HARARE INSTITUTE OF TECHNOLOGY

ISOLATION AND PURIFICATION OF POTENTIAL CYANIDE DEGRADING BACTERIA FROM A CONTAMINATED GOLD MINING SITEBYVISITOR ZVINOKONA

A RESEARCH PROJECT PROPOSAL SUBMITTED TO HARARE INSTITUTE OF TECHNOLOGY

2015

COPYRIGHTAll 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 Page1Copyright2Declaration3Acknowledgements4Table of contents5

CHAPTER 1INTRODUCTION

Background of the study6Aims7Research objectives7Research questions7Experimental hypotheses7Problem statement8Significance of the study8Scope and limitations (delimitations) of the study8

CHAPTER 2LITERATURE REVIEW

2.1 Generality of cyanide92.2 Cyanide treatment technology92.2.1 Sulfur processes92.2.2 Alkaline chlorination process102.2.3 Hydrogen peroxide process102.2.4 Cyanide biodegradation11

CHAPTER 3RESEARCH METHODOLOGY

3.1 Materials and method143.1.1 Sample collection143.1.2 Tolerance of cyanide14

CHAPTER 4EXPECTED RESULTS

Expected results15

References 15

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 cyano-metalic 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 QUESTIONS1. 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 HYPOTHESESIf contaminated samples harbor micro flora then cyanide degrading bacterial can be isolated.1.6PROBLEM STATEMENTDue 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 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 determination of optimum parameters for maximum degradation of cyanides remains to be studied.

CHAPTER 2. LITERATURE REVIEW2.1 GENERALITY ON CYANIDECyanide 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 TECHNOLOGYThis 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 SO2 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 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.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 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- + NH3SCN- + 2H2O + 2 O2-> SO42-+ HCO3-+ NH3

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 METHODOLOGYA multi-methods approach is proposed in this research and it will that include the use of interviews,sample tests and field observations.

Table 1 shows the personnel and reasons for interviews undertaken by key informants at the mine.

Personnel to be interviewedReasons for interview

Mine managerInformation on activities at the mine.

Safety, health and environmental officerThe health impacts of effluent disposal and the measures put in place to mitigate the impacts.

Environmental Management Agency officialsInformation on the environmental policy in place to monitor and regulate mine activities.

Plant manager 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, 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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