HARARE INSTITUTE OF TECHNOLOGY

ISOLATION AND PURIFICATION OF POTENTIAL CYANIDE DEGRADING

BACTERIA FROM A CONTAMINATED GOLD MINING SITE
BY
VISITOR ZVINOKONA

A RESEARCH PROJECT PROPOSAL SUBMITTED TO HARARE INSTITUTE OF

TECHNOLOGY

2015

COPYRIGHT
All rights reserved. No part of this dissertation may be reproduced, stored in any retrieval
system, or transmitted in any form or by any means, electronic, mechanical, photocopying,
recording or otherwise from scholarly purpose, without the prior written permission of the author
or of Harare Institute of Technology on behalf of the author.

DECLARATION
This research project proposal is my original work except where sources have been
acknowledged.
.
STUDENT
DATE

SUPERVISOR DATE

ACKNOWLEDGEMENTS
I wish to acknowledge the assistance received from the following people who made it possible
for this document to be put together.
My family and friends who gave me the encouragement to finish this document.
I also want to express my gratitude to lectures and staff at Harare Institute of Technology for
their advice and support. To all I want to say, most sincerely, thank you!

Table of Contents
Cover Page
Copyright
Declaration
Acknowledgements
Table of contents

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CHAPTER 1INTRODUCTION
Background of the study
Aims
Research objectives
Research questions
Experimental hypotheses
Problem statement
Significance of the study
Scope and limitations (delimitations) of the study

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CHAPTER 2LITERATURE REVIEW

2.1 Generality of cyanide
2.2 Cyanide treatment technology
2.2.1 Sulfur processes
2.2.2 Alkaline chlorination process
2.2.3 Hydrogen peroxide process
2.2.4 Cyanide biodegradation

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CHAPTER 3 RESEARCH METHODOLOGY

3.1 Materials and method
3.1.1 Sample collection
3.1.2 Tolerance of cyanide

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CHAPTER 4EXPECTED RESULTS

Expected results

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References

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CHAPTER 1. INTRODUCTION
1.1 BACKGROUND OF THE STUDY
The general term cyanide refers to numerous compounds, natural and manmade having
chemical group CN. Several plants, some soil bacteria and several species of invertebrate
organisms produce natural cyanide and related compounds at low levels (Dixon 1999). However
public attention has been drawn to the use of cyanides in industrial processes because cyanide is
a potent inhibitor of cellular metabolism. Potassium or sodium cyanide is generally used in gold
recovery, extraction of metal, pharmaceutical, dye manufacturing industries (Mudder and Botz,
2004; Pope, 1993). The unutilized cyanide compounds is released in the effluent coming out
from the industries involved in making these organic compounds causing contamination of soil
as well as water bodies which affect the biogeochemical cycle. Current chemical methods of
waste water treatment include alkaline chlorination, wet-air oxidation and sulfur-based
technologies (Palmer et al., 1988) and each of these technologies has its own cost and disposal
considerations (Saarela and Kuokkanen, 2004). Biodegradation technologies are particularly
appealing for cyanide wastes with additional organic components to serve as substrates for
microbial growth as well as production of eco-friendly products like formate and formamide
(Dubey and Holmes, 1995; Raybuck, 1992).
Microbial degradation of cyanide at neutral or acidic conditions has been reported but under this
condition a high concentration of cyanide evaporates as hydrocyanic acid (HCN), a weak acid
with a pKa value of 9.2. Thus, it is very important to isolate cyanotrophic microorganisms that
work at alkaline pH. In this sense, this research aims to isolated a native bacterium, from a
contaminated gold mine site (water or heaps), which is able to degrade free cyanide and cyanometalic complexes under alkaline conditions (up to an initial pH of 10).Information about the
optimized parameters of growth in follow up studies (if any) can be utilized in the successful
improvement and propagation of microbes leading to bioremediation of contaminated sites.
Isolation of bacterial strains will lay a foundation for both microbiological and molecular
characterization which may be helpful in proper identification of strains.

1.2 AIMS
To isolate and purify the most cyanide tolerant bacterium able to metabolize cyanide at
alkaline pH.
1.3 RESEARCH OBJECTIVES

To collect a soil samples from a contaminated gold mine site.

To formulate a basic media of nutrient agar enriched with potassium cyanide.
To culture the bacteria resident in spent heaps/water.
To isolate and purify bacteria capable of utilizing cyanide as the sole source of carbon

and nitrogen.
To study bacterium tolerance at different cyanide concentrations.

1.4 RESEARCH QUESTIONS

1. Are the indigenous microbial populations capable of cyanide oxidation and what are their
concentrations?
2. How fast will it work?
3. What volumes and flow rates can be processed?

1.5 EXPERIMENTAL HYPOTHESES

If contaminated samples harbor micro flora then cyanide degrading bacterial can be isolated.
1.6PROBLEM STATEMENT
Due to its composition cyanide is widely used in the mining industries primarily for leaching
gold and silver from ores. Therefore cyanide is present in wastes and slurries produced by these
industries; posing potential hazard to both human health and the environment. The cyanide ion is
one of the potent inhibitor of growth and cellular metabolism, including respiratory, nitrogen
phosphate metabolism. It can also cause rapid breathing, tremors under short terms exposure,
thyroid effects, nerve damage and death for long-term exposure. When exposed in the
environment cyanide compounds are toxic to fish, invertebrates, mammals, algae and
macrophyts by causing disrupted respiration, osmoregulatory disturbances and altered growth
patterns. Mining activities in Zimbabwe, especially gold production, are likely to increase due to
the present situation whereby efforts are underway to increase foreign investment in the mining
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sector. This may mean that without further studies, the effects of mining on the social and
economic lives of the whole local community in the affected areas are devastating. It is against
such concerns that the government and other interested parties are called to pitch in and address
these issues and find cleaner alternatives to traditional practices.
1.7. SIGNIFICANCE OF THE STUDY
The primordial purpose of this study is to provide knowledge on the ability of indigenous micro
flora to tolerate and degrade cyanide up to certain levels at alkaline pH. This is key to the
development of a potential biological treatment process to address environmental remediation
concerns. If bioremediation is successfully implemented and achieved it can present an
ecofriendly and low cost alternative way of dealing with cyanide wastes from not only mining
operations but manufacturing industries as well. The need for easy to apply and fast remediation
strategies has also been evident after the recent Hwange National Park cyanide disaster left
hundreds of wild animals dead. Cyanide poses a health hazard not only to wildlife but also
humans and plants. Furthermore, this study will serve as a theoretical model for future studies of
the same or related nature. Future researchers will benefit from this study, and it will provide
them the facts needed to advance and compare their study during their respective time and
usability. Future research can be done to establish the optimum conditions and requirements
needed to enhance the efficiency and effectiveness of the isolated bacterium in carrying out both
in situ and ex situ reclamation. This is important since the occurrence of cyanide compounds in
industrial wastes presents a major environmental and ecological hazard as most of these
compounds are highly toxic and some are mutagenic as well as carcinogenic.
1.8. SCOPE AND LIMITATIONS OF THE STUDY
This work involves isolation of cyanide tolerant bacteria starting from contaminated sample
collection, media formulation, bacterial culturing, isolation and purification. The ultimate
objective of the whole proposed is provide a basis for further research in potential application of
bioremediation technology at national level using indigenous bacterial species. This study is
limited to isolation of the most cyanide bacteria hence characterization of bacteria and
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determination of optimum parameters for maximum degradation of cyanides remains to be

studied.

CHAPTER 2. LITERATURE REVIEW

2.1 GENERALITY ON CYANIDE
Cyanide is a carbon nitrogen radical, which may be found in a wide variety of life forms and
their large scale presence in the environment is attributed to the manufactured sources which are
used extensively in industries (Maegala et al., 2011).Cyanide is highly toxic to living organisms,
particularly in inactivating the respiration system by tightly binding to terminal oxidase (Porter et
al.,1983). Cyanide was toxic to humans and mammals because it binds to key iron containing
enzyme Cytochrome oxidase required for cells to use oxygen (Kang and Park, 1997). The
occurrence of cyanide compounds in industrial wastes presents a major environmental and
ecological hazard as most of these compounds are highly toxic and some are mutagenic as well
as carcinogenic (Nawaz et al., 1989). However, large amounts of cyanide are used in industries
involved in the metal-plating, pharmaceuticals, synthetic fibers, plastics, coal gasification; coal
coking, ore leaching, gold mining, and electroplating (Knowles and Bunch, 1986). Exposure to
high level of cyanide, a powerful and rapid-acting poison might harm brain and heart leading to
coma and death (Das and Santra, 2011).

2.2 CYANIDE TREATMENT TECHNOLOGY

This section discusses various treatment methods for neutralizing or detoxifying cyanide
solutions, spentleached ores, and tailings. Treatment methods range from rinsing heaps with
water to more complex techniques such as alkaline chlorination and sulfur dioxide processes,
which treat both solutions (spent cyanide solutions and heap rinsate) and slurries (tailings), to
recovery of cyanides. Natural degradation and biological treatment of cyanide is also discussed.

2.2.1 SULFUR PROCESSES

In the sulfur processes, cyanide in solution is oxidized to cyanate using sulfur dioxide or ferrous sulfate
and air in the presence of copper ion:
-

CN + SO2 + O2 + H2O --> CNO + H2SO4

The sulfuric acid formed in the reaction is neutralized with lime. Cyanate may be less toxic than
cyanide to fish, animals, and humans. Higgs and Associates (1992) report that CNO- is 3000 to
5000 times less toxic than CN-. The International Nickel Company's (INCO) SO -air process is
one of two patented sulfur dioxide treatment processes. The other is patented by Noranda Inc.
The INCO process can be applied both to barren solutions and to cyanide-bearing tailings.
Reagent requirements may be higher for tailings. The Noranda Process has been used for
wastewater solutions, but may be applicable to heaps.
Limitations to the SO2 process appear to be that the reaction proceeds more slowly at low
temperatures. A drop in temperature from 25 oC to 5 oC can cause a tenfold decrease in reaction
rate. Correspondingly larger residence times and tank volumes would be required to achieve the
same CN- removal efficiencies at lower temperatures. The SO 2 process generally does not
remove thiocyanate, cyanate, or ammonia. Cyanate can be transformed into ammonia by
microbial action; ammonia is toxic to fish. In addition, removal of toxic metals may not be
sufficient to meet permit requirements.
2.2.2 ALKALINE CHLORINATION PROCESS
The alkaline chlorination process is one of the oldest cyanide destruction methods (Higgs 1992).
In this process, cyanide in solution is oxidized to cyanate using chlorine or hypochlorite in
solution:
CN-+ Cl2 --> CNCl + ClCNCl + 2OH- --> CNO- + Cl-+H2O
Alkaline chlorination can be applied to both clear wastewaters and slurries. Equipment
requirements for the alkaline chlorination process are similar to those for the other two oxidation
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processes (hydrogen peroxide, sulfur dioxide). Wastewater to be treated is introduced into a

mixing vessel, where it is reacted with chlorine or hypochlorite (Figure 2). The pH is maintained
in the alkaline range by addition of lime. Precipitated metals are removed in a clarifier before the
wastewater is discharged.
Smith and Mudder (1991) state that the first-stage reaction (cyanide to cyanate) requires
approximately 15 minutes at pH 10.5. Hydrolysis of the cyanate to ammonia and carbonate
requires an additional 1-2 hours.
Limitations of this process are that it does not remove iron cyanides, and chloramines and free
chlorineremain in solution; these are toxic to fish.
2.2.3 HYDROGEN PEROXIDE PROCESS
In the hydrogen peroxide process, cyanide in solution is oxidized to cyanate using hydrogen
peroxide in the presence of copper ion:
-

CN + H O --> CNO + H2O2

Cyanate ion hydrolyses to form ammonia and carbonate:

-

CNO + 2H2O -->CO32 + NH4+

This process can be applied to wastewaters. Reagent requirements increase when this method is
applied to slurries. Equipment requirements for the hydrogen peroxide process are similar to
those for the INCO process. Wastewater to be treated is introduced into a mixing vessel, where it
is reacted with hydrogen peroxide. Copper sulfate is added as a catalyst. The pH is controlled by
addition of lime. Hydrogen peroxide is a strong oxidizer, which can give rise to violent
explosions and fires if brought in contact with combustible organic material (wood, old cloth
rags). Specially designed storage tanks and handling equipment must be used.

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The limitations of hydrogen peroxide treatment include handling and costs. In particular,
hydrogen peroxide is a hazardous material, and can be expensive. Special equipment for
hydrogen peroxide service may increase the total capital cost. The treatment process generates
ammonia, which is toxic to fish.
2.2.4 CYANIDE BIODEGRADATION.
This is one of the most important biotechnologies to emerge in the last two decades for treating
process and tailings solutions at precious metal mining operations (Middler and Whettock,
1984). Microbial cyanide oxidation is a proven, economical technology for destroying free and
complexed cyanide in process solutions, wastewaters, and spent heaps. The current Applied
Biosciences technology uses microorganisms, indigenous and augmented, to oxidize cyanide,
thus eliminating the need for toxic or corrosive chemical oxidizers. Various scholars have
reported investigations into microbial destruction of cyanide. Hundreds of plant and microbial
species (bacteria, fungi, and algae) can detoxify cyanide quickly to environmental acceptable
levels and into less harmful by products (Middler and Whettock, 1984) and these include
actinonyces, bacillus, thiobacillus and pseudomonas. Full scale bacteria processes have been
used effectively for many years in commercial applications in North America (Middler and
Whettock, 1984).
Many microorganisms can use potassium or sodium cyanide as a sole source of carbon and
nitrogen. For example, Pseudomonas putida utilize cyanides and nitriles as the sole source of
carbon and nitrogen (Babu et al., 1996; Chapatwala et al., 1998). Bacteria like Alcaligenes spp.,
Arthrobacter spp., Burkhoderia cepacia, Bacillus pumilus, Pseudomonas aeruginosa,
Pseudomonas flourescens, Psudeomonas putida, Pseudomonas pseudoalcaligenes CECT5344
(Huertas et al., 2010, Luque-Almagro et al., 2011; Knowles, 1976) and some fungi like
Polyporus arcularius (T 438), Schizophyllum commune (T 701), Clavariadelphus truncatus (T
192), Pleurotus eryngii (M 102), Ganoderma applanatum (M 105), Trametes versicolor (D 22),
Cerrena unicolor (D 30), Schizophyllum commune (D 35) and Ganoderma lucidum (D 33) (Ozel
et al., 2010) and only few algae like Arthrospira maxima, Scenedesmus obliquus and Chlorella
spp. are able to degrade cyanide and cyanide containing compounds (Gurbuz et al., 2004&
2009). Recently a basidiomycetous yeast Cryptococcus cyanovorans sp. nov., has been isolated
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from cyanide contaminated soil (Montaung et al., 2011). Some phytopathogenic fungi like
Fusarium solani (Dumentre et al., 1997) are able to degrade cyanide but bacterial biodegradation
shows considerable advantages since bacteria are more easily manipulated both at biochemical
and genetic levels (Huertas et al., 2006). Biological treatment of cyanide containing waste
depends on the enzyme system Rhodanese or cyanide sulftransferase in the bacteria. Different
enzymes involved in the enzyme system arecyanoalanine synthase, rhodanese cyanide
hydratase, cyanase, nitrogenase and cyanide oxygenase. The enzymes present in the
microorganisms can convert the cyanide to less toxic formamide and ammonia then to
CO2.Cyanide in the effluent can be treated by either chemical or biological methods. Bacterial
detoxification is of interest both in order to understand how cyanide may be dealt with in the
environment and to evaluate the economic viability of bacterial system for cyanide
detoxification.
Documented research has shown that cyanide waste can be used for biological purposes
(Middler, 2001). Several species of bacteria can convert cyanide, under both aerobic and
anaerobic conditions using it as a primary source of nitrogen and carbon. It is known that
organisms are capable of oxidizing the cyanide related compounds of thiocyanite and ammonia
under varying conditions of pH, temperature, nutrient levels, oxygen, and metal concentrations
(Akcil and Koldas, 2006). The biological treatment of cyanide has been shown to be a viable and
robust process for destroying cyanide in the mine process water.
The classic aerobic biological process involves two separate bacterial oxidation steps to facilitate
complete assimilation of waste water (Middler and Whettock,1984). The first step is the
oxidative breakdown of cyanides and thiocyanite, and subsequent absorption and precipitation of
free metals into the bio film. Thiocyanite cyanide is degraded into a combination of ammonia,
carbonate, and sulphate (Middler, 2001).
Cu2CN + 2 H2O + O2-> Cu-biofilm + HCO3- + NH3
-

-> SO

SCN + 2H2O + 2 O2

2-

+ HCO3 + NH3

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The second step involves the decomposition of ammonia into nitrates through the nitrification
process, with nitrate as the intermediate.
NH4++ 1O2->NO2 + 2H+ + H2O
-

NO2 + O2-> NO3

-

Various pseudomonas species are responsible for complete assimilation of the water, including
oxidation of cyanide, thiocyanite, and ammonia in the destruction process. Either chemical or
biological reactions are utilized to convert cyanide into less toxic compounds. The aerobic and
nutrient rich environment promotes the growth of the microbial population, which is capable of
uptake, conversion, sorption, and /or precipitation of thiocyanite, cyanide, ammonia, nitrate, and
sulphate (Middler, 1998).In this regard, waste water is regarded as a negative issue, but it can
also be seen as a positive aspect if its nutrients are used in irrigation for agricultural soil and
aquaculture. Nitrogen, from the cyanide destruction, could probably play an interesting role in
this respect, either through the incorporation of inorganic nitrogen orenhancing primary
productivity in soil and water. The approach of increasing such valuable nutrients into the soil
and water in areas of high mining activity could be an alternative for integrated management.

CHAPTER 3.RESEARRCH METHODOLOGY

A multi-methods approach is proposed in this research and it will that include the use of
interviews,sample tests and field observations.
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Table 1 shows the personnel and reasons for interviews undertaken by key informants at the
mine.
Personnel to be interviewed
Mine manager
Safety, health and environmental officer

Reasons for interview

Information on activities at the mine.
The health impacts of effluent disposal and the

Environmental Management Agency officials

measures put in place to mitigate the impacts.

Information on the environmental policy in

Plant manager

place to monitor and regulate mine activities.

To get information regarding major chemicals,
reagents and heavy metals used in the plant
and the management of effluent water and
spent heaps.

Sample testing and field observations will be used to define the levels of cyanide in either spent
heaps or effluent disposed from the mine. In selecting sampling locations, the researcher will
select representative points.
3.1MATERIALS AND METHOD
3.1.1 SAMPLE COLLECTION
100g of contaminated soil sample will be collected in sterile glass vials, serially diluted and
streaked on sterile nutrient medium supplemented with potassium cyanide (KCN) as the sole
carbon and nitrogen source and incubated at room temperature for 2-3 days for isolation
purposes. Isolates will be further cultured in nutrient medium in order to purify them.
3.1.2 TOLERANCE OF CYANIDE MINIMUM INHIBITORY CONCENTRATION
(MIC)
The minimum inhibitory concentration (MIC) of cyanide will be determined by using mineral
salt medium. Different concentrations of filter sterilized KCN solution (100 ppm, 500 ppm,
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1000ppm, 2000ppm, 3000ppm, 4000 ppm, 5000ppm, 6000ppm) will be added to the medium
before inoculation of the isolates (2% inoculum). Potassium cyanide will serve as the principle
carbon and nitrogen source. Experiment will be conducted in sterile test tubes with 1ml of
mineral salt medium and the tubes were incubated for 48 hours before being, scored for growth
by observing turbidity.

CHAPTER 4 EXPECTED RESULTS

4.1 EXPECTED RESULTS
Presence of various cyanide degrading bacteria with different tolerance levels.

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