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: [email protected] 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