ASSESSING THE POTENTIAL OF ASPERGILLUS FLAVUS AND PENICILLIUM SP IN REMOVING MERCURY, ZINC AND CHROMIUM FROM RAW REFINERY EFFLUENTS

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ABSTRACT

 

This study was carried out to evaluate the potential of Aspergillus flavus and Penicillium sp in removing Zinc, Mercury and Chromium from raw refinery effluents. Physicochemical, heavy metal content and heavy metal removal capacity were determined, from the raw refinery effluents from the Kaduna Refinery and Petrochemical Company (KRPC) using standard procedures. Most of the physicochemical and heavy metal content were within the permissible limits set by the Federal Ministry of Environment Nigeria (FEMNV) except temperature (42.70C), Conductivity (48.10µS/cm), Chloride (835.5mg/l) and Mercury (0.0024mg/l). Two fungal species; Aspergillus flavus and Penicillium sp originally isolated from the KRPC were obtained from the Department of Microbiology, Ahmadu Bello University Zaria and re-authenticated by characterization on the basis of their macroscopic and microscopic morphologies using standard taxonomic guides. The isolates were inoculated into 100ml flask containing 50ml of the Potato Dextrose Broth (PDB) supplemented with 5, 10 and 15 µg/ml of Zn, Hg and Cr analytical grade salts, to test for the heavy metal tolerance using dry weight mycelial mat. It was observed that the two fungal species were tolerant and grew in the medium containing 5, 10 and 15ppm of the test heavy metals. The isolates removed substantial amount of Zn, Hg and Cr from the raw refinery effluent. Aspergillus flavus was able to remove 74% of Zinc, 85% of Mercury and 81% of Chromium which translated to adsorption of 0.800mg/g of Zinc, 0.530mg/g of Mercury and 0.133mg/g of chromium, after 7days Penicillum sp was able to remove 84% of Zinc, 56% of Mercury and 84% of chromium translating to the adsorption of 0.323mg/g of Zinc, 0.413mg/g of Mercury and 0.72mg/g of Chromium, over same period. It was therefore concluded that Aspergillus flavus and Penicillum sp obtained from effluent polluted environments were tolerant to the heavy metals and are good agents in treatment of refinery effluents and remediation of sites polluted with Zinc, Mercury and Chromium.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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TABLE OF CONTENTS


 

Contents


Pages


 

Cover Page……………………………………………………………………………………i

 

Fly Leaf……………………………………………………………………………………….ii

 

Title Page…………………………………………………………………………………..…iii

 

Declaration…………………………………………………………………………………....iv

 

Certification…………………………………………………………………………………...v

 

Dedication…………………………………………………………………………………….vi

 

Acknowledgement……………………………………………………………………………vii

 

Abstract……………………………………………………………………………………....viii

 

Table of Contents…………………………………………………………………………... ix-xi

 

List of Tables………………………………………………………………………………...xii

 

List of Figures………………………………………………………………………………..xiii

 

List of Plates……………………………………………………………………………….....xiv

 

CHAPTER ONE

 

INTRODUCTION …………………………………………………………………………........1

 

1.1        Statement of Research Problem…………………………………………………………..4

 

1.2        Justification for the study………………..……………………………………………......5

 

1.3        Aims of Study………………………………………………………………………….....7

 

1.4        Specific Objectives…………………………………………………………………….....7

 

CHAPTER TWO

 

LITERATURE REVIEW………………….............……………………………………………8

 

2.1        Heavy Metals……………………………………………………………………………...8

 

2.2        Environmental and Health Risks of Heavy Metals……………………………………....10

 

2.2.1     Chromium……………..………………………………………………………………....10

 

2.2.2     Mercury…………………………………………………………………………………..11

 

2.2.3     Lead……..…………………………………………………………………………….….12

 

2.2.4     Nickel……………………………………………………………………………………..13

 

2.2.5     Cadmium………………………………………………………………………………....14

 

2.2.6     Zinc……………………………………………………………………………………….15

 

2.2.7     Arsenic…………………………………………………………………………………....16

 

2.3        Industrial Wastewater Sources………………………………………………….………...17

 

2.4        Conventional Processes for Removal of Heavy Metals…………………………………..18

 

2.4.1     Chemical Perception…………..………………………………………………………….18

 

2.4.2     Ion Exchange……………………………………………………………………………..19

 

2.4.3     Electrolytic Recovery…………………………………………………………………….19

 

2.4.4     Adsorption on new adsorbents……………………………………………………………22

 

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2.4.5    Adsorption on modified natural materials……………………………………………….21

 

2.4.5.1    Zeolites……..…………………………………………………………………………..22

 

2.4.5.2     Clay–polymer composites…..…………………………………………………………22

 

2.4.5.3    Phosphate-based adsorbents……………………………………………………………22

 

2.4.6    Adsorption on industrial by-products……………………………………………………22

 

2.4.7     Adsorption on modified agriculture and biological wastes (bio-sorption)……………....24

 

2.4.8     Adsorption on modified biopolymers and hydrogels…………………………………….25

 

2.4.9     Membrane filtration……………………………………………………………………...28

 

2.4.10 Electrodialysis…………………………………………………………………………....29

 

2.4.11 Evaluation of heavy metal removal process……………………………………………..30

 

2.4.12 The role of fungi in the removal of heavy metals……………………………………….32

 

CHAPTER THREE

 

MATERIALS AND METHODS……………………………………………………………….39

 

3.1        Study Site …………………………………………………………………………….….39

 

3.2        Sample Collection……………………………………………………………………..……39

 

3.21      Sampling Site……………………………………………………………………………...39

 

3.3        Collection of Refinery Effluent…………………………………………………………...40

 

3.4        Preliminary Analysis of the Raw Refinery Effluents Sample…………………………….44

 

3.4.1     Determination of  pH……………………………………………………………………...44

 

3.4.2    Determination of temperature………………………………………………………….…44

 

3.4.3    Determination of electrical conductivity……………………………...………….………44

 

3.4.4    Determination of Total Dissolved Solids……………………………...…………..……..44

 

3.4.5    Determination of Total Suspended Solids……………………………...…………..…….45

 

3.4.6     Determination of dissolved oxygen (DO)…………………………………………..…….45

 

3.4.7    Determination of Biochemical Oxygen Demand (BOD)…………………………………46

 

3.5        Determination of Heavy Metal Content of the Effluent………………………………..…46

 

3.6        Source of fungal isolates……………………………………………………………...…...47

 

3.6.1     Re-authentication of Fungal Isolates…………………………………………………..….47

 

3.6.2     Preparation of fungal Inocula………………………………………………………..……47

 

3.7         Determination of Zinc, Mercury and Chromium Tolerance by the Fungal Isolates…………………………………………………………………………………....48

 

3.7.1    Preparation of stock solution of heavy metal ions…………………………………….….48

 

3.7.2     Procedure for Determining Heavy Metal Tolerance of Fungal Isolates………………….48

 

3.8        Assessment of the capacity of the fungal isolates in removal of Zinc,

 

Mercury and Chromium from the refinery effluents…………………………………......50

 

CHAPTER FOUR

 

RESULTS………………………………………………………………………………………...51

4.1        Physicochemical Properties of the refinery effluent……………………………………....51

 

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4.2        Cultural and Microscopic Characteristics of the Fungal isolates used for this Study…….55

 

4.3        Tolerance of the test fungal isolates to Zinc, Mercury and Chromium…………………...58

 

4.4        Removal of Zinc, Mercury and Chromium by the test Fungal isolates…………….…….62

 

4.5        Adsorption of Zinc, Mercury and Chromium by Penicillium sp.

 

and Aspergillus flavus…………………………………………………………………….62

 

 

 

 

CHAPTER FIVE

 

DISCUSSION………………………………………………………………………..…………...65

 

5.1         Physicochemical Parameters of the Refinery Effluent……………………………………65

 

5.2         Heavy Metal Content of the raw refinery effluents……………………………………….66

 

5.3         Resistance of test fungal isolates to Zinc, Mercury and Chromium………………………67

 

5.4         Heavy Metal Removal and Adsorbed by Aspergillus flavus and Penicillium sp………………….69

 

 

 

CHAPTER SIX

 

CONCLUSION AND RECOMMENDATION………………………………………………….71

 

6.0         Conclusion…………………………………………………………………………….……71

 

6.1         Recommendation……………………………………………………………………….…..72

 

6.2         Contribution to knowledge…………………………………………………………………72

 

References…………………………………………………………………………………………74

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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LIST OF TABLES

 

Table 2.1: The Acceptable Limits for most hazardous heavy metals…………………………….9

 

Table 2.2: Adsorption capacities of modified natural materials for heavy metals…………..…..23

 

Table 2.3: Adsorption capacities of some agricultural and biological wastes for heavy metals...26

 

Table 2.4: Advantages and disadvantages of the physicochemical methods

for treatment of heavy metals in wastewater…………………………………….……33

 

Table 4.1: Physicochemical properties of the refinery effluent………………………………….52

 

Table 4.2: Percentage Removal of Heavy Metal by Test Fungi from the Refinery Effluents……63

 

Table 4.3: Quantities of the Heavy Metals bioadsorbed from the

effluent by the test fungal isolates………………………………………………....….64

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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LIST OF FIGURES

 

Figure2.1: Processes of a conventional metals precipitation treatment Plant……………..…………………………..……………………………………………........ 20

 

 

Figure 2.1: Electrodialysis principles …………………………......................................................31

 

 

Figure 2.2: The Outer layers of Fungal Cell……………………………………………………….36

 

 

Figure 3.1 Southern part of Kaduna Metropolis showing NNPC Refinery………………………..41

 

 

Figure 3.2 Schematic diagram showing the position of the sampling points relative to the

 

refinery……………………………………………………………………………………………..43

 

 

Figure 4.1a: Mean Biomass Yield of the Test Fungi to various concentrations of Zinc…….……..59

 

 

 

Figure 4.1b: Mean Biomass Yield of the Test Fungi to various Concentrations of Mercury………60

 

 

 

Figure 4.2 Mean Biomass Yield of the Test Fungi to various

 

Concentrations of Chromium Ion………………………………………………………61

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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LIST OF PLATES

 

 

Plate I.a: Raw Refinery Effluent (Site A)…………………………………………………..…41

 

 

Plate I.b: Effluent in flow channel 1 (Site B)…………………………………………….…...42

 

 

Plate I.c: Effluent in flow channel 1 (Site C)…………………………………………….…...42

 

 

Plate II:  Characterization of  Aspergillus flavus……………………………………….….....56

 

Plate III.: Characterization of Penicillum sp………………………………………………....57

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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CHAPTER ONE

 

1.0                                                                                INTRODUCTION

 

Heavy metal contaminants are common features of effluents generated and released by wide range of modern industries (Singh and Sharma 2013) and refinery effluents bearing heavy metals considered to highly toxic (Ezeonuegbu et al., 2014). Heavy metal contaminated wastewater has been identified as one of the most important sources of pollution. Such waste waters are usually generated from a variety of sources such as crude oil producing and refining, other petrochemical industries, metal processing, lubricant and car washing. These sources serve as the major contributors to the problems of heavy metal pollution especially of soil and water environments (Machido, 2015). The discharge of oily wastewater to the environment has potential to cause significant environmental hazard. This is because it contains toxic substances such as petroleum hydrocarbons, phenols, polyaromatic hydrocarbons which are inhibitory to animal and plant growth and also are mutagenic and carcinogenic to human beings (Musa et al., 2015).

 

Beddri and Ismail (2007) reported that waste-water effluents from petroleum refinery and petrochemical plants contain a diverse range of pollutants including heavy metals. The effluents also contain oil and grease,and are characterized by high biochemical oxygen demand (BOD) - bearing materials, suspended solids, dissolved solids, phenols and sulfides.

 

Heavy metals in refinery effluent mainly originate from the feedstock. Others are from corrosion products of the equipment and pipes, processed chemical additives and from materials like catalysts and other chemicals used in processes downstream of the primary distillation. The most common of these metals are Nickel, Vanadium, Copper, Chromium, Lead, Cadmium, Zinc and Selenium. Mercury has also been found to appear as impurity in natural gas and crude oil (Alao et al., 2010).

 

Heavy metals particularly Zinc, Chromium, Arsenic and Mercury are environmental pollutants threatening the health of human population and natural ecosystem through direct ingestion, inhalation

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and indirect by consumption of fish, animals or plants in which the metals have accumulated (Alao et al., 2010). Other metals like gold, aluminum, cadmium, silver, lead and mercury are also potentially toxic to living organisms (Kumar et al., 2010, Ezeonuogbu et al 2014). Toxicity of these metals occurs through the displacement of essential metals from their natural binding site or through ligand interactions.

 

Methods employed for the removal of organic pollutants from refinery effluents are not applicable when heavy metal ions are at issue because, unlike the organic pollutants, they are non- biodegradable and therefore persist in effluent subjected to conventional treatments (Tran et al., 2015).

 

Over the years, several approaches including chemical precipitation, chemical oxidation, solidification, ultra filtration, flocculation, electrolyte extraction, dilution, sedimentation, evaporation, reverse osmosis, neutralization and membrane separation have been used for removal of metal ions from solution (Viraraghavan and Yan, 2000).

 

These methods present disadvantages in that they are capital intensive, may result to incomplete metal removal, high demands of reagents, high energy requirements and generation of toxic sludge or other waste products that require careful disposal (Hemambika et al., 2011). With the increase in environmental awareness and legal constraints being imposed on discharge of effluents, the need for alternative cost effective technologies is essential. In this endeavor, biological approach has a great potential for the achievement of this goal and it is economical. Microbial populations in metal polluted environment adapt to toxic concentration of heavy metals and become heavy metal resistant (Prasenjit and Sumathi, 2005). The response of microorganisms towards toxic heavy metals is of special interest with regards to their application in the reclamation of polluted sites (Shankar et al., 2007).

 

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Biosorption and bioaccumulation are biological methods of control of heavy metal pollution. These processes involve metal uptake by either non- living (biosorption) or living (bioaccumulation) biomass (Chojnacka et al., 2007). Biosorption is influenced by environmental factors like pH, temperature, ion competition, variations in the chemical composition of the cell wall, capsule, and polysaccharides. In any case, metals can be deposited around the cell as phosphates, sulphates and oxides (Giovanni, 2009). These biochemical processes could be an effective solution to the majority of the problems associated with heavy metals released in the environment by industries (Giovanni 2009). They offer many advantages over conventional methods including cost effectiveness, efficiency, minimization of chemicals and biological sludge, requirement of additional nutrients and regeneration of biosorbents with possibility of metal recovery (Ronda et al., 2007).

 

To date, a good number of biomass types have been tested for their metal binding capabilities under various conditions. The outcome of such studies provided proofs that, fungi have great potential in this regards (Ezeonuegbu et al., 2014, Machido et al., 2015).

 

Fungi are known to tolerate and detoxify metals by several mechanisms including valence transformation, extracellular, intracellular precipitation and active uptake. These mechanisms result from complexation and ion exchange reactions between metal ions and groups on their cell walls (Gupta et al., 2000; Hemambika et al., 2011). The black molds (Aspergilli) are widely distributed in the environment and are capable of metabolizing enormous variety of substances owing to the large number of diversified enzymes they produce (Angumeenal and Venkappayya, 2004).

 

 

 

 

 

 

 

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The present study is aimed at assessing the potential of Aspergillus sp and Penicillium sp in the removal of Zinc (Zn), Mercury (Hg) and Chromium (Cr) from refinery effluents released by Kaduna refinery and petrochemical Company (KRPC).

 

 

 

1.1                        STATEMENT OF RESEARCH PROBLEMS

 

 

The three major petrochemical refineries in Nigeria generate large quantities of effluents that are discharged into natural bodies of water, the quality of which has been widely reported to be tainted as a result (Otokunefor and Obiukwu, 2005; Atubi, 2011). This represents a serious threat to the environment and human health.

 

Petroleum refineries use relatively large volumes of water, especially for cooling systems. As a result, local studies in Kaduna have also shown that approximately 165,000m3/ day of untreated liquid effluents containing heavy metals is generated and heavy metals in such effluents often far exceed those permissible for environmental safety (Musa et al., 2015). Refineries also generate solid wastes and sludges (ranging from 3 to 5 kg per ton of crude oil processed), 80% of which may be considered hazardous because of the presence of toxic organics and heavy metals. Accidental and intentional discharge of large quantities of pollutants can and do occur as a result of abnormal operation in a refinery and potentially pose a major local environmental hazard (Salleh et al., 2003).

 

The law under Nigerian constitution (the national effluents and limitation regulation) is currently not upheld, which facilitate indiscriminate discharge from our refineries, because no punishment is meted out to defaulters (Obot et al., 2007).

 

 

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Heavy metals like Zinc, Mercury and Chromium constitute a high proportion of the refinery effluent. Zinc which is considered essential micronutrients is known to become toxic at high concentrations. Mercury is problematic in cryogenic petrochemical facilities and is toxic to humans. Chromium can cause nasal problems and ulcer as well as kidney and liver damage and it has also been found to be carcinogenic leading to depressed growth (Ezeonuegbu, 2014)

 

The physical and chemical methods currently used to remove heavy metals are not cost effective and often do not restore the environment to its original state. These leaves us with biological method such as adsorption, which is a more effective and sustainable alternative means of restoring the environment to its original state compared to conventional (physical and chemical) methods is adopted (Hussein et al., 2004).

 

1.2         JUSTIFICATION FOR THE STUDY

 

 

At a global scale industrial processes generate approximately 2.4 million tons per annum of liquid effluents containing heavy metals and other toxic materials (Veglio and Beolchini, 1997; Bello and Abdullahi, 2016). Local studies in Kaduna have also shown that approximate volume of 165,000m3/ day of untreated liquid effluents containing heavy metals is generated (Ezeonuogbu, 2014).

 

Hence, the speedy development, increasing sophistication, arbitrary and unregulated discharge of industrial and urban wastes into the environmental sink has become an issue of global concern (Gupta and Mahaptra, 2003; Strong and Burgess, 2008). Therefore, effort must be out on ground as soon as possible to put a stop to this menace.

 

The removal and recovery of heavy metals are necessary for the protection of the environment and human health. Conventional methods are ineffective in treatment of low concentration of metal ions

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and also generate large quantity of toxic sludge which are to be disposed in further steps. Biological methods such as biosorption and bioaccumulation of heavy metals provide an alternative to conventional (physical and chemical) methods (Hussein et al., 2004).

 

Mycoremediation is a more promising and less expensive way of cleaning up contaminated soil and water (Kamaludeen et al., 2003). Mycoremediation through the use of biosorption and bioaccumulation approaches is an emerging technology in which microbes including fungi are used to clean up contaminated soil and water and to remove or stabilize the contaminants which offer a low cost and ecologically valuable means for the mitigation of heavy metal toxicity in the environment (Strong and Burgess, 2008). Many microbial populations including fungi have been identified as superior candidates for metal mycoremediation and major advantages of fungi are its significant metal uptake ability at low anticipated price (Bai and Abraham, 2001). Microbial populations present in metal polluted environment have adapted and become resistant to toxic metal and the presence of heavy metals and their bioavailability exert selective pressure on the microbial community with selective advantage conferred on those organisms that have developed tolerance mechanism to toxic heavy metals (Jaeckle et al., 2005).

 

The cell wall of fungi present a multi-laminated architecture where up to 90% of the dry mass consist of amino and non amino polysaccharides and proteins which offers many functional groups (such as carboxyl, hydroxyl, sulphate, phosphate and amino groups) for binding metal ions. According to Ronda et al. (2007), the cell walls of fungi are also rich in glycoproteins such as glycans (β – 1,6-and β- 1,3- linked D- glucose residues), chitosan (β-1,4-linked mannose) and phosphomanans (phosphorylated manans) with various metal binding groups present in the polymers. Heavy metals like Copper, Cadmium, Mercury, Zinc, Manganese, and Lead present in refinery effluent can be

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removed by indigenous microbes isolated from the effluents itself (Hakeem and Bhatnagar, 2010). Aspergillus and Penicillium species accumulate and absorb micronutrient and heavy metals.These organisms are exposed to a lot of health hazards, it is therefore important to remove these toxic heavy metals from waste water before its disposal.

 

 

 

 

1.3                         AIM AND OBJECTIVES

 

 

The aim was to assess the potential of Aspergillus flavus and Penicillum sp in removing Zinc, Mercury and Chromium from raw refinery effluents

 

1.4         The Specific Objectives were to;

 

 

1.      Determine the physicochemical properties of the refinery effluent.

 

 

2.      Re-authenticate the fungi isolates to be used in the study.

 

 

3.      Determine the tolerance of the fungi isolates to Zinc, Mercury and Chromium invitro.

 

 

4.      Determine the capacity of the fungi isolates in the removal of zinc, mercury and chromium from the raw refinery effluents.

 

 

 

 

 

 

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