POLLUTION CONTROL BIOTECHNOLOGY

FromWastewater Microbiology Book

by Gabriel Bitton.

• Application of genetic engineering and immobilization techniques could be used in waste treatment as well as the development and improvement of bioreactors for wastewater treatment.

• The use of commercial enzyme and microbial blends, immobilized microorganisms, and recombinant DNA technology for enhancing biological wastewater treatment is discussed.

• Also, we will explore the role of microorganisms in removal of metals in wastewater treatment plants.

INTRODUCTION

1. Use of Microbes in Pollution Control• Microbial strains are generally isolated from

environmental samples (wastewater, sludge, compost, soil).

• They are selected by conventional enrichment techniques.

• They are grown in nutrient media that contain specific organic chemical as the sole source of C and energy or source of N.

• Strains that can handle high concentrations of target chemical are selected.

• Some of the microbial strains may be subsequently irradiated to obtain a desirable mutation.

USE OF COMMERCIAAL BLENDS OF MO’s AND ENZYMES IN WWT

1. Use of Microbes in Pollution Control (lanjutan...)• Before using them, biodegradability of chemical

pollutant must be known: By searching literature By studying under laboratory conditions. By doing bioassay.

• Example: enrichment culture techniques have been used to isolate commercial strains of bacteria that degrade petroleum H-C. Mixtures of bacterial isolates belonging to genus

Pseudomonas are marketed for the in situ biorestoration of aquifer contaminated with aliphatic or aromatic H-C.

The selected strains are grown in large fermenters and then concentrated by centrifugation or filtration.

They are then preserved by lyophilization, drying, or freezing.

They must withstand the desired env conditions: T, pH, DO, nutrient, toxicity potential.

2. Use of Bioaugmentation in WWT• Bioaugmentation is the use of selected strains

of microbes isolated from the environment to improve some of the processes involved in traditional water treatment.

• Some applications of bioaugmentation are:a. Increased BOD Removal.b. Reduction of sludge volumec. Used of mixed cultures in sludge digestion.d. Biotreatment of hydrocarbon wastes.e. Biotretamnet of hazardous wastes.

3. Use of Enzymes in Waste Treatment• Enzymes play a key role in the hydrolysis and

biotransformation of organic in WWTP (e.g., phosphatases, aminopeptidases, esterases).

• Enzymes can be added to wastewaters to improve the treatability of xenobiotic compounds.

• Microbial exoenzymes are used to detoxify pesticides in soils: e.g. carbaryl is transformed to 1-naphtol.

• Parathion hydrolases (produced by Pseudomonas sp and Flavobacterium species) degrade parathion to diethylthiophosphoric acid and p-nitrophenol; these enzymes have been used to clean up parathion contaminated container, detoxification of wastes containing high concentrations of organophosphates, and in soil cleanup operation.

3. Use of Enzymes in Waste Treatment (lanjutan...)• Phage-induced depolymerases have been isolated

from a sludge sample and found to readily increase the degradation of exopolysaccharides during wastewater treatment (viscosity reduction).

• The addition of this enzyme could be used to improve sludge dewatering.

• Specific enzymes such as horse-radish peroxidase could catalyze polymerization and precipitation of aromatic compounds (e.g., substituted phenols and anilines) in drinking water and wastewater.

• Aggregated cells in activated sludge systems as well as cells attached to rock or plastic surfaces in trickling filters are good examples of exploitation of immobilized cells in waste treatment.

• Fluidized bed reactors as used in WWTP are other examples of cell immobilization on surfaces such as sand or other particles.

USE OF IMMOBILIZED CELLS IN WASTE TREATMENT

• The most popular approach to immobilization is entrapment of cells in polymeric materials such as alginate, carrageenan, or polyacrylamide.Carrageenan is an algal polysaccharide extracted from

algae of the class Rhodophyceae.Alginate is extracted from algae belonging to the class

Phaeophyceae. Alginate reacts with most divalent cations (Ca2+) to form

gels. The cells are mixed with a solution of sodium alginate and

the mixture is poured dropwise over a solution of CaCl2. Beads form instaneously and are left in the CaCl2 solution

for approximately 1 hr for complete gel formation.

Immobilizaation techniques

• Other techniques include the immobilization of activated sludge microorganisms in polyvinyl alcohol (PVA) dropped in saturated boric acid solution or refrigerated for gel formation.

• Cells can also be co-immobilized with magnetic particles in a polyacrylamide gel.

• Encapsulation of microorganisms within polymers generally produces relatively large beads (2-3mm), in which immobilized microorganisms may be subjected to oxygen limitations.

• These beads would not be suitable for certain environmental applications (e.g., groundwater restoration).

Immobilizaation techniques (lanjutan...)

• Brown lignin compounds found in paper mill effluents can be removed by immobilized white-rot fungus (Coriolus versicolor).

• Cyanide degradation by immobilized fungi producing an enzyme (cyanide hydratase) that transforms cyanide into formamide.

• CH4 production is actually done by immobilized methanogens.

• Dehalogenation of chloroaromatics (e.g., monochlorobenzoates, 2,4-dichlorophenoxy-acetic acid) by immobilized Pseudomonas spp.cells.

Some examples of the use of immobilized cells in waste treatment.

• Use of immobilized nitrifiers and denitrifiers to tackle nitrogen problems.

Nitrifying bacteria are immobilized in polyethylene glycol resin and added to activated sludge as suspended beads, enhance nitrification in activated sludge, thus reducing the time for complete nitrification.

Cells immobilized in polyvinyl alcohol could enhance the nitrification.• Immobilized activated sludge microorganisms; a two-step process,

a reactor containing immobilized activated sludge microorganisms and a biofilm reactor, achieved a high treatment efficiency.

• Use of immobilized algae to remove micronutrients from wastewater effluents. Phormodium immobilized on the surface of chitosan flakes could remove nitrogen and phosphorus from WW effluents.

Some examples of the use of immobilized cells in waste treatment (lanjutan...).

• Biodegradation of phenolic compounds.Clorinated phenols are degraded by bacteria (Pseudomonas,

Arthrobacter) immobilized in chitin surfaces by covalent bonding, by Alcaligenes spp.entrapped in polyacrylamide-hydrazide, or by Rhodococcus immobilized on a polyurethane carrier.

The biodegradation of 2-chlorophenol by activated sludge microorganisms immbolized in calcium alginate was demonstrated.

Pentachlorophenol can be degraded by Flavobacterium immoblized in calcium alginate.

Chlorophenols, methoxyphenols, and cresols can be removed from waste water by tyrosinase immobilized on magnitite.

Some examples of the use of immobilized cells in waste treatment (lanjutan...).

• Continuous reactor operation without risk of cell washout.• Ease of cell separation from reaction mixture.• High cell density.• Ability to reuse cells.• Ability of different microbial species spacially separated to

perform different functions.• Enhanced overall productivity.• Enhanced stability of immobilized mmicroorganism or

enzymes.• Decrease in volume of bioreactors.• Tolerance by immobilized microrganisms of higher levels of

toxicants.

Advantages of Immobilized cells

• Diffusion problems due to high cell density and low solubility of oxygen in water.

• Changes in cell physiology that may affect productivity.

• Changes in composition of the microbial population, which can be a problem in wastewater that harbors mixed populations of microorganisms.

• Cost of immobilization.

Disadvantages of immobilized cells

1. Removal of metal by wastewater mo’s.• Several types of bacteria (e.g., Zooglea ramigera, Bacillus

lincheniformis) from activated sludge, produce extracellular polymers that are able to complex and accumulate metals such as iron, copper, cadmium, nickel and uranium.

• A proprietary product, called Algasorb, consits of algae cells embedded in a silican gel polymer material, and can remove heavy metals including uranium.

• Fungal mycelia (e..g, Aspergillus and Penicilium) could remove metals from waste water.

• Immobilized Aspergillus oryzae remove Cd efficeintly from solution.

ROLE OF MO’s IN METAL REMOVAL

Table: Some mo’s involved in metal removal

Microorganisms Metals removed

Zooglea ramigeraSaccharomyces cerevisieaeRhizopus arrchizusChlorella vulgarisAspergillus orhizaeAspergillus nigerPecicillium spinulosumTrichoderma virideAMT-Bioclaim™

CopperUranium and other metalsUraniumGold, zinc, copper, mercuryCadmiumCopper, cadmium, zincCopper, cadmium, zincCopperBiotechnology-based use of granulated product derived from biomass

Adsorption to cell surfaces• Microorganisms bind metals as a result of interactions between

metal ions and the negatively charge microbe surfaces.• Gram positive bacteria, fungal and algal cells.Complexation• Microorganisms can produce organic acids (e..g., citric acid),

which may chelate toxic metals, resulting in the formation of metallorganic molecules.

• Metals may also be complexed by carboxyl groups found in microbial polysaccharides and other polymers.

• Pseudomonas putida has a cystein-rich protein that binds cadmium.

Mechanisms of metal removal by mo’s

Precipitation• Some bacteria ppromote metal precipitation by producing ammonia, organic

bases, or hydrogen sulfide, which precipitate metals as hydroxides or sulfides.• Sulfate reducing bacteria transform SO4 to H2S, which promotes the

extracellular precipitation of metals from solution.• Klebsiella aerogenes is able to detoxify cadmium to a cadmium sulfide (CdS),

which precipitate as electron-dense granules at the cell surface.

Volatilization• Bacterially mediated methylation converts Hg2+ to dimethyl mercury (volatile

compund).• Some bacteria detoxify mercury by transforming Hg2+ into Hg0 (a volatile

species).• Mercuric reductase enzyme cataalyzes the transformation Hg2+ to Hg0 .

Intracellular accumulation of metals• Microbial cells can accumulate metals, which gain entry into the cell by specific

transport ssystem..

Mechanisms of metal removal by mo’s

• Major applications of recombinant DNA technology:enhancement of biodegradation of xenobiotics,

andthe use of nucleic acid probes to detect

pathogens and parasites in WWTP and other env. samples.

POTENTIAL APPLICATION OF RECOMBINANT DNA TECHNOLOGY

1. Nucleic acid probes (NAP)• A gene probe is made of a piece of DNA controlling a

desirable function in a cell (e.g., biodegradation of a given xenobiotic), and labeled with a radioactive element, such as 32P or with an enzyme (e.g, ß-galactosidase, alkali phosphatase).

• The probe can hybridize with a complementary strand of target DNA isolated from a given environmental sample or a bacterial colony.

• NAP can be used to detect mo’s that are capable of degrading specific chemicals in WW samples.

• Some of NAPs that can be combined with polymerase chain reaction (PCR) technology are available for detecting bacterial, viral and protozoan pathogens and parasites, and for tracking genetically engineered mo’s.

Some Tools in Recombinant DNA Technology

2. Polymerase chain reaction (PCR)• This technique simulates in vitro the DNA

replication process that occurs in vivo.• It consists of amplifying discreet fragment of DNA

by generating millions of copies of target DNA.• During cell division, 2 new copies of DNA are made

and 1 set of genes is passed on to each daughter cell.

• Copies of genes increase exponentially as the number of generations increases.

• PCR simulates in vitro the DNA duplication process and can create millions of copies of the target DNA sequence.

Some Tools in Recombinant DNA Technology

1. Detection of specific bacteria in environmental samples.

2. Environmental monitoring of geneticalyy engineered microbes (GEMs), e.g., GEMs that perform certain useful function (pesticide degradation) can be traced with PCR.

3. Detection of indicator and pathogenic microorganisms.

Environmental application of PCR

Successful use of recombinant DNA technology in waste treatment are:• Enhance denitrification• Increase growth rates of nitrifying bacteria, so speeding

up nitrification and reducing the sludge age• Improve bacterial flocculation in activated sludge• Improve performance of biofilm processes• Enhance biological removal of phosphorus• Improve performance of methanogens• Better control of activated sludge bulking.

Potential Application of Recombinant DNA Technology to Waste treatment

• The existence of multistep pathways in the degradation of xenobitics raises the possibility thaat an engineered mo may not be able to completely mineralize the target xenobiotic.

• The engineered mo maay be capable of degrading only 1 or 2 compounds.

• Knowledge about the degraadative pathways of interest is limited.

• The recombinant strain of interest may be unstable in the naatural environment.

• Public concern about the accidental or deliberate release of GEMs into the environment.

Some Limitation of Recombinant DNA Technology to Waste treatment