Biological Treatment of Cyanide by Using Klebsiella pneumoniae Species Nermin Hande Avcioglu * and

Untreated effl uents of industrial processes are mainly responsible for environmental pollution with various forms of toxic substances, especially free cyanides and metal cyanide complexes (1–4). Since cyanide is highly reactive and forms diff erent complexes with transitional metals, it is a dangerous toxic compound for living organisms (4–6). Additionally, cyanide is also the inhibitor of cytochrome oxidases in electron transport chain, which is a crucial pathway in the respiratory system of bacteria and other organisms (1,3,5,7,8). In this respect, treatment of cyanide in wastewaters and in contaminated areas is an important issue for scientists.


Introduction
Untreated effl uents of industrial processes are mainly responsible for environmental pollution with various forms of toxic substances, especially free cyanides and metal cyanide complexes (1)(2)(3)(4).Since cyanide is highly reactive and forms diff erent complexes with transitional metals, it is a dangerous toxic compound for living organisms (4)(5)(6).Additionally, cyanide is also the inhibitor of cytochrome oxidases in electron transport chain, which is a crucial pathway in the respiratory system of bacteria and other organisms (1,3,5,7,8).In this respect, treatment of cyanide in wastewaters and in contaminated areas is an important issue for scientists.
Klebsiella sp., which is a natural colonizer of humans, vertebrates, birds, reptiles and even insects, is also isolated from contaminated areas such as soil and wastewaters (14)(15)(16)(17).Accordingly, Klebsiella sp. is used as a remediation agent in research of the removal of azo dyes, phenols, hexahydro-1,3,5-trinitro-1,3,5-triazine, organochlorine insecticides, aromatic amines and many other toxic substances (16)(17)(18)(19).However, biodegradation of diff erent cyanide sources, optimization of medium containing cyanide and eff ect of diff erent ions on cyanide biodegradation using K. pneumoniae have not been studied yet.Therefore, in this study, the aim is to investigate the cyanide biodegradation abilities of 17 diff erent K. pneumoniae strains and to select the most effi cient one, as well as to determine the eff ects of optimization conditions for cyanide biodegradation process.Additionally, biodegradation effi ciency of K. pneumoniae in the presence of diff erent cyanide compounds (potassium hexacyanoferrate(II) tri hydrate, potassium tetracyanonickelate(II) hydrate and sodium ferrocyanide decahydrate) was also determined.Furthermore, the eff ects of diff erent ions as contaminants in the biodegradation medium and the effi ciency of sterile crude extract of K. pneumoniae strain as an extracellular product on cyanide biodegradation were also investigated.

Klebsiella pneumoniae strains and growth conditions
Seventeen diff erent Klebsiella pneumoniae strains were investigated according to their cyanide degradation efficiencies.Accordingly, K. pneumoniae strains were inoculated into the biodegradation medium and incubated at 30 °C and 150 rpm for 5 days (Certomat BS-I; Sartorius, Tokyo, Japan).At the end of the incubation, the most eff ective strain in cyanide degradation was identifi ed by using 16S rRNA analysis which showed similarity to K. pneumoniae ATCC 13883 strain (Refgen, Ankara, Turkey).Accordingly, this Kp2 strain was used for the rest of the study.

Analysis of potassium cyanide biodegradation products
Residual cyanide concentration was assessed by using modifi ed picric acid method (21) as follows: 0.5 mL of 0.5 % (by mass per volume) picric acid solution and 0.5 mL of 0.25 M Na 2 CO 3 solution were added into 0.5 mL of culture supernatant.This mixture was boiled for 5 min, then diluted to 10 mL with 8.5 mL of distilled water and cooled under tap water for 30 min, aft er which its absorbance was measured spectrophotometrically at λ=520 nm (model UV 1700; Shimadzu).
Modifi ed nesslerization method (22) was also used to evaluate the concentration of ammonia in the biodegradation medium.Accordingly, 0.5 mL of culture supernatant was diluted with 0.5 mL of distilled water, 0.05 mL of EDTA (0.01 mol) and 2 mL of Nessler's reagent (Merck, Darmstadt, Germany) were added into the diluted sample and aft er 10 min the absorbance of the solution was measured spectrophotometrically at λ=425 nm (UV 1700; Shimadzu).Finally, growth of K. pneumoniae Kp2 strain in the biodegradation medium was measured spectrophotometrically at λ=600 nm (UV 1700; Shimadzu).

Eff ects of physiological conditions on potassium cyanide biodegradation
Eff ects of incubation period (1 to 4 days), initial pH value (3 to 10), incubation temperature (20 to 50 °C), initial KCN concentration (0.25 to 2 mM) and rotation speed (0 to 200 rpm) were investigated in order to determine optimal potassium cyanide biodegradation conditions by K. pneumoniae Kp2 strain.The experiments were performed in triplicate.

Eff ect of ions on potassium cyanide biodegradation process
The eff ect of magnesium, nickel, cobalt, iron, chromium, arsenic, copper and zinc ions on the biodegradation of potassium cyanide was also investigated.In this respect, ion concentrations of 0.1, 0.25 and 0.5 mM were added into the biodegradation medium separately and K. pneumoniae Kp2 strain was inoculated into it at the volume ratio 1:10.Incubation was carried out at 25 °C and 150 rpm for 3 days (Certomat BS-I; Sartorius).The experiment was performed in triplicate.

Biodegradation of diff erent cyanide sources by K. pneumoniae
In order to investigate the biodegradation ability of K. pneumoniae against diff erent cyanide compounds (potas sium hexacyanoferrate(II) trihydrate, potassium tetracyanonickelate(II) hydrate and sodium ferrocyanide decahydrate), biodegradation medium was prepared by using 0.5 mM of each cyanide compound separately.K. pneumoniae Kp2 was inoculated into this medium at the volume ratio 1:10 and incubation was performed at 25 °C and 150 rpm for 3 days (Certomat BS-I; Sartorius).The experiment was performed in triplicate.
Eff ect of K. pneumoniae crude extract on potassium cyanide biodegradation K. pneumoniae Kp2 was incubated into the biodegradation medium at the volume ratio 1:10 and incubation was performed at 25 °C and 150 rpm for 3 days (Certomat BS-I; Sartorius).Aft er incubation, cultures were centrifuged at 2200×g for 5 min (Eppendorf Centrifuge 5417R; Hamburg, Germany).Culture supernatant was taken and sterilized by using 0.45 μM cellulose acetate fi lter (Sartorius) to obtain sterile crude extract of K. pneumoniae Kp2.Accordingly, diff erent mass per volume ratios of this sterile crude extract (10, 20, 30, 40 and 50 %) were inoculated into the biodegradation medium separately and incubation was performed at 25 °C and 150 rpm (Certomat BS-I; Sartorius) in order to examine the biodegradation efficiency of sterile crude extract.The experiment was performed in triplicate.

Results and Discussion
Industrial discharge of cyanide and cyanogen compounds may have serious eff ect on living organisms (3,4,23).Therefore, the contaminated wastewaters or soils must be treated before disposal in order to protect the environment (5).Accordingly, diff erent chemical treatments are used in order to detoxify cyanide and its derivatives.However, when chemical treatments are used, additional methods are required to detoxify the by-products that are produced.In contrast to chemical treatment, biological treatments form nontoxic end products such as ammonia, so they are seen to be more advantageous and successful than the chemical ones (1,4,5,7,10,(24)(25)(26).In this respect, biodegradation of cyanide by using diff erent microorganisms including bacteria, fungi and plants are used in cyanide biodegradation processes (27,28).
In this study, biodegradation abilities of seventeen diff erent K. pneumoniae strains were investigated and Kp2 was selected as the most eff ective one (Fig. 1).This strain was determined to be similar to K. pneumoniae ATCC 13883 by 16S rRNA analysis.
Optimization conditions for cyanide degradation by K. pneumoniae Kp2 were investigated for application in diff erent contaminated soils and wastewaters including cyanogen compounds.As a result, complete degradation of potassium cyanide was observed on the third day (Fig. 2) under the optimal conditions of 0.5 mM initial potassium cyanide concentration (Fig. 3), initial pH=7 (Fig. 4), rotation speed of 150 rpm (Fig. 5) and incubation temperature of 25 °C (Fig. 6).Growth of K. pneumoniae Kp2 under diff erent conditions and formation of ammonia by degradation of potassium cyanide are also shown in Figs.2-6.
Additionally, in this study K. pneumoniae Kp2 degraded potassium hexacyanoferrate(II) trihydrate and sodium ferrocyanide decahydrate with the effi ciencies of 85 and 87.5 % in three days, respectively (data not shown).Therefore, these results indicate that K. pneumoniae Kp2 can be used as alternative bacteria in the biotreatment of wastewaters contaminated with diff erent cyanide sources.
In addition to cyanide, arsenic, chromium, copper, iron, nickel and zinc in gold mining industry, cadmium, chromium, copper, nickel and zinc in metal and electroplating industries, are the main contaminants of untreated industrial effl uents (3,33).These ions bind with cya-  nide and form various compounds that exhibit diff erent stability and toxicity (31,20).In this respect, diff erent concentrations of ions (0.1, 0.25 and 0.5 mM) were added into the biodegradation medium and the correlation between cyanide biodegradation and the growth of K. pneumoniae Kp2 in the presence of ions was investigated.As a result, K. pneumoniae Kp2 was found to degrade KCN in the presence of diff erent ions except for copper at concentration of 0.5 mM.Additionally, in accordance with the other studies the amount of the bacteria in the biodegradation medium was found to decrease with the increase of ion concentration (Fig. 7).However, this drop does not inhibit the biodegradation effi ciency of K. pneumoniae, except for media containing 0.5 mM of arsenic (2 %), zinc (6 %) and chromium (8 %) (Table 1).Therefore, these results indicated that K. pneumoniae Kp2 strain can be used as a remediation agent in the treatment of cyanogen wastes.
Lastly, apart from full biodegradation of potassium cyanide by sterile crude extract of K. pneumoniae observed on the third day in this study, it was observed that 50 % of crude extract fully degraded potassium cyanide on the fi ft h day (Fig. 8).This result indicates that not only the culture of K. pneumoniae but also its crude extract have the ability to degrade KCN.

Conclusions
The results of this research demonstrated the effi ciency of Klebsiella pneumoniae and its sterile crude extract in the biodegradation of diff erent cyanogen compounds.Additionally, K. pneumoniae Kp2 is a useful strain in the treatment of cyanogen wastes in the presence of diff erent ions (magnesium, nickel, cobalt, iron, chromium, arsenic, copper and zinc) that are principal pollutants besides cyanide in wastes.Therefore, this strain and its sterile crude extract seem to be convenient agents for cyanide removal from wastewaters and soils.

Table 1 .
Cyanide biodegradation in the presence of diff erent concentrations of ions