Environmental Microbial Biotechnology: Bioremediation PDF

ENVIRONMENTAL MICROBIAL BIOTECHNOLOGY (MICR 307) BIOREMEDIATION PROF. A.O. OLANIRAN F3 03-028 Discipline of Microbiology University of KwaZulu-Natal Westville Campus INTRODUCTION ◼ The quality of life on Earth is linked to the overall...

ENVIRONMENTAL MICROBIAL BIOTECHNOLOGY (MICR 307) BIOREMEDIATION PROF. A.O. OLANIRAN F3 03-028 Discipline of Microbiology University of KwaZulu-Natal Westville Campus INTRODUCTION ◼ The quality of life on Earth is linked to the overall quality of the environment. ◼ Carelessness and negligence in the use of land. ◼ Increase in the problems associated with contaminated sites in many countries results from; Industrial activities Use of pesticides, insecticides, etc. in agriculture Solvents in dry-cleaning Of human origin From activities of living organisms, e.g. fermentation ◼ Conventional techniques for pollutants’ removal Landfill Cap and contain ◼ Better approaches: to completely destroy the pollutants or transform them into innocuous products include; High temperature incineration Chemical decomposition (e.g., base-catalyzed dechlorination, UV oxidation). BIOREMEDIATION ◼ Use of natural biological activity to destroy or render harmless various contaminants ◼ Bioremediation is defined as the process whereby organic wastes are biologically degraded under controlled conditions to an innocuous state, or to levels below concentration limits established by regulatory authorities. ◼ By definition, bioremediation is the use of living organisms, primarily microorganisms, to degrade the environmental contaminants into less toxic forms.  Exxon Valdez Oil Spillage ◼ On March 24, 1989, the tanker Exxon Valdez deviated from the shipping lane in Prince William Sound (PWS), Alaska to avoid icebergs and grounded on Bligh Reef. ◼ This resulted in the release of 37,000 tons (10.9 million gallons) of Alaska North Slope (ANS) crude oil. ◼ This was about 20% of the 180,000 tons of crude oil the vessel was carrying when it struck the reef. ◼ The salvage effort that took place immediately after the grounding saved the vessel from sinking, thus preventing a far larger oil spill from happening. THE SPILLAGE Source: http://en.wikipedia.org/wiki/Exxon_Valdez The salvage operation prevented the vessel from sinking and saved 80% of the cargo, thus preventing a far larger oil spill. Source: http://www.valdezscience.com  Environmental impact Wildlife was severely affected by the oil spill DISASTER TO AQUATIC LIFE ◼ Thousands of animals died immediately; the best estimates include; 250,000–500,000 seabirds 2,800–5,000 sea otters approximately 12 river otters 300 harbour seals 250 bald eagles 22 orcas Billions of salmon and herring eggs www.openlearn.open.ac.uk ◼ Due to a thorough cleanup, little visual evidence of the event remained in areas frequented by humans just one year later, but the effects of the spill continue to be felt today. ◼ In the long term, reductions in population have been seen in various ocean animals, including stunted growth in pink salmon populations. ◼ Sea otters and ducks also showed higher death rates in following years, partly because they ingested contaminated creatures. ◼ Many animals were also exposed to oil when they dug up their prey in dirty soil. Pink salmon runs (millions of fish) in Prince William Sound for the period 1970-1999 (Data source: Alaska Department of Fish and Game, Division of Commercial Fisheries annual fin fish management reports for the PWS area.). Ranking of major oil spills by amount of oil spilled The dramatic recovery of a heavily oiled shoreline from 1989-1992. The 1989 picture shows pools of oil on an exposed boulder beach. The 1992 picture of the same beach shows no oil. Many exposed north facing shores on the islands in PWS were in the direct path of the spill and were heavily oiled in 1989. Through a combination of natural and human processes, most of the oil from the spill was gone from PWS by 1992.  Recent Oil Spill ◼ Tanker truck rollover - United States, Santa Maria, Cuyama River - 21 March 2020 - 19.5 tons ◼ Willowton Oil – August 2019 – near PMB & Durban, SA ◼ Keystone Pipeline 2019 spill - 29 October 2019 - 1,240 tons ◼ United States, Walsh County, North Dakota ◼ Refugio oil spill on May 19, 2015 at Santa Barbara Country, California ◼ Deposited 142,800 U.S. gallons (3,400 barrels) of crude oil onto one of the most biologically diverse coastlines of the west coast ◼ The Rayong oil spill occurred on July 27, 2013, in the Gulf of Thailand, 50,000 L (310 bbl) of oil spewing into the coastal waters Principles of Bioremediation ◼ Microorganisms play key roles ◼ Biodegradation of a compound is often a result of the actions of multiple organisms. ◼ Environmental Impact Assessment – needed to determine best bioremediation option ◼ Bioattenuation – process in which biodegradation occurs naturally without human intervention ◼ Biostimulation – adjustment of sites in order to provide bacterial communities with a favourable environment to effectively degrade contaminants. ◼ Bioaugmentation – importing of MOS to a contaminated site to enhance degradation. ◼ For bioremediation to be effective, microorganisms must enzymatically attack the pollutants and convert them to harmless products. ◼ Some contaminants, such as chlorinated organic or high aromatic hydrocarbons, are resistant to microbial attack. ◼ It is not easy to predict the rates of clean-up for a bioremediation exercise; there are no rules to predict if a contaminant can be degraded. ◼ Table 1 shows a list of contaminants potentially suitable for bioremediation. FACTORS OF BIOREMEDIATION FACTORS OF BIOREMEDIATION ◼ The control and optimization of bioremediation processes is a complex system of many factors. ◼ These factors include: the existence of a microbial population capable of degrading the pollutants; the availability of contaminants to the microbial population; the environmental factors (type of soil, pH, temperature, the presence of oxygen or other electron acceptors, and nutrients). MICROBIAL POPULATIONS FOR BIOREMEDIATION PROCESSES ◼ Microorganisms can be isolated from almost any environmental conditions. ◼ Microbes will adapt and grow at subzero temperatures, as well as extreme heat, desert conditions, in water, with an excess of oxygen, and in anaerobic conditions, with the presence of hazardous compounds or on any waste stream. ◼ The main requirements are an energy source and a carbon source. ◼ Because of the adaptability of microbes and other biological systems, these can be used to degrade or remediate environmental hazards. ◼ These microorganisms can be sub-divided into the following groups: Aerobic: ◼ Examples of aerobic bacteria recognized for their degradative abilities are Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and Mycobacterium. ◼ These microbes have often been reported to degrade pesticides and hydrocarbons, both alkanes and polyaromatic compounds. ◼ Many of these bacteria use the contaminant as the sole source of carbon and energy. Anaerobic: ◼ Anaerobic bacteria are not as frequently used as aerobic bacteria. ◼ There is an increasing interest in anaerobic bacteria used for bioremediation of polychlorinated biphenyls (PCBs) in river sediments, dechlorination of the solvent; tetrachloroethene (PCE), trichloroethylene (TCE), and chloroform (CHCl3). Ligninolytic fungi: ◼ Fungi such as the white rot fungus, Phanaerochaete chrysosporium have the ability to degrade an extremely diverse range of persistent or toxic environmental pollutants. ◼ Common substrates used include straw, saw dust, or corn cobs. ◼ They produce enzymes like peroxidases (Lignin and Manganese peroxidase) and lacasses which have wide substrate range. Methylotrophs: ◼ Aerobic bacteria that grow utilizing methane for carbon and energy. ◼ The initial enzyme in the pathway for aerobic degradation is methane monooxygenase. ◼ It has a broad substrate range and is active against a wide range of compounds, including the chlorinated aliphatics; trichloroethylene and 1,2-dichloroethane. AVAILABILITY OF CONTAMINANTS ◼ For degradation, it is necessary that bacteria and the contaminants be in contact. ◼ This is not easily achieved, as neither the microbes nor contaminants are uniformly spread in the soil. ◼ Some bacteria are mobile and exhibit a chemotactic response, sensing the contaminant and moving toward it. ◼ Other microbes such as fungi grow in a filamentous form toward the contaminant. ◼ It is possible to enhance the mobilization of the contaminant using some surfactants such as sodium dodecyl sulphate (SDS), while some organisms produce biosurfactants.  Nutrients ◼ MOS in contaminated soil may not necessarily be there in the numbers required for bioremediation of the site. Their growth and activity must be stimulated. ◼ Biostimulation usually involves the addition of nutrients and oxygen to help indigenous microorganisms. ◼ These nutrients are the basic building blocks of life and allow microbes to create the necessary enzymes to break down the contaminants. ◼ All of them will need nitrogen, phosphorous, and carbon ◼ Carbon is the most basic element of living forms and is needed in greater quantities than other elements. ◼ In addition to hydrogen, oxygen, and nitrogen, it constitutes about 95% of the weight of cells. ◼ Phosphorous and sulfur contribute with 70% of the remainders. ◼ The nutritional requirement of carbon to nitrogen ratio is 10:1, and carbon to phosphorous is 30:1. Environmental requirements ◼ Optimum environmental conditions for the degradation of contaminants are shown in Table 3. ◼ Microbial growth and activity are readily affected by pH, temperature, and moisture. ◼ It is important to achieve optimal conditions. ◼ Temperature affects biochemical reactions rates, and the rates of many of them double for each 10 °C rise in temperature. ◼ Available water is essential for all the living organisms. ◼ The amount of available oxygen will determine whether the system is aerobic or anaerobic. ◼ To increase the oxygen amount in the soil it is possible to till or sparge air. ◼ In some cases, hydrogen peroxide or magnesium peroxide (oxygen releasing compounds) can be introduced. ◼ Soil structure controls the effective delivery of air, water, and nutrients. ◼ Materials such as gypsum or organic matter can be applied for improvement. ◼ Low soil permeability can impede movement of water, nutrients, and oxygen. ◼ Hence, soils with low permeability may not be appropriate for in situ clean-up techniques. BIOREMEDIATION STRATEGIES Different techniques are employed depending on the degree of saturation and aeration of an area. In situ techniques are defined as those that are applied to soil and groundwater at the site with minimal disturbance. Ex situ techniques are those that are applied to soil and groundwater at the site which has been removed from the site via excavation (soil) or pumping (water).  Classification of bioremediation processes Bioaugmentation In situ biodegradation Adapted from Oyetibo et al. (2017) In situ bioremediation ◼ The most desirable options due to lower cost and less disturbance. ◼ In situ treatment is limited by the depth of the soil that can be effectively treated. ◼ In many soils, effective oxygen diffusion for desirable rates of bioremediation extend to a range of only a few centimeters to about 30 cm into the soil, although depths of 60 cm and greater have been effectively treated in some cases. Bioventing the most common in situ treatment involves supplying air and nutrients through wells to contaminated soil to stimulate the indigenous bacteria. ◼ Bioventing employs low air flow rates and provides only the amount of oxygen necessary for the biodegradation while minimizing volatilization and release of contaminants to the atmosphere. ◼ It works for simple hydrocarbons and can be used where the contamination is deep under the surface. In situ biodegradation ◼ involves supplying oxygen and nutrients by circulating aqueous solutions through contaminated soils to stimulate naturally occurring bacteria to degrade organic contaminants. ◼ It can be used for soil and groundwater. ◼ Generally, this technique includes conditions such as the infiltration of water-containing nutrients and oxygen or other electron acceptors for groundwater treatment.  Biosparging: ◼ It involves the injection of air under pressure below the water table to; increase groundwater oxygen concentrations and enhance the rate of biological degradation of contaminants by naturally occurring bacteria. ◼ Biosparging increases the mixing in the saturated zone and thereby increases the contact between soil and groundwater. ◼ The ease and low cost of installing small-diameter air injection points allows considerable flexibility in the design and construction of the system.  Bioslurping ◼ Commonly used for treating soil and groundwater contaminated with volatile and semi-volatile organic compounds. ◼ Combines the efficiency of vacuum-enhanced pumping, soil vapor extraction together with bioventing.  Bioaugmentation: ◼ Involves the addition of microorganisms indigenous or exogenous to the contaminated sites. ◼ Two factors limit the use of added microbial cultures in a land treatment unit: non-indigenous cultures rarely compete well enough with an indigenous population to develop and sustain useful population levels and most soils with long-term exposure to biodegradable waste have indigenous microorganisms that are effective degrades if the land treatment unit is well managed. Ex situ bioremediation These techniques involve the excavation or removal of contaminated soil from ground.  Landfarming ◼ contaminated soil is excavated and spread over a prepared bed and periodically tilled until pollutants are degraded. ◼ The goal is to stimulate indigenous biodegradative microorganisms and facilitate their aerobic degradation of contaminants. ◼ In general, the practice is limited to the treatment of superficial 10–35 cm of soil. ◼ Landfarming has received much attention as a disposal alternative owing to reduced monitoring and maintenance costs, as well as clean-up liabilities. Composting ◼ involves combining contaminated soil with non-hazardous organic amendants such as manure or agricultural wastes. ◼ The presence of these organic materials supports the development of a rich microbial population and elevated temperature characteristic of composting.  Biopiles ◼ A hybrid of landfarming and composting. ◼ Essentially, engineered cells are constructed as aerated composted piles. ◼ Typically used for treatment of surface contamination with petroleum hydrocarbons. ◼ They are a refined version of landfarming that tend to control physical losses of the contaminants by leaching and volatilization. ◼ Biopiles provide a favorable environment for indigenous aerobic and anaerobic microorganisms.  Bioreactors: ◼ Bioremediation in reactors involves the processing of contaminated solid material (soil, sediment, sludge) or water through an engineered containment system. ◼ A slurry bioreactor may be defined as a containment vessel and apparatus used to create a three-phase (solid, liquid, and gas) mixing condition to increase the bioremediation rate of soil-bound and water-soluble pollutants as a water slurry of the contaminated soil and biomass (usually indigenous microorganisms) capable of degrading target contaminants. ◼ In general, the rate and extent of biodegradation are greater in a bioreactor system than in situ or in solid- phase systems because the contained environment is more manageable and hence more controllable and predictable. ◼ However, the contaminated soil requires pre treatment (e.g., excavation) or alternatively the contaminant can be stripped from the soil via soil washing or physical extraction (e.g., vacuum extraction) before being placed in a bioreactor. Example of a slurry bioreactor  Advantages of bioremediation ◼ Public acceptance. ◼ Theoretically, bioremediation is useful for the complete destruction of a wide variety of contaminants. Many compounds that are legally considered to be hazardous can be transformed to harmless products. ◼ On-site application. ◼ It can prove less expensive than other technologies that are used for clean-up of hazardous waste. Disadvantages of bioremediation ◼ Limited to those compounds that are biodegradable. ◼ There are some concerns that the products of biodegradation may be more persistent or toxic than the parent compound. ◼ Biological processes are often highly specific and require certain important factors (nutritional and environmental). ◼ It is difficult to extrapolate from bench and pilot- scale studies to full-scale field operations. ◼ Sites with complex mixtures of contaminants that are not evenly dispersed in the environment are often problematic. ◼ Bioremediation often takes longer than other treatment options, such as excavation and removal of soil or incineration. ◼ Regulatory uncertainty remains regarding acceptable performance criteria for bioremediation. PHYTOREMEDIATION PHYTOREMEDIATION ◼ Phytoremediation involves depolluting contaminated soils, water or air with plants able to contain, degrade or eliminate metals, pesticides, solvents, explosives, crude oil and its derivatives, and various other contaminants, from the mediums that contain them. ◼ It is clean, efficient, inexpensive and non- environmentally disruptive, as opposed to processes that require excavation of soil. Phytoremediation Techniques Phytoextraction or phytoaccumulation ◼ Process used by the plants to accumulate contaminants into the roots and aboveground shoots or leaves (biomass). ◼ Saves tremendous remediation cost. ◼ Unlike the degradation mechanisms, this process produces a mass of plants and contaminants (usually metals) that can be transported for disposal or recycling. Two versions of phytoextraction: ◼ Natural hyper-accumulation, where plants naturally take up the contaminants in soil unassisted, and ◼ Induced or assisted hyper-accumulation, in which a conditioning fluid containing a chelator or another agent is added to soil to increase metal solubility or mobilization so that the plants can absorb them more easily. ◼ The three hallmarks that differentiate hyperaccumulators from related non-hyperaccumulator taxa are: ◼ a much greater capability to tolerate and take up heavy metals from the soil; ◼ a faster and effective root-to-shoot translocation of metals; and ◼ a much greater ability to detoxify and sequester huge amounts of heavy metals in the leaves Examples of phytoextraction from soils ◼ Arsenic, using the Sunflower (Helianthus annuus), or the Chinese Brake fern ("Pteris spp"], a hyperaccumulator. ◼ Chinese Brake fern stores arsenic ◼ in its leaves. ◼ Cadmium and zinc, using alpine pennycress (Thlaspi caerulescens), a hyperaccumulator of these metals at levels that would be toxic to many plants. ◼ Lead, using the following plants which sequester lead in the biomass. Indian Mustard (Brassica juncea) Ragweed (Ambrosia artemisiifolia) Hemp Dogbane (Apocynum cannabinum) Poplar trees ◼ Salt-tolerant (moderately halophytic) barley and/or sugar beets are commonly used for the extraction of Sodium chloride (common salt) to reclaim fields that were previously flooded by sea water. barley Sugar beets ◼ Uranium, using sunflowers, as used after the Chernobyl accident (a nuclear reactor accident in the Chernobyl Nuclear Power Plant in the Soviet Union). Phytotransformation ◼ Uptake of organic contaminants from soil, sediments, or water and, their subsequent transformation to more stable, less toxic, or less mobile form. ◼ Chromium can be reduced from hexavalent (Cr6+) to trivalent chromium (Cr3+), which is a less mobile and non-carcinogenic form. ◼ Certain plants, such as Cannas, can render organic pollutants, such as pesticides, explosives, solvents, industrial chemicals, and other xenobiotic substances, non-toxic by their metabolism. Phytostabilization ◼ A technique in which plants reduce the mobility and migration of contaminated soil. E.g. metal precipitation ◼ Leachable constituents are adsorbed and bound into the plant structure ◼ They then form a stable mass of plant from which the contaminants will not re-enter the environment. Phytodegradation or rhizodegradation ◼ The breakdown of contaminants through the activity existing in the rhizosphere. ◼ This activity is due to the presence of proteins and enzymes produced by the plants or by soil organisms such as bacteria, yeast, and fungi. ◼ Rhizodegradation is a symbiotic relationship that has evolved between plants and microbes. ◼ Plants provide nutrients necessary for the microbes to thrive, while microbes provide a healthier soil environment. Rhizofiltration ◼ Filtering water through a mass of roots to remove toxic substances or excess nutrients. ◼ A water remediation technique that involves the uptake of contaminants by plant roots. ◼ The pollutants remain absorbed in or adsorbed to the roots. ◼ Used to reduce contamination in natural wetlands and estuary areas. ◼ An overview of phytoremediation applications is shown in Table 5. Phytoremediation is well suited for use: at very large field sites where other methods of remediation are not practicable or cost effective. at sites with a low concentration of contaminants where only polish treatment is required over long periods of time. in conjunction with other technologies where vegetation is used as a final cap and closure of the site. Advantages ◼ The cost of the phytoremediation is lower than that of traditional processes both in situ and ex situ ◼ Easy monitoring of the plants ◼ It is the least harmful method because it uses naturally occurring organisms and preserves the natural state of the environment. ◼ The possibility of the recovery and re-use of valuable metals (by companies specializing in “phytomining”)  Phytomining ◼ Phytomining describes the exploitation of sub- economic ore bodies using plants. ◼ Phytominers grow a crop of a metal-hyper- accumulating plant species ◼ Harvest the biomass and burn it to produce a bio-ore. ◼ The final bio-ore is smelted to yield the metal. ◼ Smelting – a process of applying heat to an ore (usually beyond its melting temperature, to extract a base metal ◼ The first phytomining experiments were carried out using the nickel (Ni) hyperaccumulator, Streptanthus polygaloides ◼ It was found that a yield of 100 kg/ha of sulphur-free Ni could be produced. ◼ The Ni-hyperaccumulators Alyssum bertolonii from Italy and Berkheya coddii from South Africa have even greater potential to extract Ni. ◼ Soil conditioners, particularly N and P amendments, greatly enhance Ni phytomining.  Phytomining has the following unique features: ◼ It offers the possibility of exploiting ores or mineralised soils that are uneconomic by conventional mining methods. ◼ ‘Bio-ores’ are virtually sulphur-free, and their smelting requires less energy than sulphidic ores. ◼ The metal content of a bio-ore is usually much greater than that of a conventional ore and therefore requires less storage space despite the lower density of a bio- ore. ◼ Phytomining is a ‘green’ technology that should appeal to the conservation movement as an alternative to opencast mining of low-grade ores. Limitations: ◼ Phytoremediation is limited to the surface area and depth occupied by the roots. ◼ Slow growth and low biomass require a long- term commitment ◼ With plant-based systems of remediation, it is not possible to completely prevent the leaching of contaminants into the groundwater ◼ The survival of the plants is affected by the toxicity of the contaminated land and the general condition of the soil. ◼ Difficulty establishing and maintaining vegetation at some sites with high toxic levels. ◼ Potential contamination of the vegetation and food chain. ◼ Possible bio-accumulation of contaminants which then pass into the food chain, from primary level consumers upwards.