1. Introduction
The accumulation of cyanide-containing wastewaters in the environment is a serious hazard for ecosystems and human health due to the toxicity of cyanide. Thus, before the discharge of these effluents into the environment, they need to be remediated, either by physical, chemical, and/or biological methods [
1,
2,
3,
4,
5]. The general amount of cyanide that is released from industrial activities has been estimated to be approximately 14 million kg/yr. Three forms of cyanide, including hydrogen cyanide and sodium or potassium cyanide (NaCN or KCN, solid), are the main man-made cyanide compounds [
6]. Cyanide compounds are toxic and hazardous for most living organisms because of their strong binding to metalloproteins, which ultimately results in the inactivation of the electron transport chain [
7,
8]. In fact, cyanide can form complexes with toxic metals, such as nickel, copper, zinc, and iron. The stability and resistance to the biodegradation of such cyano-metal complexes are comparable with free cyanide [
9]. Natural and anthropogenic activities are a result of cyanide production and presence in the environment, and these compounds can be in different forms, i.e., liquid, solid, and gas phases [
6,
10]. Organisms such as plants, bacteria, and fungi are some of the natural cyanide producers; however, the main source of cyanides in the environment are from anthropogenic sources, such as in metal extraction, electroplating, polymer synthesis, steel manufacturing, carbonization, organic chemical production, pharmaceutical, and agricultural and gold mining industries [
2,
11]. These industries generate free- and metal-cyanide complexes at different concentrations and volumes, thus affecting the living organisms that thrive in aquatic environments when cyanide-contaminated waters are discharged into surface waters [
12].
Cyanide can be removed from industrial wastewater by biodegradation, using physical and chemical methods. Alkaline chlorination, barren water rinse, ozonation, adsorption through granulated activated carbon, and ion exchange are some examples of the physical and chemical methods [
13]. Physicochemical degradation methods for the removal of cyanide compounds are expensive and generate additional poisonous products. Moreover, these techniques cannot completely treat some of the cyanide complexes [
14]; however, cyanide biodegradation provides an appropriate alternative [
15]. The biological degradation process has been studied extensively since it is economical and environmentally friendly [
1,
16]. Some aerobic micro-organisms are able to utilize thiocyanate (SCN
−) and cyanideas a nitrogen source or as a sulfur source, in the case of SCN
− [
17]. Several species of bacteria, fungi, and algae have been reported to be cyanide degraders. Some microbial strains, such as
Klebsiella oxytoca,
Pseudomonas fluorescens,
Escherichia coli,
Fusarium solani,
Stemphylium loti,
Rhizopous oryzae, etc., are capable of useing these compounds as a source of nitrogen and carbon for their own growth [
18]. Five common pathways (Hydrolytic, Oxidative, Reductive, Substitution/transfer, Syntheses) are involved in the biodegradation of cyanide and different organisms are able to use one or more pathways for cyanide biodegradation [
13].
However, major focus has been directed to the hydrolytic pathway for cyanide biodegradation, and this is due to: (a) the specific activity of hydrolytic pathway enzymes is high, (b) the enzymes do not need any cofactors, and (c) the triple nonpeptide bonds in cyanide compounds are cleaved directly and the products have low toxicity and can be degraded further [
19]. Cyanide hydratase (CHT), nitrile hydratase (NH), thiocyanate hydrolase (TCH, carbonyl pathways), nitrilase, and cyanidase are the enzymes that are involved in hydrolytic pathways. Some phytopathogenic fungi, such as
S. loti;
Leptosphaeria maculans;
Gloeocercospora sorghi; the
Fusarium species; and saprotrophic fungi, such as
Neurospora crassa,
Aspergillus nidulans, and bacterial species, have an intracellular and inducible enzyme that is known as cyanide hydratase (formamide hydrolyase E.C. 4.2.1.66), which catalyzes the hydration of cyanide to produce formamide [
11,
20]. Rhodanese (EC 2.8.1.1) and cyanide hydratase (EC.4.2.1.66) are the cyanide-degrading enzymes that are produced by
Trichoderma sp. [
21]. The biodegradation of metal-cyanide compound by Fusarium oxysporum N-10 was aided by cyanide hydratase and the enzyme appears to exist as a homotetramer [
22]. The cyanide hydratase of
Aspergillus niger K10 was expressed in
E.coli, and it has been shown that HCN and nitriles can be degraded using this enzyme. Additionally, cyanide hydratase and nitrilase activity were not observed in the truncated enzymes that did not contain 18–34 C-terminal amino acids [
20].
These studies demonstrate that fungal organisms are capable of degrading cyanide species. However, there are minimal studies that have studied various filamentous fungal organisms’ biodegradative capabilities of cyanide, including one study that focused on the detection and sequence alignment of the cyanide hydratase gene. Hence, this present study focused on the assessment of various filamentous fungi for the biodegradation of cyanide, including the detection of the cyanide hydratase gene. Sequence alignment of the cyanide hydratase gene from the assessed fungal organisms and their gene networks with respect to Trichoderma were also undertaken.
3. Discussion
In this study, the selected fungi,
T. harzianum, not only had a high tolerance to cyanide but was also able to use cyanide as a nitrogen since its dry weight increased in the 15 mM cyanide concentration medium in the 7-day culture. This notion is deciphered from the fact that the PDB media contained carbon-based compounds without a nitrogen source. Previous studies have demonstrated that a number of microorganisms, such as
Pseudomonas pseudoalcaligenes,
F. solani, and
Trichoderma sp., are able to convert cyanide to ammonia and bicarbonate and use these products as sources of nitrogen and carbon, respectively. The reaction mechanism of cyanide hydratase in
L. maculans produces formamide, which can also be utilized as a source of nitrogen [
21,
24,
25]. The maximum cyanide degradation that was observed using indigenous
T. harzianum was 75% at a concentration of 15 mM in the 7-day culture in comparison to other studies with lower cyanide concentrations (see
Table 4). The cyanide degradation of 15 mM cyanide in the 5-day and 3-day cultures was greater than the other tested concentrations. However, in the concentration of 20, 25, and 30 mM cyanide, the cyanide degradation efficiency decreased. The decrease in this concentration can be attributed to substrate inhibition and toxicity. It has been reported that different organisms have different tolerances to cyanide concentrations; however, at a particular concentration, cyanide becomes toxic to the organism and can lead to the suppression of the growth of an organism [
4].
Previous studies observed the highest free cyanide biodegradation efficiency of 77% for
F. oxysporum at 22 °C and pH 11 [
26]. The cloning of cyanide hydratase, which is one of the cyanide-degrading enzymes that are present in
Trichoderma sp., and the confirmation of its presence showed its key role in cyanide degradation.
A. niger K10 was observed to possess the cyanide hydratase gene, which was expressed in
E.coli, where the
E.coli that was expressing the cht gene was able to degrade nitriles, thus demonstrating the role that cyanide hydratase plays in cyanide biodegradation [
20]. Besides this microorganism, the cyanide hydratase gene exists in some phytopathogenic fungi, such as
S. loti,
L. maculans,
G. sorghi, and the
Fusarium genus, and saprophytic fungi, such as
N. crassa and
A. nidulans, which are organisms that were tested in this study [
20]. The sequence alignment of cyanide hydratase isolated from
T. harzianum using the BLAST tool [
27] revealed a 94.01% identity and 99% coverage with cyanide hydratase of
Trichoderma simmonsii. Additionally, the multiple sequence alignment of the cyanide hydratase of six species showed a 94.01% to 79.01% identity with our query (sequence) and the catalytic triad, C-K-E, was conserved. Liu et al. (2013) carried out multiple sequence alignments of nitrilase sequences and the catalytic triad, which was similar to the one observed in this study, was revealed [
28]. The phylogenic tree indicates the same origin for the
Trichoderma harzianum strain CBS 229.95 and the indigenous
T. harzianum at the same time, before the other genes emerged. Gene network analysis demonstrated that different proteins are involved in cyanide bioremediation, and these include cyanide hydratase (EC 4.2.1.66), formamidase (EC 3.5.1.49), L-3-cyanoalanine synthase (EC 4.4.1.9), and nitrilase (EC 3.5.5.1). The analysis also revealed that Cyanide hydratase (G0RNY5_HYPJQ), Dipeptidase (G0R9H3_HYPJQ), and Dipeptidase (G0RV68_ HYPJQ) are found in the directed networks, while Carbon–nitrogen hydrolase-like protein (G0RW75_HYPJQ) and ATP adenylyltransferase (G0RJ02_HYPJQ) are found in the undirected networks; these are known as hub (central) proteins. Since the beta subunit of assimilatory sulfite reductase has a key role in growth regulation [
29], it can help in the fungal resistance and growth of
Trichoderma organisms.