneration

Ann Microbiol (2015) 65:553–563 DOI 10.1007/s13213-014-0891-2

ORIGINAL ARTICLE

Isolation of sulfide remover strain Thermithiobacillus tepidarius JNU-2, and scale-up bioreaction for sulfur regeneration

Hailin Yang & Kai Gao & Shoushuai Feng & Ling Zhang & Wu Wang

Received: 18 November 2013 /Accepted: 2 April 2014 /Published online: 8 May 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract A sulfur removing bacterium named JNU-2 was to imparting severe effects on ecosystems even at very low isolated from an industrial anaerobic sludge pool. Its 16S concentrations (Wiessner et al. 2005). Various toxicological rRNA phylogeny showed high homology to the species effects of sulfide upon human health have been described Thermithiobacillus tepidarius. The strain Thermithiobacillus (Mahmood et al. 2009). The removal of malodorous reduced tepidarius JNU-2 was initially cultivated for removing sulfide sulfur emissions has been traditionally accomplished using from sulfate-containing waste water for recycling sulfur. physical or chemical methods, such as vapor scrubbing, in- Thiosulfate removal and elemental sulfur production ratios cineration or adsorption. However, these control technologies reached 98.0 % and 83.06 %, respectively, after incubating are usually uneconomical with the large flow rates and low for 24 h. Furthermore, based on the traits of JNU-2, a novel contaminant concentrations of waste air streams (Wani et al. sulfur regeneration reactor—an internal airlift loop reactor 1998). To overcome these difficulties, biological treatment (IALR)—was constructed and applied successfully. The key processes are often desirable and serve as suitable alternatives operating conditions of ventilatory capacity (VC) and hydrau- to physico-chemical systems (Yang 1992; Ferrea et al. 2004; lic retention time (HRT) were also investigated. The produc- Gadekar et al. 2006). tion ratio of elemental sulfur was about 60.0 % and the The biological sulfate removal process consists of two maximal yield was 75 mg L−1 h−1 at 60 mL min−1 VC and serial biological processes: namely, reduction of sulfate to 10 h HRT. All results indicated that the strain JNU-2 and the sulfide (anaerobic stage); followed by oxidation of sulfide to novel reactor would be of great potential for producing sulfur elemental sulfur (aerobic stage) in order to remove the excess sources at industrial sites. sulfide of the treated stream before discharging it into the environment (Lohwacharin and Annachhatre 2010). Keywords Thermithiobacillus tepidarius . Sulfide removal . Anaerobic reduction of sulfate or sulfite to sulfide has been Internal airlift loop reactor . Sulfur regeneration adopted as a traditional microbial process to remove sulfate for several years (Lee and Sublette 1991;Selvarajand Sublette 1995;Azabouetal.2007; Rao et al. 2007). However, very little useful information is available about the Introduction biological oxidation of sulfide, especially for generating elemental sulfur source. The low loading rate and the difficulties The emission of sulfide is a major problem associated with in separating sulfur produced by bacteria are the major prob- anaerobic treatment of sulfate- and sulfite-containing waste lems in this process. Most studies on sulfide removal are still waters. Hydrogen sulfide (H S) is toxic to humans in addition 2 at the stage of laboratory-scale tests, which are also difficult for practical application (Chan and Suzuki 1993; Pagella et al. H. Yang (*) : K. Gao : S. Feng : L. Zhang : W. Wang The Key Laboratory of Industrial Biotechnology, Ministry of 1996a, 1996b; Wiessner et al. 2005). Therefore, isolation of Education, School of Biotechnology, Jiangnan University, 1800 Lihu sulfide-oxidizing bacteria and the design of simple and effi- Road, Wuxi 214122, People’s Republic of China cient reactors is of great importance for promoting the process e-mail: [email protected] of sulfide bio-oxidation into elemental sulfur. H. Yang Sulfide can be generally oxidized by three different micro- e-mail: [email protected] organisms: photosynthetic bacteria, filamentous sulfur 554 Ann Microbiol (2015) 65:553–563 bacteria and colorless sulfur bacteria (Kuenen and Robertson 121 °C for 20 min, and some components not suitable for 1992). Of these bacteria, colorless sulfur-oxidizing bacteria high temperature sterilization were filter-sterilized. are most widely used to oxidize sulfide, using oxygen as an electron acceptor (Mahmood et al. 2007, 2009). Thiobacillus, Bacterial enrichment and isolation which is the most common colorless sulfur bacteria in the soil and natural water (Ketly and Harrison 1989), has the charac- Municipal wastewater sludge samples were collected in June teristics of producing elemental sulfur efficiently, discharging 2012, from an anaerobic sludge pool in a sewage treatment sulfur from the cell, mild reaction conditions, etc. (Kuenen plant in Wuxi (Jiangsu, China). The operating temperature and et al. 1985). With these traits, strains of Thiobacillus are pH of the pool were about 10–40 °C and 6.0–8.0, respectively. selected as the significant species for studies on the industrial The bacterium was enriched from the sample described above applications of the biological oxidation of sulfide into elemen- with the improved thiosulfate medium. Enrichment culture tal sulfur (Xian and Yang 1984; Pagella et al. 1996a; Ramírez conditions as follows: 37 °C, pH 7.0, in shake bottles at et al. 2009). Recently, several kinds of aerobic reactors, such 170 rpm. When the enrichment sample liquid became turbid as batch-fed reactor (Pagella et al. 1996b), biotrickling filter and the pH reduced to around 4, serial dilution was adopted to (Jiang and Tay 2010), immobilized cell biofilter (Wani isolate the strain on solid medium. After 4 days of incubation et al.1998), airlift bioreactor (Lohwacharin and Annachhatre at 37 °C, single milky colonies began to develop, and the 2010), etc., have been used in sulfide removal, but the prob- isolation process was repeated five times to ensure strain lems mentioned above have not been fully resolved. purity. In this study, the moderate thermophilic, neutrophil, acid- produced sulfur-oxidizing strain Thermithiobacillus Morphological and phylogenetic analysis tepidarius JNU-2 was isolated from an anaerobic pond in a sewage treatment plant, China, and tested in sulfide removal. Morphology of the pure culture strain JNU-2 was observed by To further understand the role ofThermithiobacillus tepidarius light microscopy (DM-2500, Leica, China) and transmission strain JNU-2 in sulfide oxidation, its physiological and mo- electron microscopy (TEM) (H-7000, Hitachi, Japan). The lecular traits and its role in removing sulfide produced from total genomic DNA of the culture was extracted using a sulfate bio-reduction were studied. Using strain JNU-2, a Small Bacteria Genomic DNA Fast Extraction Kit laboratory scale internal airlift loop reactor (IALR) was de- (FastaGen, Shanghai, China). The 16S ribosomal RNA signed for oxidizing sulfide to elemental sulfur. The ventila- (rRNA) gene was amplified by polymerase chain reaction tory capacity (VC) and hydraulic retention time (HRT) were (PCR) using forward primer 27F 5′-AGAGTTTGATCCTG investigated to optimize optimal operating conditions for sul- GCTCAG-3′ and reverse primer 1492R 5′-GGTTACCTTG fide oxidation and elemental sulfur formation. TTACGACTT-3′ (DeSantis et al. 2007). The PCR program was 94 °C for 4 min, 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 90 s, with a final extension step of 72 °C for 10 min after 30 cycles. The PCR product was purified using B-type small Materials and methods DNA fragment Gel Extraction Kit (BioDev-Tech, Beijing, China). After sequencing of 16S rRNA, the sequence was Media for microbial growth analyzed with those of similar types of strains in the National Center for Biotechnology Information using the The improved medium from Atlas and Parks was used in BLAST software (http://blast.ncbi.nlm.nih.gov/Blast.cgi/). A biochemical and physiological characterization studies. The phylogenetic tree was constructed using CLUSTAL X version medium consists of the following components: 1.81 and MEGA version 4.0 programs to determine the

Na2S2O3·5H2O10.0g,KH2PO4 4.0 g, K2HPO4 4.0 g, relationship of strain JNU-2 with other Thiobacillus micro- MgSO4·7H2O 0.8 g, NH4Cl 0.4 g, trace element solution organisms. Details of the relative strains used in this study are 10 mL, distilled water 1,000 mL (Atlas and Parks 1993). listed in Table 1. The trace element solution was composed of the following components: EDTA-disodium 50.0 g, ZnSO4·7H2O22.0g, Culture conditions and growth curve CaCl2 5.54 g, MnCl2·4H2O 5.06 g, FeSO4·7H2O 4.99 g, (NH4)6Mo7O24·4H2O1.10g,CuSO4·5H2O1.57g, The medium and culture conditions used for the initial sulfide CoCl2·6H2O 1.61 g, distilled water 1,000 mL. The medium removal experiment were described above. Active strain JNU- was adjusted to pH 7.0 with 1 M NaOH. The solid medium for 2 was inoculated at 5 % into fresh medium for obtaining its 2− separation and purification was prepared by addition of 1.8 % growth curve. Cell concentration, pH, S2O3 concentration 2− agar powder. The basal medium was autoclaved using a high- and SO4 concentration were detected every 12 h. To confirm pressure steam sterilizer (LDZX-40II, Shenan, China) at the optimal carbon and energy sources of JNU-2, the Ann Microbiol (2015) 65:553–563 555

Table 1 Reference strains in the analysis of molecular phylogeny

Species Strain Sequence number Similarity % Description and source

Acidithiobacillus thiooxidans ATCC 19377 NR044920 92 Coventry, UK Acidithiobacillus thiooxidans BY-s DQ676511 92 Isolated from diverse habitats of China. Lanzhou, Gansu Acidithiobacillus thiooxidans SZS DQ676508 92 Isolated from diverse habitats of China. Lanzhou, Gansu Acidithiobacillus ferrooxidans GD1-3 FJ194542 92 Changsha, Hunan, China Acidithiobacillus ferrooxidans GD-2 FJ194541 92 Changsha, Hunan, China Acidithiobacillus caldus N39-30-02 EU499920 92 A spent copper sulfide heap, Australia Acidithiobacillus caldus DX-2 DQ470072 92 Changsha, Hunan, China Acidithiobacillus caldus MTH-04 AY427958 92 Jinan, Shandong, China Acidithiobacillus caldus MT1 AF513711 92 Isolated from a pilot plant stirred-tank bioleaching operation.Wales, UK Acidithiobacillus albertensis BY-0506 GQ254658 92 Acid mine drainage. Changsha, Hunan, China Acidithiobacillus albertensis DSM 14366 NR028982 92 Bethesda, USA Halothiobacillus neapolitanus C153 AB308268 84 Cattle manure compost, Sapporo, Japan Halothiobacillus neapolitanus NCIMB 8539(X44) JF416645 85 Type strain of Halothiobacillus neapolitanus,Paker. Halothiobacillus halophilus DSM 6132 NR026015 85 Type strain of Halo Bethesda,MD20894,USA, Thiobacillus halophilus. Halothiobacillus hydrothermalis R3 NR025943 84 Type strain of Halothiobacillus hydrothermalis. Deep-sea hydrothermal vent in Fiji Basin. Thermithiobacillus tepidarius DSM 3134 NR042145 100 DSM 3134 Thermithiobacillus sp. ParkerM HM173631 99 NCIMB 8349 Thiobacillus thioparus ATCC 8158 NR044755 84 Type strain of Thiobacillus thioparus. Bethesda, USA Thiobacillus thioparus THI 111 HM535226 85 Coventry, England Thiobacillus denitrificans NCIMB 9548 NR025358 84 Type strain of Thiobacillus denitrificans. Bethesda, MD, USA Thiobacillus denitrificans NBY57 HQ851082 84 Yumthang hot spring, India Thermithiobacillus tepidarius JNU-2 KC493561 100 Sewage treatment plant, Wuxi, Jiangsu, China inoculation quantity was 2 % and all cultures were incubated same amount of both ingredients together were added to the at 37 °C, 170 rpm for 3 days. Inocula were washed twice with medium. All experiments were carried out at least in duplicate. basal medium after harvesting via centrifugation. The Na2S2O3·5H2O in the medium with trace elements was re- Conditions for sulfide removal placed by one of the following compounds (g L−1): yeast extract 1.0; peptone 1.0; glucose 1.0; fructose 1.0; sucrose These experiments used Na2S2O3—the main oxidized product 1.0; lactose 1.0; galactose 1.0; maltose 1.0; methionine 1.0; of Na2S in the air. In shake flask experiments, a 5 % inoculum glycine 1.0; lysine 1.0; Na2S2O3·5H2O10.0;Na2S1.0;ele- size was inoculated in 100 mL sulfur-containing liquid. CFU, 2− 2− mental sulfur 1.0; yeast extract 1.0, Na2S2O3·5H2O 10.0; S2O3 concentration and SO4 concentration were deter- peptone 1.0, Na2 S 2 O 3 ·5H2 O 10.0; glucose 1.0, mined under optimal culture conditions. Using strain JNU-2, Na2S2O3·5H2O 10.0. Colony-forming unit (CFU) data were a lab scale IALR was designed for oxidizing sulfide to ele- determined, and all experiments were carried out at least in mental sulfur (Fig. 1).Thereactorwasmadeofglassandits duplicate. To determine the optimum pH and temperature for internal diameter and height were 5 cm and 30 cm, respec- growth and sulfide oxidized to elemental sulfur, 5 % of active tively. The reactor structure was simple and effective, it was strain JNU-2 was inoculated in fresh medium and grown at an divided into a liquid circulation reaction area and a solid initial pH range of 4–9 and temperature range of 30–50 °C. particle precipitation area by a guide tube. This design realized 2− 2− CFU, S2O3 concentration and SO4 concentration were the combination of reaction and separation. Under conditions detected every 12 h for determining the optimal culture of VC for 360 and 60 mL min−1, respectively, HRT was conditions. regulated to obtain the optimal culture conditions for sulfide In the optimal culture conditions determined above, removal. pH was adjusted to 6.0 every 12 h and the temper- − − 3.0 g L 1 sodium acetate, 1.0 g L 1 sodium lactate, or the ature was controlled at 37.0±3.0 °C. 556 Ann Microbiol (2015) 65:553–563

determined by the iodometric method (Eaton et al. 1998) and sulfate was measured by the turbidimetric method (Pagella et al. 1996a). Elemental sulfur was determined by sulfur balance:

0 CS ¼ 64:13=112:13ΔC 2− −32:13=96:13ΔC 2− ð1Þ S2O3 SO4 Where ΔC 2− is reduction in thiosulfate concentration; and S2O3 ΔC 2− is increase in sulfate concentration. SO4 The reaction is as follows: 2− þ = ¼ 0 þ 2− ð Þ S2O3 1 2O2 S SO4 2 0 þ ¼ 2− ð Þ S 2O2 SO4 3

Results and discussion

Fig. 1 Schematic diagram of the internal airlift loop reactor (IALR) for Screening of a sulfide-oxidizing microbes and colony oxidation of sulfide to elemental sulfur. 1 IALR; 2 medium storage tanks; 3, 4 liquid pumps; 5 air pump morphology

Na2S2O3·5H2O was employed as the sole energy source for Analytical methods screening sulfur-oxidizing microbes. A faint yellow granular deposit was visible in the enrichment culture broth, and was The pH of was adjusted and determined by a pH meter (PHB- identified as elemental sulfur by combustion reaction. Five 10, Sartorius, Göttingen, Germany). Thiosulfate was strains, named JNU-1, 2, 3, 4 and 5 were isolated from municipal

Fig. 2a–d Collection site and morphological characterization of JNU-2 cells. a Anaerobic treatment tank at sewage treatment plant in Wuxi. b Transmission electron micrograph (TEM) of clustered JNU-2 cells. c Colony of isolate JNU-2. d TEM micrograph of a single JNU-2 cell. Images of JNU-2 cells were viewed on a solid agar plate, and strain JNU-2 was cultivated at 37 °C for 4 days Ann Microbiol (2015) 65:553–563 557

Fig. 3 Neighbor-joining tree Acidithiobacillus ferrooxidans (FJ194542) based on 16S ribosomal RNA Acidithiobacillus ferrooxidans (FJ194541) gene sequences of strain JNU-2 66 and related Thiobacillus isolates. Acidithiobacillus albertensis (NR028982) 100 Numbers in parentheses are Acidithiobacillus albertensis (GQ254658) GenBank accession numbers. Bar Acidithiobacillus thiooxidans (DQ676508) Evolutionary distance 100 53 Acidithiobacillus thiooxidans (NR044920) Group 1 54 Acidithiobacillus thiooxidans (DQ676511) Acidithiobacillus caldus (EU499920) Acidithiobacillus caldus (AF513711) 100 100 Acidithiobacillus caldus (DQ470072) 70 Acidithiobacillus caldus (AY427958) Thermithiobacillus tepidarius (NR042145)

100 Thermithiobacillus sp. (HM173631) Group 2 79 JNU-2 (KC493561)

100 Halothiobacillus neapolitanus (AB308268) Halothiobacillus neapolitanus (JF416645) Group 3 100 Halothiobacillus halophilus (NR026015) 100 Halothiobacillus hydrothermalis (NR025943)

99 Thiobacillus thioparus (NR044755) Thiobacillus thioparus (HM535226) Group 4 100 Thiobacillus denitrificans (NR025358) 100 Thiobacillus denitrificans (HQ851082)

0.02

wastewater sludge, the removal ratios of NaS2O3 assayed after Analysis on phylogeny of strain JNU-2 36 h was 77.4 %, 99.4 %, 81.3 %, 59.6 % and 72.6 %, respectively. Thus, JNU-2 was chosen to explore the potential of After reclassification of some species of Thiobacillus to newly removal sulfur. The bacteria grew on thiosulfate-agar medium designated genera (Kelly and Wood 2000), a genus of at 37 °C, and formed colonies with a diameter of 0.5–1.0 mm Thermithiobacillus comprising only one common species after 4 days. The edge of the colonies was regular and pellucid. was named Thermithiobacillus tepidarius. The 16S rRNA The center was yellowish, which indicated extracellular deposi- gene of strain JNU-2 (1,452 bp) was sequenced and submitted tion of sulfur. Cells of strain JNU-2 were measured as 2.33± to GenBank with the accession number KC493561. The 16S 0.15 μminlengthand0.87±0.10μm in width (n=50), with a rRNA sequences of strain JNU-2 were then submitted to a short rod shape, and were gram negative. Capsule was also Blast search of GenBank with 22 other related strains, and a observed around the cells under TEM (Fig. 2), which might help phylogenetic tree based on their 16S rRNA gene sequences strain JNU-2 resist the hostile acidic environment with high was constructed (Fig. 3). Descriptions of referenced strains are concentrations of sulfur compounds. listed in Table 1. The results indicated that the phylogenetic

Fig. 4 Initial profiles of changed biomass and chemical parameters by JNU-2 bio-oxidation 558 Ann Microbiol (2015) 65:553–563 Ann Microbiol (2015) 65:553–563 559

Fig. 5a–f Effect of pH and temperature on cell growth, thiosulfate Strain JNU-2 exhibited a range of tolerant pH (5–8), and oxidation and elemental sulfur accumulation of strain JNU-2. a Effect the optimum pH value for growth was 7–8(Fig.5a). of pH on cell growth. b Effect of pH on thiosulfate oxidation. c Effect of pH on elemental sulfur accumulation. d Effect of temperature on cell Nevertheless, the purpose of this reaction was oxidization of growth. e Effect of temperature on thiosulfate oxidation. f Effect of sulfide and accumulation of elemental sulfur, rathern than temperature on elemental sulfur accumulation biomass production. The most appropriate pH value for the thiosulfate oxidation and elemental sulfur accumulation was tree was divided into four groups, and that strain JNU-2 was 5–6(Fig.5b,c). Similarly, the optimum temperature for cell located in the same clade as Thermithiobacillus.StrainJNU-2 growth was different from that of thiosulfate oxidation and had a rather high sequence identity with strains belonging to elemental sulfur accumulation. At the optimum thiosulfate the species Thermithiobacillus tepidarius (NR042145, 100 % oxidation and elemental sulfur accumulation conditions similarity), Thermithiobacillus sp. (HM173631, 99 %). (pH 6, 37 °C), about 98.0 % of thiosulfate conversion ratio Strains of group 2, belonging to genera of was achieved, and 83.06 % of reduced state sulfur (thiosulfate Thermithiobacillus, showed the highest homology with contains a reduced state sulfur and a oxidized state sulfur) was JNU-2. The genera of Acidithiobacillus, Halothiobacillus oxidized to elemental sulfur after 24 h (Fig. 5d–f). The rela- and Thiobacillus were respectively included in groups 1, 3 tionship between biomass and accumulation of elemental and 4, which showed only 92 % genetic similarity with JNU-2 sulfur was not strictly a positive correlation when the CFU − (Fig. 3). Among Thermithiobacillus species, strain JNU-2 was exceeded 1.5×106 cells mL 1. Strous et al. (1997)reported initially exploited to be used for sulfide removal. that colorless sulfur bacteria (CSB) could oxidize sulfide, even elemental sulfur, outside cells into sulfate even in the case of Conditions affecting JNU-2 growth and sulfide oxidation almost no significant cell growth in nutrient limited conditions. It was considered that a pH value close to neutral and The growth pattern and chemical parameters of strain moderate temperature were advantages for desulfurization JNU-2 in thiosulfate medium are shown in Fig. 4.The reactor design and operation. lag phase of JNU-2 was about 12 h, and it then entered a logarithmic phase for the next 24 h. During cultiva- Tolerance to lactate and acetate of sulfide oxidation of JNU-2 tion, the thiosulfate concentration decreased along with increasing biomass and sulfate concentration. Both ele- Since the sulfide bio-oxidation process occurs immediately mental sulfur accumulation and pH values experienced a after sulfate bio-reduction, the profile of substrate-lactate and rise and fall process. These results indicated that thio- bio-conversion product acetate in the bio-reduction process sulfate was oxidized to sulfate by strain JNU-2, with must be investigated, and the related impacts on sulfide oxi- elemental sulfur as its intermediate. This related path- dation must be evaluated. The reaction in the sulfate bio- way of sulfide oxidation had been reported previously reduction was confirmed as the following (Miu 2004): (Chung et al. 1996;Kimetal.2002); the reaction 2 − þ 2− ¼ − þ þ þ 2− mechanism was listed as follows: C3H5O3 SO4 2CH3COO 2CO2 2H2O S ð7Þ − 0 − 2HS þ O2 ¼ 2S þ 2OH ð4Þ Figure 6 shows the tolerance of acetate and lactate on thiosulfate oxidation and elemental sulfur accumulation of 2 0 þ þ − ¼ 2− þ þ ð Þ S 3O2 2OH 2SO4 2H 5 strain JNU-2. Compared with more than 98 % thiosulfate þ ¼ S 2− þ þ ð Þ conversion ratio and 83.06 % elemental sulfur production H2S 2O2 O4 2H 6 ratio (the reduced state sulfur of thiosulfate was converted to 2− In the thiosulfate medium, CO2 and S2O3 were the sole elemental sulfur) of the blank control after 24 h, the presence carbon and energy sources for the growth of strain JNU-2, of acetate strongly inhibited the oxidation of thiosulfate respectively. Therefore, this strain could grow (22.88 % thiosulfate conversion ratio after being cultivated chemolithotrophically by utilizing energy from the oxidation for 84 h). Correspondingly, there was hardly any accumula- of thiosulfate in the medium. It was observed that JNU-2 cells tion of elemental sulfur (1.38 % elemental sulfur production −1 grewwhen1gL elemental sulfur and Na2S was supplied ratio after cultivating 24 h). Due to the presence of lactate, the individually into the medium instead of 10 g L−1 same thiosulfate oxidation and elemental sulfur accumulation

Na2S2O3·5H2O, and none of the sole organic medium compo- results lagged 12 h behind the control. This was due to the lag nents stimulated cell growth. Addition of 1 g L−1 yeast extract, phase of strain JNU-2 being prolonged by lactate. The same peptone and glucose to the medium with 10 g L−1 results as in the presence of acetate were obtained when both

Na2S2O3·5H2O had no positive effect on growth, indicating acetate and lactate were added to the system. A significant that strain JNU-2 is an obligatory chemolithotrophic sulfur- decline in carboxypeptidase activity on organic substrates was oxidizing microorganism. observed in Thiobacillus intermedius and Thiobacillus 560 Ann Microbiol (2015) 65:553–563

Fig. 6a,b Effect on thiosulfate oxidation and elemental sulfur accumulation of substrate and product of sulfate reduction of strain JNU-2. a Effect of lactate and acetate on thiosulfate oxidation, b effect of lactate and acetate on elemental sulfur accumulation novellus by London and Rittenberg (1966), and Aleen and maximal, the thiosulfate was converted almost completely Huang (1965), respectively. The results of JNU-2 cell toler- and the yield of elemental sulfur reached its highest peak. ance to acetate and lactate were also closely consistent with When the pH value reached a constant minimum, the concen- the above reports. The sulfide generated from anaerobic re- tration of sulfate rose to the top and the yield of elemental duction reaction should be extracted in order to avoid feed- sulfur dropped to the minimum (Fig. 7). This is likely due to back inhibition by the reductive products. the fact that the reactions (Eqs. 4– 6) reacted simultaneously (thiosulfate and elemental sulfur were simultaneously oxi- Initial analysis of biochemical indexes of sulfide removal dized), and then the growth and metabolism of strain JNU-2 cells was restrained by the extreme low pH (< 3.8). The sulfur removal experiment was carried out based on the Considering the activity of JNU-2, an initial pH 6.0 was superiority of JNU-2. After incubating for 24 h in the sulfur determined for stimulating the alkaligenous reaction (Eq. 4). removal system with pure strain JNU-2, the almost total thiosul- Correspondingly, the production of elemental sulfur was en- fate oxidation ratio (98.0 %) was higher than that of Thiobacillus hanced and the efficacy of sulfur removal was sequentially thioparus (90 %) (Xian and Yang 1984), with 83.06 % of improved. reduced state sulfur converted into elemental sulfur. In the first 12 h, the yield of elemental sulfur rose slowly The pH value increased slightly and then dropped dramat- with the bio-oxidation of thiosulfate. Subsequently, a rapid ically through the culture cycle. When the pH value was increasing trend was seen (Fig. 7) due to bacteria entering a

Fig. 7 Changes in various parameters in the sulfur removal shake flask experiment of strain JNU-2 Ann Microbiol (2015) 65:553–563 561

Fig. 8 Changes in various parameters in sulfur removal experiments of strain JNU-2 in the IALR at different ventilatory capacity (VC): a 360 mL min−1, b 60 mL min−1

rapidgrowthphaseafteralagperiodinthesystem. 60 mL min−1 (Fig. 8b) were used, respectively, for sulfide Thiosulfate was utilized extensively as an electron donor by removal in the IALR used with the JNU-2 strain. JNU-2 for obtaining energy, and a large proportion of thiosul- In this reaction, it was important to first ensure the largest fate was converted to elemental sulfur during this phase. sulfide removal ratio, and then, with the yield of elemental Studying the characteristics of the environment and bacteria sulfur guaranteed, the maximal ratio of generation of elemen- in rapid growth phase is helpful for further improving the tal sulfur was also pursued. When thiosulfate was removed efficiency of sulfide removal and elemental sulfur production. almost completely (more than 98 %), the maximal elemental sulfur yield was about 90 mg L−1 h−1 at higher VC (at HRT = − − Removal of sulfide using an internal airlift loop reactor 6 h) and 75 mg L 1 h 1 at lower VC (HRT = h). However, at these times, a 60.0 % generation ratio of elemental sulfur Oxygen is one of the most significant factors for the oxidation (reduced state sulfur of thiosulfate converted to elemental of sulfide to elemental sulfur. Janssen et al. (1997)demon- sulfur) at lower VC was higher than 45 % at higher VC. The strated that the molar oxygen/sulfide ratio needed to produce results was superior to those obtained using Thiobacillus elemental sulfur in a fed-batch reactor was 0.6–1.0. novellus in a biofilter for H2S oxidation under mixotrophic Ventilatory capacity values of 360 mL min−1 (Fig. 8a)and conditions (Chung et al. 1997), in which a removal efficiency 562 Ann Microbiol (2015) 65:553–563 of 99.6 % was achieved and the products were sulfate The isolated strain JNU-2 and the IALR model are of great (83.6 %) and sulfite (12.6 %), and little conversion of sulfide potential in the application of sulfide removal from sulfate to elemental sulfur was achieved. Although the elemental waste water. sulfur yield was higher at higher VC, more sulfate ion was also produced, which requires further processing. The efficacy of sulfur and quantity of sulfate ion was better balanced at the Acknowledgments This work was supported by grants from the Na- optimal VC 60 mL min−1. tional High Technology Research and Development Program of China (863 Program) (No. 2012AA021201 and 2012AA021302), the Program As described above, the initial pH 6.0 was determined for of Innovation Projects Plan of Jiangsu Province (No. CXZZ11_0481), stimulating the alkaligenous reaction (reaction generating el- Doctor Candidate Foundation of Jiangnan University (No. emental sulfur), so the pH was adjusted to 6 every 12 h. At a JUDCF11013), the Priority Academic Program Development of Jiangsu VC of 360 mL min−1, the pH became acidic (minimum below Higher Education Institutions, the 111 Project (No. 111-2-06). 3) at almost every HRT after pH adjustment (Fig. 8a). This may be because the higher VC initiated a high oxygen consumption rate (Eqs. 5, 6); hydrogen ion was produced and sulfur was also significantly consumed. At a VC of References 60 mL min−1, the pH became alkaline in most cases after pH adjustment (Fig. 8b). This probably because the lower VC Aleen MIH, Huang E (1965) Carbon dioxide fixation and Thiobacillus novellus caused a lack of oxygen for the occurrence of high oxygen carboxydismutase in . Biochem Biophys Res Commun 20:515–520 consumption reactions. The alkaligenous reaction was thus Atlas RM, Parks LC (1993) Handbook of microbiological media. CRC, activated (Eq. 4) and was positive for elemental sulfur pro- Boca Raton duction. So on the whole, the elemental sulfur generated ratio Azabou S, Mechichi T, Patel BKC, Sayadi S (2007) Isolation and char- with the lower VC was higher than that with the higher VC. acterization of amesophilic heavy-metals-tolerant sulfate-reducing bacterium Desulfomicrobium sp. from an enrichment culture using The pH at the lower VC was not always changed to alkaline. phosphogypsum as a sulfate source. J Hazard Mater 140:264–270 In the second half of the 40 h HRT and the whole period of the Chan CW, Suzuki I (1993) Quantitative exaction and determination of 20 h HRT, the pH became acidic. Accordingly, elemental elemental sulfur and stoichiometric oxidation sulfide to elemental Thiobacillus thiooxidans – sulfur production was lowest under these conditions. sulfur by . Can J Microbiol 12:1166 1168 Chung YC, Huang C, Tseng CP (1996) Operation optimization of Interestingly, the cell concentration was highest during the Thiobacillus thioparus CH11 biofilter for hydrogen sulfide removal. period of operation (Fig. 8b). The high concentration of the J Biotechnol 52:31–38 JNU-2 strain was considered to give rise to more thiosulfate, Chung YC, Huang C, Li CF (1997) Removal characteristics of H2Sby Thiobacillus novellus and more elemental sulfur oxidized into sulfate (Eqs. 4, 5). CH3 biofilter in autotrophic and mixotrophic environments. J Environ Sci Health A32:1435–1450 Starting time is also a significant factor in the continuous DeSantis TZ, Brodie EL, Moberg JP, Zubieta IX, Piceno YM, Andersen bio-process. In order to shorten the starting time in the IALR, GL (2007) High-density universal 16S rRNA microarray analysis 5 % pure culture of strain JNU-2 at the logarithmic phase was reveals broader diversity than typical clone library when sampling – inoculated into IALR filled with 500 mL sulfur-containing the environment. Microb Ecol 53:371 383 Eaton AD, Clesceri LS, Rice WE, Greenberg AE (1998) Standard broth. The differences in starting time at different VC are methods for the examination of water and wastewater, 20th edn. shown in Fig. 8; higher VC caused the starting time to be American Public Health Association (APHA), New York shortened to 24 h; meanwhile, lower VC caused a delay of cell Ferrea I, Massana R, Casamayor EO, Balague V,Sanchez O, Pedros-Alio growth until 48 h. The start-up operating conditions were C, Mas J (2004) High-diversity bio-film for the oxidation of sulfide- containing effluents. Appl Microbiol Biotechnol 64:726–734 generally different from that of the continuous stage. Gadekar S, Nemati M, Hill GA (2006) Batch and continuous bio- oxidation of sulphide by Thiomicrospira sp. CVO: reaction kinetics and stoichiometry. Water Res 40:2436–2446 Conclusions Janssen AJH, Ma SC, Lens P, Lettinga G (1997) Performance of sulfide- oxidizing expanded-be reactor supplied with dissolved oxygen. Biotechnol Bioeng 53:32–40 A highly efficient sulfide-oxidizer bacterium JNU-2 was Jiang X, Tay JH (2010) Microbial community structures in a horizontal screened from an anaerobic sludge pool, and identified taxo- biotrickling filter degrading H2SandNH3. Bioresour Technol 101: nomically as Thermithiobacillus tepidarius.Thisstrainwas 1635–1641 Kelly DP, Wood AP (2000) Reclassification of some species of then employed for removal of sulfide from sulfate waste Thiobacillus to the newly designated genera Acidithiobacillus gen. water, with ratios of thiosulfate removal and elemental sulfur nov., Halothiobacillus gen. nov. and Thermithiobacillus gen.nov. Int generation of 98.0 % and 83.06 %, respectively. Moreover, a J Syst Evol Microbiol 50:511–516 novel and efficient model of sulfur regeneration reactor—an Ketly DP, Harrison AP (1989) Bergey’s manual of systematic bacteriol- ogy. Williams & Wilkins, Baltimore internal airlift loop reactor (IALR) was designed and con- Kim H, Kim YJ, Chung JS, Xie Q (2002) Long-term operation of a structed based on the traits of JNU-2, and applied to the biofilter for simultaneous removal of H2S and NH3.JAirWaste oxidation of reduced sulfur compounds into elemental sulfur. 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