1

New insights into the genetic and metabolic diversity of thiocyanate-degrading 1

microbial consortia 2

3

Mathew P. Watts and John W. Moreau 4

School of Earth Sciences, University of Melbourne, Carlton, Victoria 3010, AUSTRALIA 5

6

Keywords: thiocyanate, biodegradation, bioremediation, metagenomics, geomicrobiology, 7

mine contamination 8

9

Submitted to Applied Microbiology and Biotechnology on Sept. 28th, 2015. 10

Revised and resubmitted on Nov. 6th, 2015. 11

Mini-review format 12

13

14

Correspondence: jmoreau@unimelb.edu.au 15

16

2

Abstract 17

Thiocyanate is a common contaminant of the gold mining and coal-coking industries for 18

which biological degradation generally represents the most viable approach to remediation. 19

Recent studies of thiocyanate-degrading bioreactor systems have revealed new information 20

on the structure and metabolic activity of thiocyanate-degrading microbial consortia. 21

Previous knowledge was limited primarily to pure-culture or co-culture studies in which the 22

effects of linked carbon, sulfur and nitrogen cycling could not be fully understood. High 23

throughput sequencing, DNA fingerprinting and targeted gene amplification have now 24

elucidated the genetic and metabolic diversity of these complex microbial consortia. 25

Specifically, this has highlighted the roles of key consortium members involved in sulfur 26

oxidation and nitrification. New insights into the biogeochemical cycling of sulfur and 27

nitrogen in bioreactor systems, allows tailoring of the microbial metabolism towards meeting 28

effluent composition requirements. Here we review these rapidly advancing studies and 29

synthesize a conceptual model to inform new biotechnologies for thiocyanate remediation. 30

31

3

Introduction 32

Thiocyanate (SCN-) is a common contaminant in gold mining and coal coking wastewaters 33

(Dash et al. 2009). The chemical stability and environmental toxicity of this compound 34

(Bhunia et al. 2000; Wald et al. 1939) have driven a search for new and more effective 35

remediation technologies aimed at degradation of SCN- to benign or inert reaction products. 36

Abiotic remediation methods currently consist mainly of chemical oxidation (Breuer et al. 37

2011; Jensen and Tuan 1993; Mudder et al. 2001; Wilson and Harris 1960) or 38

sorption/separation processes (Aguirre et al. 2010) that are expensive to implement and may 39

require high reagent inputs or produce significant quantities of hazardous solid waste (Akcil 40

2003; Dash et al. 2009). A more viable alternative to abiotic thiocyanate degradation 41

involves the biotechnological harnessing of environmental microorganisms capable of 42

metabolising thiocyanate (van Zyl et al. 2011; Whitlock 1990) . 43

44

Bacteria possessing a SCN--degrading metabolism have been enriched and isolated 45

from diverse environments, including activated sludge (Katayama et al. 1995; Patil 46

2014), soda lakes (Sorokin et al. 2004; Sorokin et al. 2001), soils (Vu et al. 2013; 47

Wood et al. 1998) and gold mine tailings (Stott et al. 2001). These bacteria belong to 48

a range of metabolic niches, and can use SCN- as an energy, carbon, nitrogen or sulfur 49

source (Gould et al. 2012; Sorokin et al. 2001). Several studies have used traditional 50

cultivation-based and/or polymerase chain reaction (PCR) amplification-based (and 51

therefore unavoidably biased) ribosomal gene sequencing to identify specific 52

members of SCN--degrading microbial consortia (Huddy et al. 2015; Lee et al. 2008; 53

Shoji et al. 2014; van Zyl et al. 2014; Villemur et al. 2015). These studies have 54

revealed much about the physiological capabilities of microbial species or 55

populations, but have not been able to explore the full metabolic potential and 56

4

biogeochemical linkages of active consortia or whole microbial communities involved 57

in SCN- degradation. 58

59

An earlier paper reviewed the general knowledge about microbial SCN- degradation at that 60

time (Gould et al. 2012). Since then, however, new studies involving both gene-targeting and 61

metagenomic DNA sequencing have uncovered new information about the diversity, 62

phylogeny, metabolism and interspecies dependencies of SCN--degrading consortia. Also, 63

new experiments have unravelled the interwoven dynamics of microbial sulfur-, nitrogen- 64

and carbon-cycling in such a way as to yield important new implications for the design of 65

SCN- bioremediation technologies. Here, we aim with this mini-review to consolidate 66

current knowledge of the key components and processes associated with microbial SCN- 67

degradation. We first discuss the physiological constraints of pure cultures of SCN--68

degrading microorganisms in processing SCN- and its degradation products, and then 69

examine what has recently been learned from cultivation-independent SCN--degrading 70

microbial community studies, largely from experimental bioreactor systems. We focus our 71

analysis on two key microbially mediated processes: sulfide oxidation and 72

nitrification/denitrification. We further discuss the impact of different carbon cycling 73

pathways on SCN- degradation, with potential for dependent interactions between autotrophs 74

and heterotrophs, or for syntrophic interactions with eukaryotes such as algae and fungi. 75

Finally, we synthesize these topics into an updated conceptual model for microbial SCN- 76

degradation from which the key elements for effective SCN- bioremediation technologies can 77

be drawn. 78

79

SCN- degradation by specific microorganisms/metabolisms 80

5

A variety of autotrophic bacteria have been isolated that are capable of using SCN- as their 81

sole energy source via sulfur oxidation. These chemolithotrophic bacteria utilise the reduced 82

sulfur released from the initial step of SCN- degradation as an energy source for growth, 83

oxidizing sulfide to sulfate (Katayama et al. 1992). They occupy a continuum of 84

environmental settings, with neutrophilic Thiobacillus thioparus (Happold et al. 1958), , 85

Paracoccus spp. (Katayama et al. 1995) and strain specific SCN- degradation proposed for 86

Thiobacillus denitrificans (Kelly and Wood 2000; Beller et al. 2006);; halophilic 87

Thiohalophilus thiocyanoxidans (Bezsudnova et al. 2007) and Halothiobacillus sp. (Sorokin 88

et al. 2014) and the haloalkaliphilic Thioalkalivibrio paradoxus (Sorokin et al. 2002), 89

Thioalkalivibrio thiocyanoxidans (Sorokin et al. 2002) and Thioalkalivibrio 90

thiocyanodenitrificans (Sorokin et al. 2004). All of these bacteria can also directly utilize 91

other reduced sulfur species for growth, such as sulfide, polysulfide, elemental sulfur or 92

thiosulfate (Sorokin et al. 2004; Sorokin et al. 2002), sometimes preferentially over SCN- 93

(Katayama and Kuraishi 1978). In addition to utilizing sulfur as an energy source by these 94

bacteria, utilisation of nitrogen released from thiocyanate as ammonia has also been reported 95

(Youatt 1954). However, the activity of alkaliphilic sulfur oxidizers belonging to the 96

Thioalkalivibrio genus was actually inhibited in the presence of 2-3 mM ammonia at pH ~10, 97

due to the increased toxicity of NH3 at such high pH values (Sorokin et al. 2001). While 98

most SCN--degrading bacteria oxidise sulfur aerobically, some species, such as T. 99

denitrificans and T. thiocyanodenitrificans, are facultative anaerobes able to reduce nitrate or 100

nitrite as well (Kelly and Wood 2000; Sorokin et al. 2004), although possibly at lowered 101

growth rates (Sorokin et al., 2004). 102

103

In addition to the chemolithotrophic bacteria, a number of heterotrophs have been isolated 104

capable of SCN- degradation. The biochemistry and metabolic capability of this group have 105

6

not been studied in the level of detail of the sulfur oxidizing bacteria, but these species are 106

unified by their requirement for an organic carbon source. The heterotrophic bacteria include 107

the diverse genera Ralstonia (du Plessis et al. 2001), Sphingomonas (du Plessis et al. 2001), 108

Klebsiella (Lee et al. 2003), Pseudomonas (Stratford et al. 1994), Arthrobacter (Betts et al. 109

1979) and Methylobacterium (Wood et al. 1998). These genera primarily utilize SCN- as a 110

source of nitrogen, obtaining their energy from the organic carbon instead of the liberated 111

reduced sulfur. The presence of alternative nitrogen sources, such as ammonium, has been 112

reported to inhibit SCN- degradation in some strains (Stafford and Callely 1969), while not in 113

others (Betts et al. 1979). A mixotrophic bacterium, Burkholderia phytofirmans, has also 114

been reported to degrade SCN-, requiring a carbon source and utilizing SCN- as a sole 115

nitrogen source, while apparently oxidizing the sulfur from SCN- (Vu et al. 2013). 116

117

In addition to the above bacteria, eukaryotic SCN- degradation has also been noted in a 118

species of fungus, Acremonium strictum (Kwon et al. 2002). This fungus, isolated from coke-119

oven wastewater, was able to degrade SCN- under circum-neutral pH conditions in the 120

presence of high concentrations of phenol, and alternatively ammonia and nitrate, but 121

exhibited inhibition by nitrite and cyanide. 122

123

Enzymatic pathways for SCN- biodegradation 124

Thiocyanate biodegradation requires the initial hydrolysis of SCN- via specific enzymes to 125

ammonia, sulfide and CO2 (Sorokin et al. 2014), depending on whether the microorganism 126

employs the carbonyl sulfide (COS) pathway (Eqns. 1, 2) or cyanate (CNO-) pathway (Eqns. 127

3, 4) (Kelly and Baker 1990). These two pathways constitute the primary recognized 128

biological mechanisms for SCN- degradation, and are both aerobic. The former occurs via 129

the hydrolysis of SCN-, forming ammonia along with carbonyl sulfide (Ebbs 2004), an 130

7

intermediate that can be further hydrolysed to hydrogen sulfide (H2S) via carbonyl sulfide 131

hydrolase (Ogawa et al. 2013). At this stage, the sulfide is available as an electron donor for 132

sulfur-oxidizing bacteria (Bezsudnova et al. 2007). As a reaction intermediate, carbonyl 133

sulfide readily diffuses out of the cell, and can be detected in the gaseous phase during 134

biodegradation (Kim and Katayama 2000). 135

136

The specific enzyme that mediates this reaction, SCN- hydrolase (SCNase), first identified in 137

the chemolithotrophic bacterium T. thioparus THI 115 (Katayama et al. 1992), exhibits 138

significant homology to bacterial nitrile hydratases (Katayama et al. 1998). The latter study 139

was able to clone and sequence the genes encoding the β, ɑ and γ subunits of this enzyme; the 140

scnB, scnA and scnC genes respectively. Enzymes with homology to this SCNase, or genes 141

encoding its production, have since been identified in other SCN--degrading cultures; 142

T.thiocyanoxidans (Bezsudnova et al. 2007) and a lake water enrichment (Yamasaki et al. 143

2002). A further novel SCNase was isolated from a mesophilic SCN--degrading isolate, strain 144

THI201, with little homology to the previously identified SCNase of T. thioparus THI 115 or 145

T.thiocyanoxidans (Hussain et al. 2013). 146

147

SCN�

� H�O COS � NH� (Eqn. 1) 148

COS H�S � CO� (Eqn. 2) 149

150

The cyanate pathway was first proposed for Thiobacillus thiocyanoxidans (Youatt 1954), 151

now taxonomically included in the T. thioparus (Katayama et al. 1992). Despite no evident 152

accumulation of cyanate as an intermediate, the pathway was proposed due to the presence of 153

an enzyme, cyanase (Anderson 1980), capable of hydrolysing cyanate to ammonia and 154

carbon dioxide (CO2). However, cyanase activity has since been found to be widely 155

8

expressed in bacteria and plants not capable of thiocyanate degradation (Anderson et al. 156

1990), and its presence does not necessarily indicate an ability to metabolize thiocyanate. 157

Indeed, the most substantial evidence for a cyanate pathway has come from the accumulation 158

of cyanate by members of the Thioalkalivibrio genus that lack, or suppress, cyanase activity 159

(Sorokin et al. 2002). This study also found no evidence for the production of sulfide, but 160

rather detected elemental sulfur as a reaction product, deviating from the proposed equation 161

(Eqn.3). Despite this evidence for the utilisation of the cyanate pathway, the enzyme for the 162

conversion of thiocyanate to cyanate remains unidentified. 163

164

SCN�

� H�O CNO�

� H�S (Eqn. 3) 165

CNO�

NH� � CO� (Eqn. 4) 166

167

SCN--degradation by microbial consortia 168

Although a number of microorganisms have been found that can degrade SCN- in isolation, 169

most bioremediation systems rely on consortia, due to the ability of different species or 170

populations to tolerate small to moderate environmental changes, which likely increases the 171

robustness of the system. The co-presence of other non-SCN--degrading microbes may also 172

yield a benefit through metabolising undesirable by-products such as ammonium. Several 173

such co-culture or consortia-based bioreactors have been demonstrated at laboratory, pilot 174

and field scales (van Zyl et al. 2011; Whitlock 1990). A variety of bioreactor designs have 175

been developed, typically in the form of continuously stirred tank reactor systems (Lee et al. 176

2008) or moving bed bioreactors (Stott et al. 2001). The former are typically employed in 177

combination with a settling tank to retain and re-use biomass (van Zyl et al. 2011) or contain 178

some type of solid substrate to provide a surface for biofilm growth (Villemur et al. 2015). As 179

the focus of this review is the metabolic capabilities of the microbial community within the 180

9

reactors, a comprehensive review on bioreactor design is not provided here, and has been 181

discussed in more detail previously (Gould et al. 2012). 182

183

Sulfur oxidising consortia 184

The chemolithotrophic sulfur-oxidizing bacteria previously discussed typically dominate 185

SCN--degrading bioreactor consortia (Fig 1). This dominance results from their activity in 186

SCN- contaminated waste or activated sludge, often used as inocula for bioreactors, as well as 187

from their adaptation to the moderately saline and circumneutral to slightly alkaline pH 188

conditions of typical bioreactor feedstocks. Of the sulfur-oxidizing bacteria, those belonging 189

to the genus Thiobacillus are often identified in bioreactor communities (Huddy et al. 2015; 190

Kantor et al. 2015; Lee et al. 2008; Ryu et al. 2015; Villemur et al. 2015). For example, a co-191

culture of sulfur-oxidising Thiobacillus or Halomonas capable of SCN- degradation was 192

enriched from slightly alkaline and moderately saline gold mine tailings (Stott et al. 2001). 193

These bacteria were used to inoculate a lab scale moving bed bioreactor with a total surface 194

area of 20 m2, and were able to degrade 2800 mg L-1 SCN- to 1 mg L-1 at a flow rate of 30 195

mL min-1. Increased salinity in bioreactors may result in dominance of Halothiobacillus spp. 196

(Sorokin et al. 2014). While moderately halophilic members of this clade are not known to 197

degrade SCN-, Halothiobacillus halophilus/hydrothermalis SCN-R1 can degrade SCN- via 198

the cyanate pathway, and is the only Halothiobacillus species known to be capable of this 199

process. 200

201

Interestingly, despite their ability to fix carbon from CO2, Thiobacillus spp. are still often the 202

dominant in the presence of an organic carbon source, such as with molasses supplied in the 203

well-known ASTERTM biodegradation system (Huddy et al. 2015; Kantor et al. 2015). 204

Investigations of the 16S rRNA gene sequences affiliated with Thiobacillus in another 205

10

bioreactor study found that the majority of sequences grouped with T. thioparus, T. 206

thiophilus, T. denitrificans or Thiobacillus sajenensis (Villemur et al. 2015). As previously 207

discussed, T. thioparus and some strains of T. denitrificans have been found to degrade SCN- 208

in pure cultures (Happold et al. 1958; Kelly and Wood 2000). More recently, a metagenomic 209

study investigated two different SCN--degrading bioreactors, one of which received an 210

influent containing SCN- and the other a mixture of SCN- and CN-, both dominated by 211

Thiobacilli (Kantor et al. 2015). The SCN--only bioreactor metagenome revealed complete 212

genes encoding for SCN- hydrolase (SCNase) were present and associated with Thiobacillus 213

spp. These genes were co-located in a conserved operon containing the gene encoding for the 214

cyanase enzyme, alongside three other genes with possible roles in sulfur metabolism. The 215

co-localisation of genes encoding SCNase and cyanase potentially explains the co-expression 216

of these enzymes during pure culture studies (Bezsudnova et al. 2007). Another SCNase 217

gene, recently also identified in Afipia spp. strain TH201 (Hussain et al. 2013), was found in 218

two Thiobacillus spp. genomes from the CN- and SCN- bioreactor, along with novel and 219

previously unrecognized SCNases associated with Thiobacillus spp. and Pseudonocardia 220

spp. genomes. 221

222

Other non-SCN--degrading sulfur-oxidizing bacteria have also been detected in SCN--223

degrading microbial communities (Shoji et al. 2014; Kantor et al. 2015). The presence of the 224

non-SCN--degrading sulfur-oxidizing bacterium, Thiomicrospira thermophila, has been 225

reported, in this case metabolising thiosulfate present in the influent, and again potentially 226

oxidizing sulfide released by SCN--degrading microorganisms (Shoji et al. 2014). A number 227

of sulfur-oxidizing bacteria not capable of SCN- degradation were also found to be present, at 228

low abundance, in the complex bioreactor community studied by Kantor et al. (2015). 229

Intriguingly, the latter study found no gene encoding for carbonyl sulfide hydrolase (COSase) 230

11

in either of the bioreactors analysed, suggesting the potential for expression of other sulfur-231

oxidising genes (e.g., sox, rdsr, APS reductase, ATP sulfurylase) to further oxidise the sulfur 232

released from the breakdown of COS. Thus, several sulfur oxidation pathways were 233

identified using a metagenomic approach, although only Thiobacillus spp. possessed both sox 234

and rdsr alongside SCNase, suggesting this genus still played the primary role in coupled 235

SCN- degradation and sulfur cycling. 236

237

Nitrogen cycling consortia 238

Heterotrophic bacteria capable of SCN- degradation typically utilize the nitrogen released as 239

ammonia as their nitrogen source . Indeed, some consortia from SCN- treatments are 240

principally made up of these heterotrophic nitrogen assimilators, such as the culture 241

documented in van Zyl et al. (2011) and du Plessis et al. (2001). This consortium contained 242

the heterotrophs Ralstonia eutropha, Sphingomonas paucimobilis and Pseudomonas sp., 243

which are known to degrade SCN-. Heterotrophic SCN- degraders are also identified as less 244

abundant members of other SCN- degrading consortia, for example the genus Sphingomonas 245

in (Felföldi et al. 2010) and (Kantor et al. 2015). 246

247

In addition to SCN--degrading bacteria that utilize released nitrogen, a number of non-SCN--248

degrading nitrogen cycling microbes are also often found in SCN--degrading consortia (Fig 249

1). Associated with the conversion of SCN- to ammonia in aerobic bioreactors, a number of 250

nitrifying consortium members have been identified (Kantor et al. 2015; Ryu et al. 2015; 251

Villemur et al. 2015). In fact, the two stage treatment reactor used in Ryu et al. (2015) 252

produced nitrification occurring simultaneously with SCN- degradation, resulting in an 253

increase in nitrite and nitrate, likely mediated by Nitrospira spp. present in the inoculum. 254

Interestingly, the abundance of this genus decreased upon exposure to SCN-, an observation 255

12

interpreted as a potential toxicity response to either SCN- or nitrous acid generated from 256

ammonium oxidation. Nonetheless, harnessing the action of nitrifying bacteria, typically 257

affiliated with Nitrobacter, Nitrosospira, Nitrosomonas and Nitrospira, can enable the 258

removal of ammonium and nitrite from bioreactors (Villemur et al. 2015). Kantor et al. 259

(2015) detected genes encoding for the ammonia monooxygenase and hydroxylamine 260

oxidoreductase of Nitrosospira multiformis in their metagenomic bioreactor study, supporting 261

the potential for cycling and removal of nitrogen. Significantly, however, no nitrite oxidation 262

genes were detected, indicating a possible limitation to the ability of nitrifying bacteria to 263

offset the action of ammonium as an inhibitor to SCN--degraders. 264

265

Active denitrification has also been identified in some bioreactor systems (Fig 1), as either an 266

intentionally promoted process (Villemur et al. 2015), or an unintentional effect of the 267

activity of microbial consortia (Kantor et al. 2015). The former study consisted of a series of 268

bioreactors, one of which was targeted to promote denitrification by creating anaerobic 269

conditions. The presence of T. denitrificans in these reactors is inferred to indicate an active 270

denitrifying population. In the latter study, genes for denitrification from nitrite were found 271

to be present within five members of a bioreactor consortium. Members of the 272

Xanthomonadaceae were found to denitrify to N2O, while other members could complete 273

denitrification from this intermediate. Complete denitrification genes were also found to be 274

present on the genomes of Thiobacillus spp. and other autotrophs. Interestingly, several 275

genomes contained genes encoding for cyanase, suggesting bioavailability of this 276

intermediate as a carbon or nitrogen source. Recent work has also highlighted the role of 277

archaeal ammonia-oxidizers in cyanate degradation in natural environments, suggesting the 278

possibility their growth could be stimulated in thiocyanate contaminated groundwaters 279

(Palatinszky et al. 2015). 280

13

281

Other recent studies have examined potential syntrophic links with eukaryotes for nitrogen 282

removal in SCN--degrading bioreactors. A paper by (Ryu et al. 2015) used a mixed 283

bacterial/microalgal consortium, in which microalgae assimilated nitrogen accumulated as 284

ammonia. Notably, this process was maintained in the same bioreactor as SCN- degradation, 285

using operational parameters to promote the desired metabolism, switching from 286

lithoautotrophic conditions supportive of Thiobacillus spp. in the SCN--degrading stage to 287

photoautotrophy stimulated by LED light activation, after which the abundance of 288

Microactinium and cyanobacteria increased. Alongside the increased abundance of 289

microalgae, bacteria previously known as symbionts in algal cultures also flourished. The 290

most significant of these was a Rhizobium-like microorganism, which typically requires a 291

plant host and is capable of N2 fixation, highlighting the potential supporting role of these 292

bacteria for microalgal activity. Interestingly, Kantor et al. (2015) also detected genes from a 293

eukaryote (genus Rhizaria) encoding for nitrite reduction, suggesting a role for this eukaryote 294

similar to that of the fungus Fusarium oxysporum (Kim et al. 2009). 295

296

Carbon cycling in thiocyanate-degrading consortia 297

The presence of organic carbon can support heterotrophic bacteria that utilize SCN- as a 298

nitrogen source (du Plessis et al. 2001; van Zyl et al. 2011). Organic carbon is initially input 299

into SCN--degrading bioreactors in a number of ways, for example through addition of a 300

labile carbon source such as molasses (van Zyl et al. 2011) to promote heterotrophic growth. 301

A complex milieu of carbon compounds is also present in SCN- coal coking waste, including 302

phenol (Staib and Lant 2007) that can be degraded by a number of heterotrophic bacteria 303

(Felföldi et al. 2010). Finally, inputs of SCN- and air also provide CO2 to autotrophic 304

bacteria for conversion to biomass that can be recycled within the bioreactor. Genes used in 305

14

carbon fixation, including Calvin–Benson–Bassham (CBB) cycle and RuBisCO genes, were 306

detected in a bioreactor metagenome, with the latter belonging to the predominant 307

Thiobacillus and Thiomonas spp. (Kantor et al. 2015). A number of heterotrophic eukaryotes 308

have also been identified in carbon rich bioreactors (Huddy et al. 2015; van Zyl et al. 2011). 309

These include fungi, yeasts and amoebae that are likely to be metabolizing organics or dead 310

biomass. Some of these eukaryotes are closely related to species which are known to degrade 311

CN-; e.g., Fusarium oxysporium (Huddy et al. 2015). 312

313

Implications for bioreactor design 314

The efficiency of SCN- biodegradation is a function of multiple operational variables, 315

involving the chemistry of the influent waste stream, and the composition and activity of a 316

potentially complex microbial community. Numerous lab scale bioreactor studies are 317

principally aimed at optimisation of these parameters and identifying potential problems with 318

inhibition, prior to pilot or field scale operation. Larger scale SCN- bioremediation efforts 319

have typically focussed upon mixed consortia, typically due to the increased robustness from 320

the metabolic diversity of this approach. The bacterial species present in these consortia are a 321

function of both the original inoculum and the culture conditions employed. As previously 322

discussed, these systems are initially inoculated with a complex bacterial consortium from 323

activated sludge or contaminated waste, while amendments and exposure to SCN- and other 324

contaminants likely modify the microbial ecology of the bioreactor. 325

326

Recent studies have yielded new insights into how an understanding of bioreactor microbial 327

community structure can lead to better designs incorporating coupled biogeochemical 328

processes, either within the same reactor or across several reactors in sequence. Most of 329

these processes involve controlling the spatial and temporal growth and metabolic activity of 330

15

intermixed or segregated sulfur-oxidising and nitrogen-cycling microorganisms. Recent 331

work has demonstrated that a series of bioreactors can be quite effective (Banerjee 1996; 332

Staib and Lant 2007; van Zyl et al. 2014), typically where other compounds alongside SCN- 333

were targeted for degradation. One such system (Banerjee 1996) contained four identical 334

reactors in series and the co-contaminant phenol inhibiting SCN- degradation in the first two 335

reactors. After phenol biodegradation occurred, however, SCN- was effectively degraded in 336

the second two reactors. Other reactor designs (Villemur et al. 2015) have employed 337

connected arrays of moving bed bioreactors for comparison of SCN-, cyanate and ammonia 338

removal efficiencies. One array employed all aerobic bioreactors for SCN- degradation, while 339

the other employed an anaerobic bioreactor at the start of the series aimed at promoting 340

denitrification. This denitrification step significantly improved removal of SCN-, cyanate and 341

ammonia over the course of the array. 342

343

Variation in phenotypes of bioreactor microbial communities with and without suspended 344

solids has been observed (van Zyl et al. 2014). Interestingly, in the absence of suspended 345

solids, copious amounts of biofilm were formed. In the presence of solids, the authors found 346

Bosea, Microbacterium and Thiobacillus spp. to be major constituents of the SCN--degrading 347

consortium, results consistent with those of van Buuren et al. (2011). Also detected were 348

four fungi (two filamentous and two yeasts). Fusarium oxysporum, a known cyanide 349

degrader that was present in the ASTERTM consortium characterized by du Plessis et al. 350

(2001) was not detected, however. Interestingly, increased solid loading was found to 351

correspond with a longer adaptation period for microbial growth. In the presence of solids 352

(i.e. the absence of biofilm), greatly reduced microbial diversity was found, interpreted to 353

represent a lack of potential anaerobic and microaerophilic consortium members. 354

355

16

If effluent from SCN- degrading bioreactors is to be used as return flow to ore or tailings 356

processing plants, or BIOX® bioleaching reactors (http://www.biomin.co.za/#), the potential 357

combined effects of various microbial metabolic processes and efficiencies must be taken 358

into consideration. The van Hille et al. (2014) study describes the inhibitory effects of SCN- 359

on two isolates (Leptospirillum ferriphilum and Acidithiobacillus caldus) obtained from the 360

BIOX® consortium. These bacteria exhibited complete inhibition of iron oxidation in the 361

former, and almost complete inhibition of sulphur oxidation in the latter, above 1.25 mg L-1 362

SCN-. The treatment of SCN- to below these values, via bioremediation, is therefore a pre-363

requisite for enabling the re-use of tailings water in the BIOX® process. 364

365

Concluding remarks 366

Utilizing SCN- metabolizing microorganisms in a bioreactor approach is becoming a more 367

widely adopted practice, due to its effectiveness compared to abiotic chemical approaches. 368

Earlier studies focused upon the metabolic potential of single isolates or co-cultures of SCN- 369

degraders, using SCN- as a sulfur or nitrogen source. Although informative for culturable 370

isolates, this approach is limited when applied to the complex microbial consortia typically 371

present within a bioreactor system. The recently increased accessibility of high throughput 372

sequencing techniques has, however, enabled insights into the true metabolic and genetic 373

diversity of these systems. The information garnered from this approach has revealed 374

fundamental information on nutrient cycling in SCN- contaminated systems, relevant to 375

natural systems. In addition, this information helps inform improvement of current or future 376

biotechnological approaches, through understanding the key processes limiting SCN- 377

biodegradation, or though optimisation of redox cycling of its constituent elements; carbon, 378

sulfur and nitrogen (Fig 1). 379

380

17

Advances in molecular sequencing techniques have highlighted the dominance of the genus 381

Thiobacillus, in a number of SCN--degrading bioreactors, noting the importance of sulfur 382

cycling for SCN- biodegradation. Nitrogen cycling has also been found to be a key metabolic 383

process, where ammonium released from SCN- degradation can be assimilated in to biomass, 384

nitrified or denitrified, depending upon the conditions in the bioreactor. Understanding the 385

constraints upon these processes has enabled the development of novel bioreactor designs 386

aimed at removal of nitrogen species, by encouraging the proliferation of denitrifying 387

bacteria (Villemur et al. 2015) or nitrogen-assimilating microalgae (Ryu et al. 2015). 388

Understanding the often complex cycling of carbon in bioreactor systems has also revealed 389

interesting insights into the roles of heterotrophic and autotrophic microorganisms. 390

Significantly, the dominance of autotrophic SCN- degrading bacteria suggests that the 391

addition of a labile carbon source may not be needed for effective SCN- degradation (Kantor 392

et al. 2015). 393

394

This review therefore serves to highlight that the utilisation of high throughput DNA 395

sequencing has greatly improved our understanding of the microbial community dynamics 396

and genetic capability within SCN- degrading bioreactors. This aids the development of more 397

efficient and effective bioremediation approaches, which have the metabolic versatility to 398

tailor the effluent chemical composition. This approach has significance not just for SCN- but 399

also for the development and optimisation of other biotechnological approaches to 400

contaminant remediation. 401

402

403

404

Compliance with Ethical Standards 405

18

406

Conflict of Interest: Authors declare that they have no conflict of interest. 407

408

Ethical approval: This article does not contain any studies with animals performed by any of 409

the authors. 410

411

412

REFERENCES 413

Aguirre NV, Vivas BP, Montes-Morán MA, Ania CO (2010) Adsorption of thiocyanate anions from 414

aqueous solution onto adsorbents of various origin. Adsorption Science & Technology 415

28(8):705-716 416

Akcil A (2003) Destruction of cyanide in gold mill effluents: biological versus chemical treatments. 417

Biotechnol Adv 21(6):501-511 doi:http://dx.doi.org/10.1016/S0734-9750(03)00099-5 418

Anderson PM (1980) Purification and properties of the inducible enzyme cyanase. Biochemistry 419

19(13):2882-2888 420

Anderson PM, Sung Y-c, Fuchs JA (1990) The cyanase operon and cyanate metabolism. FEMS 421

Microbiol Rev 7(3-4):247-252 422

Banerjee G (1996) Phenol-and thiocyanate-based wastewater treatment in RBC reactor. J Environ Eng 423

122(10):941-948 424

Beller HR, Chain PS, Letain TE, Chakicherla A, Larimer FW, Richardson PM, Coleman MA, Wood 425

AP, Kelly DP (2006) The genome sequence of the obligately chemolithoautotrophic, 426

facultatively anaerobic bacterium Thiobacillus denitrificans. J Bacteriol 188(4):1473-1488 427

Betts P, Rinder D, Fleeker J (1979) Thiocyanate utilization by an Arthrobacter. Canadian journal of 428

microbiology 25(11):1277-1282 429

Bezsudnova EY, Sorokin DY, Tikhonova TV, Popov VO (2007) Thiocyanate hydrolase, the primary 430

enzyme initiating thiocyanate degradation in the novel obligately chemolithoautotrophic 431

19

halophilic sulfur-oxidizing bacterium Thiohalophilus thiocyanoxidans. Biochimica et 432

Biophysica Acta (BBA)-Proteins and Proteomics 1774(12):1563-1570 433

Bhunia F, Saha N, Kaviraj A (2000) Toxicity of thiocyanate to fish, plankton, worm, and aquatic 434

ecosystem. Bull Environ Contam Toxicol 64(2):197-204 435

Breuer P, Jeffery C, Meakin R Fundamental investigations of the SO2/air, peroxide and Caro's acid 436

cyanide destruction processes. In: Proc ALTA 2011 Gold Conf, Perth WA, Australia, ALTA 437

Metallurgical Services, 2011. p 154-168 438

Dash RR, Gaur A, Balomajumder C (2009) Cyanide in industrial wastewaters and its removal: A 439

review on biotreatment. J Hazard Mater 163(1):1-11 440

du Plessis C, Barnard P, Muhlbauer R, Naldrett K (2001) Empirical model for the autotrophic 441

biodegradation of thiocyanate in an activated sludge reactor. Lett Appl Microbiol 32(2):103-442

107 443

Ebbs S (2004) Biological degradation of cyanide compounds. Curr Opin Biotechnol 15(3):231-236 444

doi:http://dx.doi.org/10.1016/j.copbio.2004.03.006 445

Felföldi T, Székely AJ, Gorál R, Barkács K, Scheirich G, András J, Rácz A, Márialigeti K (2010) 446

Polyphasic bacterial community analysis of an aerobic activated sludge removing phenols and 447

thiocyanate from coke plant effluent. Bioresour Technol 101(10):3406-3414 448

Gould WD, King M, Mohapatra BR, Cameron RA, Kapoor A, Koren DW (2012) A critical review on 449

destruction of thiocyanate in mining effluents. Minerals Engineering 34:38-47 450

Happold F, Jones G, Pratt D (1958) Utilization of thiocyanate by Thiobacillus thioparus and T. 451

thiocyanoxidans. Nature 182: 266-267. 452

Huddy RJ, van Zyl AW, van Hille RP, Harrison ST (2015) Characterisation of the complex microbial 453

community associated with the ASTER™ thiocyanate biodegradation system. Minerals 454

Engineering 76:65-71 455

Hussain A, Ogawa T, Saito M, Sekine T, Nameki M, Matsushita Y, Hayashi T, Katayama Y (2013) 456

Cloning and expression of a gene encoding a novel thermostable thiocyanate-degrading 457

enzyme from a mesophilic alphaproteobacteria strain THI201. Microbiology 159(Pt 458

11):2294-2302 459

20

Jensen JN, Tuan Y-J (1993) Chemical oxidation of thiocyanate ion by ozone. 460

Kantor RS, Zyl AW, Hille RP, Thomas BC, Harrison ST, Banfield JF (2015) Bioreactor microbial 461

ecosystems for thiocyanate and cyanide degradation unravelled with genome‐resolved 462

metagenomics. Environ Microbiol 463

Katayama Y, Hiraishi A, Kuraishi H (1995) Paracoccus thiocyanatus sp. nov., a new species of 464

thiocyanate-utilizing facultative chemolithotroph, and transfer of Thiobacillus versutus to the 465

genus Paracoccus as Paracoccus versutus comb. nov. with emendation of the genus. 466

Microbiology 141(6):1469-1477 467

Katayama Y, Kuraishi H (1978) Characteristics of Thiobacillus thioparus and its thiocyanate 468

assimilation. Canadian journal of microbiology 24(7):804-810 469

Katayama Y, Matsushita Y, Kaneko M, Kondo M, Mizuno T, Nyunoya H (1998) Cloning of genes 470

coding for the three subunits of thiocyanate hydrolase of Thiobacillus thioparus THI 115 and 471

their evolutionary relationships to nitrile hydratase. J Bacteriol 180(10):2583-2589 472

Katayama Y, Narahara Y, Inoue Y, Amano F, Kanagawa T, Kuraishi H (1992) A thiocyanate 473

hydrolase of Thiobacillus thioparus. A novel enzyme catalyzing the formation of carbonyl 474

sulfide from thiocyanate. J Biol Chem 267(13):9170-9175 475

Kelly DP, Baker SC (1990) The organosulphur cycle: aerobic and anaerobic processes leading to 476

turnover of C1-sulphur compounds. FEMS Microbiol Rev 7(3-4):241-246 477

Kelly DP, Wood AP (2000) Confirmation of Thiobacillus denitrificans as a species of the genus 478

Thiobacillus, in the beta-subclass of the Proteobacteria, with strain NCIMB 9548 as the type 479

strain. Int J Syst Evol Microbiol 50(2):547-550 480

Kim S-J, Katayama Y (2000) Effect of growth conditions on thiocyanate degradation and emission of 481

carbonyl sulfide by Thiobacillus thioparus THI115. Water Res 34(11):2887-2894 482

Kim S-W, Fushinobu S, Zhou S, Wakagi T, Shoun H (2009) Eukaryotic nirK genes encoding copper-483

containing nitrite reductase: originating from the protomitochondrion? Appl Environ 484

Microbiol 75(9):2652-2658 485

Kwon HK, Woo SH, Park JM (2002) Thiocyanate degradation by Acremonium strictum and inhibition 486

by secondary toxicants. Biotechnol Lett 24(16):1347-1351 487

21

Lee C, Kim J, Chang J, Hwang S (2003) Isolation and identification of thiocyanate utilizing 488

chemolithotrophs from gold mine soils. Biodegradation 14(3):183-188 489

Lee C, Kim J, Do H, Hwang S (2008) Monitoring thiocyanate-degrading microbial community in 490

relation to changes in process performance in mixed culture systems near washout. Water Res 491

42(4–5):1254-1262 doi:http://dx.doi.org/10.1016/j.watres.2007.09.017 492

Mudder TI, Botz M, Smith A (2001) Chemistry and treatment of cyanidation wastes. Mining Journal 493

Books, London, UK 494

Ogawa T, Noguchi K, Saito M, Nagahata Y, Kato H, Ohtaki A, Nakayama H, Dohmae N, Matsushita 495

Y, Odaka M (2013) Carbonyl sulfide hydrolase from Thiobacillus thioparus strain THI115 is 496

one of the β-carbonic anhydrase family enzymes. J Am Chem Soc 135(10):3818-3825 497

Palatinszky M, Herbold C, Jehmlich N, Pogoda M, Han P, von Bergen M, Lagkouvardos I, Karst SM, 498

Galushko A, Koch H (2015) Cyanate as an energy source for nitrifiers. Nature 499

524(7563):105-108 500

Patil Y (2014) Development of a bioremediation technology for the removal of thiocyanate from 501

aqueous industrial wastes using metabolically active microorganisms. Patil Yogesh B (2013) 502

Development of a bioremediation technology for the removal of thiocyanate from aqueous 503

industrial wastes using metabolically active microorganisms In: Applied Bioremediation-504

Active and Passive Approaches (Editors Yogesh B Patil and Prakash Rao), Intech Open 505

Science Publisher 506

Ryu B-G, Kim W, Nam K, Kim S, Lee B, Park MS, Yang J-W (2015) A comprehensive study on 507

algal–bacterial communities shift during thiocyanate degradation in a microalga-mediated 508

process. Bioresour Technol 509

Shoji T, Sueoka K, Satoh H, Mino T (2014) Identification of the microbial community responsible for 510

thiocyanate and thiosulfate degradation in an activated sludge process. Process Biochem 511

49(7):1176-1181 512

Sorokin DY, Abbas B, van Zessen E, Muyzer G (2014) Isolation and characterization of an obligately 513

chemolithoautotrophic Halothiobacillus strain capable of growth on thiocyanate as an energy 514

source. FEMS Microbiol Lett 354(1):69-74 515

22

Sorokin DY, Antipov AN, Muyzer G, Kuenen JG (2004) Anaerobic growth of the haloalkaliphilic 516

denitrifying sulfur-oxidizing bacterium Thialkalivibrio thiocyanodenitrificans sp. nov. with 517

thiocyanate. Microbiology 150(7):2435-2442 518

Sorokin DY, Tourova TP, Lysenko AM, Kuenen JG (2001) Microbial thiocyanate utilization under 519

highly alkaline conditions. Appl Environ Microbiol 67(2):528-538 520

Sorokin DY, Tourova TP, Lysenko AM, Mityushina LL, Kuenen JG (2002) Thioalkalivibrio 521

thiocyanoxidans sp. nov. and Thioalkalivibrio paradoxus sp. nov., novel alkaliphilic, 522

obligately autotrophic, sulfur-oxidizing bacteria capable of growth on thiocyanate, from soda 523

lakes. Int J Syst Evol Microbiol 52(2):657-664 524

Stafford D, Callely A (1969) The utilization of thiocyanate by a heterotrophic bacterium. Journal of 525

General Microbiology 55(2):285-289 526

Staib C, Lant P (2007) Thiocyanate degradation during activated sludge treatment of coke-ovens 527

wastewater. Biochem Eng J 34(2):122-130 528

Stott M, Franzmann P, Zappia L, Watling H, Quan L, Clark B, Houchin M, Miller P, Williams T 529

(2001) Thiocyanate removal from saline CIP process water by a rotating biological contactor, 530

with reuse of the water for bioleaching. Hydrometallurgy 62(2):93-105 531

Stratford J, Dias AEO, Knowles CJ (1994) The utilization of thiocyanate as a nitrogen source by a 532

heterotrophic bacterium: the degradative pathway involves formation of ammonia and 533

tetrathionate. Microbiology 140(10):2657-2662 534

van Hille RP, Dawson E, Edward C, Harrison ST (2014) Effect of thiocyanate on BIOX® organisms: 535

Inhibition and adaptation. Minerals Engineering 536

van Zyl AW, Harrison ST, van Hille RP (2011) Biodegradation of thiocyanate by a mixed microbial 537

population. Mine Water–Managing the Challenges (IMWA 2011), Aachen, Germany 538

van Zyl AW, Huddy R, Harrison STL, van Hille RP (2014) Evaluation of the ASTERTM process in the 539

presence of suspended solids. Minerals Engineering(0) 540

doi:http://dx.doi.org/10.1016/j.mineng.2014.11.007 541

23

Villemur R, Juteau P, Bougie V, Ménard J, Déziel E (2015) Development of four-stage moving bed 542

biofilm reactor train with a pre-denitrification configuration for the removal of thiocyanate 543

and cyanate. Bioresour Technol 544

Vu H, Mu A, Moreau J (2013) Biodegradation of thiocyanate by a novel strain of Burkholderia 545

phytofirmans from soil contaminated by gold mine tailings. Lett Appl Microbiol 57(4):368-546

372 547

Wald MH, Lindberg HA, BARKER MH (1939) The toxic manifestations of the thiocyanates. Journal 548

of the American Medical Association 112(12):1120-1124 549

Whitlock JL (1990) Biological detoxification of precious metal processing wastewaters. Geomicrobiol 550

J 8(3-4):241-249 551

Wilson I, Harris G (1960) The oxidation of thiocyanate ion by hydrogen peroxide. I. The pH-552

independent reaction. J Am Chem Soc 82(17):4515-4517 553

Wood AP, Kelly DP, McDonald IR, Jordan SL, Morgan TD, Khan S, Murrell JC, Borodina E (1998) 554

A novel pink-pigmented facultative methylotroph, Methylobacterium thiocyanatum sp. nov., 555

capable of growth on thiocyanate or cyanate as sole nitrogen sources. Arch Microbiol 556

169(2):148-158 557

Yamasaki M, Matsushita Y, Namura M, Nyunoya H, Katayama Y (2002) Genetic and 558

immunochemical characterization of thiocyanate-degrading bacteria in lake water. Appl 559

Environ Microbiol 68(2):942-946 560

Youatt JB (1954) Studies on the metabolism of Thiobacillus thiocyanoxidans. Journal of General 561

Microbiology 11(2):139-149 562

563

564

Figure Caption 565

Fig 1. Conceptual model of bioreactor thiocyanate degradation and subsequent sulfur and 566

nitrogen cycling consortia, in the presence of both autotrophic and heterotrophic consortium 567

members, in a thiocyanate-degrading bioreactor system. After (Kantor et al. 2015). 568

24

569

25

570

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s:

Watts, MP; Moreau, JW

Title:

New insights into the genetic and metabolic diversity of thiocyanate-degrading microbial

consortia

Date:

2016-02-01

Citation:

Watts, M. P. & Moreau, J. W. (2016). New insights into the genetic and metabolic diversity

of thiocyanate-degrading microbial consortia. APPLIED MICROBIOLOGY AND

BIOTECHNOLOGY, 100 (3), pp.1101-1108. https://doi.org/10.1007/s00253-015-7161-5.

Persistent Link:

http://hdl.handle.net/11343/56700

File Description:

Accepted version