ABSTRACT

Tanneries are potential economic sector that uses both natural elements and synthetic chemicals concurrently. The operations releases large volume of effluents containing part and remain of these chemicals that leachate, persist downward and affects water, food crops and human health. Fungi (in its slurry form) as biological agents were used and carry out bioremediation of tannery waste polluted soil. Fungi were isolated from soil polluted with tannery waste and soil devoid of tannery practices. Culture plate techniques including cultural and morphological characteristics were employed and Microbes were isolated, enumerated and purified using serial dilution and spread plate technique. From polluted soil, microbial and physicochemical characteristics were determined where Nutrient agar (NA) was used for total heterotrophic bacterial count (THBC), Sabouraud dextrose agar (SDA) for total heterotrophic fungal counts (THFC), minimal salt agar (MSA) for total tannery waste utilizing bacterial counts (TtWUBC) and total tannery waste utilizing fungal counts (TtWUFC). The isolated fungi were screened in minimal salt medium (MSM) added with tannery effluents, at 530nm spectrophotometer. The identities of successful isolate were further confirmed using molecular genome technique by fungal DNA extraction, PCR amplification, Gel electrophoresis, genomic sequencing and BLAST at NCBI database. The soils polluted with tannery waste were amended with fungi slurry and four treatments (T1, T2, T3 and T4) were constructed and tested for their effectiveness in bioremediation; changes in Physichochemical, microbial and heavy metals contents were measured periodically. The fungi isolates from tannery waste polluted soil include Aspergillus and Penicillium with (20%) occurrence each and (10%) occurrence for each of the Cladosporium, Mucor, Geotricum, Rhizopus and Microsporum sp. while in soil devoid of tannery practices include Aspergillus with (30%) occurrence, Rhizopus (10%), Fusarium (10%), Penicillium (10%), Mucor (10%) and Trichoderma (10%) occurrence. From both soil sampled, Aspergillus sp. had highest occurrence. The microbial load counts of polluted soil before amendment include THBC with (2.5×106 CFU/g), TWUBC (1.5×106 CFU/g), THFC (1.5×105 CFU/g) and TtWUFC (8×103 CFU/g) while pH 7.5, organic carbon (4.7%), Nitrogen (0.22%), Phosphorus (1.25mg/kg), Calcium (3.75mg/kg), Magnesium (3.55mg/kg), sodium (20mg/kg), potassium (12.1mg/kg), CEC (47.3mg/kg) were physicochemical properties recorded and the textural triangle classified the polluted soil as sandy loamy. The optical density of fungi isolates after 8days incubation revealed 1.330 A. fumigatus and 1.373 A. niger conventionally whereas rDNA with ITS1 and 2 primers identified A. aculeatus and A. niger molecularly. The mean range of polluted soil amended with fungi slurry revealed pH  of 7.5-8.8, organic carbon which reduced from 4.7-1.1%, Nitrogen 0.2-0.09mg/kg, phosphorus 1.25-0.69mg/kg, calcium 3.75-2.02mg/kg, magnesium 3.55-0.12mg/kg, potassium 12.1-3.6mg/kg and sodium 20-6.9mg/kg and In the THBC the treatment sets were significantly differentiated (P<0.05) which takes the form T3>T2>T1>T4 and the TtWUBC the order were T4<T1<T2<T3 while in the THFC the order of significance difference were T2>T3>T1>T4 and TtWUFC involved T3>T2>T1>T4 while heavy metals changes like Cu, Zn and Cr were decreased in T1with 9.09-6.51mg/kg, 51.10-27.75mg/kg and 115.27-19.82mg/kg as compared to control, T2 Cu, Fe, Zn and Cr with 9.33-3.57, 79.87-58.95, 55.36-29.21 and 118.12-32.66mg/kg were reduced while T3 Cu, Mn, Fe, Zn and Cr with 8.54-4.31, 10.12-2.12, 74.37-21.72, 54.99-12.09 and 113.25-22.10mg/kg  respectively were decreased. The activity of each fungi depend on time and the species depict capable agent for bioremediation of tannery waste polluted soil although, combined activity indicated much higher performance than individual agent.

CHAPTER ONE

INTRODUCTION

1.1 Background to the study

Public and environmental health quality concern raised the issue of practices that involves the use of unsafe agents on some common industrialized sectors. Among them is a tannery leather production (Azubuike et al., 2016). Pollutant had long exist in our natural environments often day to day human activities that rendered the degree of these pollutant deleterious to integrity of environment and human health (Megharaj et al., 2011). As a result of these occurrences, the environments need to be restored back to its normality. Many processes can be use including bioremediation which is more effective, efficient, eco-friendly and require less energy and time consumption (Vidali, 2010). Bioremediation is the use of living organism (Bacteria, fungi or algae) and their metabolites to bring back the polluted portion of the environment to their normal condition  by total removal or partial reduction (degrade, mineralize or transform) of the pollutant concentration to a non-injurious mode (Verma and Jaiswal, 2016). With the provision of suitable condition for growth and metabolism, the innate microbes are regarded first to solving the problem correlated with bioremediation of polluting sources (Frutos et al., 2012). Considering the site of remediation, the process may be classified as Ex situ and in situ (Azubuike et al., 2016). With regard to this research Ex situ were employed which is a bioremediation technique (applied to soil and water) that involves taking away the polluted sample elsewhere for investigation (Philp and Atlas, 2015). This process depend on many factors most of which include the type of the environment (soil, water or air), pollutant nature (heavy metals, dyes, organic/inorganic matter, hydrocarbons and others), cost and location (Megharaj et al., 2011).

Tanning can be defined as the method that transforms the proteinaceous material of unprocessed hide/skin into stable unputrescible leather product (Famielec and Wieczorek-Ciurowa, 2011). Tannery refers to a place where skins of animal (goat, sheep and others) are treated (Dargo and Ayalew, 2014). The treatments involve step by step addition or mixture of different matter (like trees and plants) or chemicals (e.g chromium salt, sodium sulphate etc) water, animal skin collagen and other tannin reagents (Krishanamoorthi et al., 2009). Treatment process use huge amount of water from the initial wash of hides or skins and/ an agent required necessary for the reaction to occur, and these are released directly or indirectly into the surrounding soil environment. The wastewater obtain as a result of treatment process are called tannery effluents (Rabah and Ibrahim, 2010). The effluents characteristically contain certain pollutants of both organic/ inorganic and metals which are toxic (Tripathi et al., 2011). Inorganic include sulphide, chloride, chromium, lead, zinc and others while protein wastes, phenols, formaldehyde are organic pollutants that may accumulate the nearby environments after effluents release (Hasegawa et al., 2011).

Large number of researchers gives more emphasis on chromium and chlorophenols because of their complexity and danger associated environment. Chromium is considered as toxic metal at elevated level that mostly occurs in the environment as the most common forms are,  Cr3+ (trivalent) and Cr6+ (hexavalent) species (Kotas and Stasicka, 2010). Chromium sulphate Cr (III) has been utilized as an agent of tanning, leading to polluting the soil environment as well as groundwater within or circling the territory and these are converted to Cr (VI) which has been indicated to affects health of living organisms including plants and animals (Tripathi et al., 2011). Chromium (III) confirmed to be essential to living organisms as it conserve nutrients metabolism energetically (Srivastava and Thakur, 2009). At alkali pH Cr (III) occur initially in the form of hydroxide precipitate and at neutral pH in the effluents, Cr (III) is been transformed into water-containing Cr (III) and oxidized to Cr (VI) (CrO42- and Cr2O7-2 form) at acidic pH (Sivakumar, 2016). Hexavalent chromium in the leather tanning industry (Anger et al., 2015) is also been used as interceptors of corrosion, Dyes and pigments production and conservation of wood (Shanker et al., 2015). Like human activity, hexavalent chromium in the tannery may carry on in the soils, reaching ground waters and are examined to be water soluble with higher transportability in soil ( Loyuax-Lawniczak et al., 2010). It has been observed that hexavalent chromium can be absorbed through inhalation and/ skin route of animals following exposure (USEPA, 2010) and these can cross cell membrane easily and can be distributed through the body to the sites of metal accumulation such like kidney, liver and bone (USEPA, 2010 ).

Chlorinated phenols like 3, 5- dichlorophenols are mostly used during the tanning process as agent to deter any harmful biological activity and can be biodegraded by microbes (Thakur et al., 2001, Yang, et al., 2006, Tripathi et al., 2011). Chlorophenols are highly toxic, carcinogenic and because of high chloride content with stable aromatic ring system, they are regarded as recalcitrant organic compound hence, are inhibitors of oxidative phosphorylation (Thakur et al., 2002; Srivastava et al., 2007; Tripathi et al., 2011). Other Heavy metals like cadmium (Cd), lead (Pb), zinc (Zn), Copper (Cu) and others are mostly known for their long term persistent nature, high toxicity, agent that induces cancer and mutation to the well being of living organisms and significantly poses great environmental problems (Kazemian and Mallah, 2008; Picardo et al., 2009). Some of the other pollutants within the tannery waste include Azodyes, Cobalt, Antimony, Barium, Selenium, Mercury, Arsenic, Nickel, Formaldehyde resins so also Pesticides residues (Mwinyihija, 2010).

Tanning in Nigeria has been practiced mostly in Northwestern region including Sokoto, especially within the metropolitan city. While the areas (of tannery practice) becomes tannery industrialized community one of which was named after the place, Majema.

 Sokoto tannery uses the oldest traditional vegetable tanning process of which natural resources like trees and some specific plants are being utilized. Many of the tanneries are small scale operations (though they are able to produce huge number of leather) and used open ground soil to release their waste (Tariq et al., 2009). These tanneries with their effluents are located in close vicinity of the residential housing areas that are in clusters otherwise called densely populated areas (Nolde, 2017). Operations of tannery in community with this pattern result to high environmental consequences and affects soil and human health (Tariq et al., 2009). As a result of conditions like time and energy, the process has been augments with advancements of chemicals used in the chrome tanning process like sodium sulphate, chromium salt and others (Lofrano et al., 2013).

The sector uses large number of synthetic chemicals while various steps of tannery operations, significant amount of portion of chemicals may not reach the targeted skin/hide site as such, may either fall on the ground soil or remain discarded via wastewater or effluents (Rabah and Ibrahim, 2010).  For the purpose of this research, tanneries are divided into two (2); traditional vegetable tannery and modern chrome tannery (Tariq et al., 2009).

Traditional vegetable are places that have been utilizing natural extracts of trees and plants for complete transformation of skin and hides of animals into leather. For absolute process, hierarchies of steps are involved to fully produce leather (Lofrano et al., 2013). Raw skin/hides are soaked at initial phase to extract away excess salts and other dense matter and to bring back the water content lost while curing (Tariq et al., 2009).

Modern chrome tannery are places that involves an anternate tanning process with significant fixation and binding, chemical stability of chromium-collagen compound arise from common mineral used and basic chromium sulfate (Lofrano et al., 2013). This method tanned leather more than vegetable tanning process with higher stability of temperature, very stable in water, require short time process of production and less energy used (Lofrano et al., 2013). Nowadays, Chrome tanning promote advancement in production being more rapid process and much preferred method because of its versatility and this has been used in developing areas.

Fungi are one group of microbes with large members encircling a range of vast form from microscopic unicellular yeast to enormous macroscopic multicellular fungi like mushroom and giant puff-ball (Bridge and Spooner, 2011). In other words, they are group of microorganism that can be found everywhere in the environment. With Their mycelia network, they are potential in controlling nutrients flow and energy. They contribute hugely in terrestrial world. The web of their mycelium can pass/spread deep inside the soil thus; fungi are truly regarded as the natural engineers of ecosystem (Singh, 2006). Fungi are organisms with true nucleus containing DNA fenced in nuclear membrane. They have cell walls that constitute a material named chitin. They digest (many fungi) nutrients before they absorb from their surrounding environment. Moldy fungi have typical mycelia with straight/branched filaments called hyphae that interwine (Tortora et al., 2010).the mycelia is the vegetative structure growing (from their tips) to form branching trees. Special thready, tubiform arrangements, that builds branched systems are mycelia. The network of hypha make one fungal mycelium. Most fungal hyphae are septate with cross wall and their pores via septa permitt transfer and relocation of nuclei, organelles and cytoplasm via mycelium to another component (singh, 2006)

Fungal mycelium in the cytoplasm release either macro or micro-vesicles for apical growth significantly. The morphologies of filamentous fungi in cultures present diverse distinct form. Mycelia that dissipate or become assemble in the medium are considered as clumps while compact that becomes massively spherical are pellets (singh, 2006)

Fungi may include yeasty, mouldy and filamentous type that can be use in the cleanup of pollutant on soil and water in the environments. Some of the organisms like yeast and mould are largely resistant to medium or surrounding with high content of alkali or acid and pH as well (Singh, 2006)

1.2: Statement of the Research Problem

Substances (chemicals) are being used in the tannery for effective process which results to complex mixture of organic matter and synthetic chemicals and their released to the environment could result to serious health problems. Physically the color of that soil environment changed; it appeared dark from one portion and the other light. The soil compacted to each other especially when dried. No living activity has been noticed (like growth of small plants, earthworm survival, and others) though there’s simultaneous use of water in the vicinity.

 1.3: Justification of the Study

Bioremediation was adopted for its chief-economic use, very rapid and easy to achieve effective result and very less negative consequence after use. Fungi were used because many researches have been done widely on the basis of bacterial remediation and fungi mycelium has the potential to enter deep into the soil contaminants thereby removing/reducing them to less toxic elements in the environments.

 1.4: Aim and Objectives of the Study

 1.4.1 Aim of the Study

The aim of the study is to utilize fungal slurry to remediate tannery waste polluted soil.

1.4.2: Objectives of the Study

The objectives of this study were:

1.      Isolate and characterize fungi from the soil using conventional and molecular techniques.

2.      Determine microbiological and physicochemical properties of tannery waste polluted soil.

3.      Screen fungi for potential to grow in tannery effluent.

4.      Remediate tannery waste polluted soil using selected slurry of fungal isolates.

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