Lectures 6 and 7- Environmental Biotech- Bioremediation 2.pdf

Lectures 6 and 7 Environmental Biotechnology BIOC 3260 Principles of Biotechnology Dr. Angela T. Alleyne 1  Define...

Lectures 6 and 7 Environmental Biotechnology BIOC 3260 Principles of Biotechnology Dr. Angela T. Alleyne 1  Define environmental biotechnology use of metagenomics in Explain biotechnology LEARNING OUTCOMES Describe use of SIP biotechnology Antimicrobial resistance Explain mechanisms- Integrons At the end of this lecture you Discuss applications of bioremediation will be able to: phytoremediation, bioremediation, Differentiate among and biostimulation bioethics and environmental Discuss biotechnology 2  Environmental Biotechnology Environmental biotechnology is the direct study of biotechnology in the environment or the applications of biotechnologies that focus on improving the environment. It includes among others: ̶ Bioremediation- use of naturally occurring biological organisms to break down hazardous substances into less toxic or non-toxic forms. ̶ Phytoremediation- use of plants and plant products for decontaminating or stabilizing contaminants and metals from soil. ̶ Biosensors ̶ Biofuels ̶ Biomarkers ̶ Biological surveys Craig Venter: Sampling the ocean's DNA | TED Talk 3  ─ Species identification ─ investigation of their metabolic capabilities, and ─ understanding of potential microbial community shifts in response to changing environmental factors Microbial communities 4  ─ Cultivation-based techniques have been shown to target only about 1% of microbes occurring in the environment. Microbial communities 5  Metagenomics Metagenomics is the study of the genomes of whole communities of microscopic life forms. ̶ Metagenomics has been used to identify new and beneficial genes from the environment, e.g., novel antibiotics, enzymes that biodegrade pollutants, and novel enzymes. ̶ Metagenomic research sometimes allows us to identify microorganisms, viruses, or free DNA that exist in the natural environment by identifying genes or DNA sequences from the organisms. 6  Metagenomics ̶ Microbes play an essential role in degradation, mineralization, or transformation of environmental pollutants and are potentially capable of restoring contaminated environmental sites. ̶ Methods include: ̶ shotgun DNA sequencing, PCR, RT-PCR, and others. 7 8 Associated Gene Environmental Sample Esterases Deep sea sample from the Mediterranean Lipases Soil from Madison Wisconsin, Agricultural Research Station, Amylases Estuarine water samples from the Delaware Bay , USA Methanogens Soil sample Pitch Lake la-Brea, Trinidad and Tobago Turbomycin Soil from Madison Wisconsin, Agricultural Research Station, Isolation of Antibiotics Turbomycin A and B from a Metagenomic Library of Soil Microbial DNA (nih.gov) Meckenstock_RU-2014- Examples of Water_droplets_in_oil_are_microhabitats_for_microbial_life.pdf (helmholtz- metagenomic gene muenchen.de) Recent application of metagenomic approaches toward the discovery of mining studies antimicrobials and other bioactive small molecules - PubMed (nih.gov)  Haemolysin is a bacterial toxin that punctures holes in susceptible cell membranes, allowing the cellular contents to leak out and the cell then dies. Haemolysin lyses red blood cells and creates a clear zone around a bacterial colony growing on blood agar plates. Antibiotic discovery: Turbomycin Some E. coli clones (P57G4, P89C8, and P214D2,) Metagenomic approach to from an E. coli library of soil had dark red or the discovery of a new orange colours. antibiotic from soil. Further investigation of these clones found two novel antibiotics, turbomycin A and B 10.1128/AEM.68.9.4301-4306.2002 10  24,546 member library (SL2) of DNA from soil cloned into a BAC vector. Both exhibited broad-spectrum antibiotic activity against gram- negative and gram-positive organisms. https://www.ncbi.nlm.nih.gov/pmc/ar icles/PMC124076/ Screening for hemolytic activities initially identified one clone, designated P57G4, that produced a zone of clearing on blood agar and also produced dark brown and orange 11 colors when grown in LB medium. Taken from: Biotechnology by Clark  Antibiotic discovery Expression of DNA extracted from soil can reveal new and novel small organic compounds with novel characteristics and antibiotic activity. Traditional culture methods from soil represent only 1% or less of the total microbial community 12 Metabolomics: Stable Isotope Probing (SIP) doi: 10.4155/bio.13.348 Stable Isotope Probing (SIP) Theory of SIP ̶ RNA-SIP focuses on isolating RNA(16S RNA) from an environmental sample. ̶ When the isotope is incorporated into the genome of an organism, it is heavier and the cDNA can be easily separated from other DNA 15  SIP uses ̶ RNA-SIP can be used to identify a variety of microorganisms in environmental samples. example, water from an aerobic industrial wastewater plant was evaluated for phenol degrading microorganisms. SIP used as a tool for the identification of active members of the microbial community and associated genes important to community processes such as biodegradation 16  SIP metagenomics Functional Sequence based screening screening Metagenomic libraries are used to identify the entire genetic complement of newly discovered life forms, without culturing them. 17 metagenomic libraries screening Functional analysis ̶ Functional approaches include expression screening with various genetic traps and phage bio panning. ̶ Expression screening depends on the choice of an expression vector. Proteins are expressed from the metagenomic DNA fragments when they are cloned into a vector with transcriptional and translational start and stop sequences. ̶ Then an assay for the target Taken from: Biotechnology by Clark function is devised 18  Stable Isotope Labeling by Amino Acids in Cell culture Traditional SILAC compares differences in protein quantities between two states. Pulse-chase SILAC examines changes in the proteome, which can then be compared across different biological states or test conditions. : 10.1039/d2mo00077f 19  SILAC- proteomics a metabolic labeling technique for proteins. Incorporates isotopically labeled amino acids into the media used for cell culture, unlabeled versus labeled cells can be differentiated by mass spectrometry ( MS). proteins can be analyzed intact by top-down analysis, or the proteins can be digested into peptides and analyzed by bottom-up proteomic analysis 20 21  integrons are mobile genetic elements associated with antibiotic resistance Integrons a large family of genetic elements, which can capture gene cassettes. 22  Integron analysis Integron analysis identifies open reading frames that are used by integrons and can identify novel unknown genes. Integrons are gene acquisition systems commonly found in bacterial genomes associated with resistance to e.g β- lactams, all aminoglycosides, chloramphenicol, trimethoprim, erythromycin, quinolones, and rifampin etc. Taken from: Biotechnology by Clark 23  Integrons structure ─ a ( att1 ) site to integrate a segment of DNA known as a gene cassette , ─ a promoter to express the gene cassette (Pc ), ─ and a gene for integrase ( int1 ), the enzyme that recombines the gene cassette into the integron. Adapted from: http://www.cell.com/cms/attachment/2021782031/2041686783/gr2_lrg.jpg 24  Gene cassette Many cassettes encode antibiotic Segments of DNA with resistance genes one or two open reading frames (ORFs) that lack promoters and are flanked by 59-base pair elements (59-be , also known as attC sites). Integrase recognizes the 59-be sites and excises the gene cassette and integrates it downstream Taken from: Biotechnology by Clark of the Pc promoter. 25  Gene cassette Many casettes encode antibiotic resistance genes This allows the open reading frame to be expressed into protein. The 59-be sequence may vary in length, but must contain a conserved, seven- nucleotide sequence. Taken from: Biotechnology by Clark 26  Bioremediation Many different man-made compounds have contaminated the environment through everyday use, accidental spillage, or intentional dumping. Many environmental biotechnologists work on “biological” means of cleaning the environment. Releasing an organism that can degrade a pollutant would provide a very easy, low-cost way of cleaning up a polluted site. 27 Bioremediation and Biotechnology microbes can degrade toxic xenobiotic compounds which were earlier believed to be resistant to the natural biological processes occurring in the soil. Microbes are genetically engineered to maximize their metabolic ability to remove contaminants from soil. it is either Removal of a material degraded from an environment takes one of two routes: immobilised into a biologically inactive compound 28 Pollutant removal metabolically versatile actinobacteria. frequently found in the environment potential for applications in bioremediation, biotransformation, and biocatalysis. Pollutant removal Rhodococci can: degrade pollutants, such as short and long-chain alkanes, aromatic molecules, and heterocyclic, and polycyclic aromatic compounds e.g., quinolone, pyridine, thiocarbamate, s -triazine herbicides, 2- mercaptobenzothiazole (a rubber vulcanization accelerator), benzothiophene, dibenzothiphene, methyl tertbutyl ether (MTBE), and ethyl tert -butyl ether (ETBE).  Rhodococcus species are found in all types of environments, including nuclear waste sediments, tropical soils, Arctic soils, and sites in Europe, Japan, and the United States. Cold-tolerant Rhodococcus strains were isolated from oil-contaminated soils in Oil Antarctica; they grew on a range of alkanes from hexane (C6) through at least eicosane bioremediation (C20). The genome of Rhodococcus sp. strain RHA1 has 9.7 Mb of DNA, including one chromosome and three large linear plasmids. The plasmids are critical because they are important for gene transfer and recombination events. 31  Some Rhodococcus strains are very efficient oil degraders and act by adhering to oil droplets. Several strains of Rhodococcus can survive in solvents such as ethanol, butanol, dodecane, and toluene, which Oil would kill many other bacteria bioremediation They have enzymes e.g., including cytochrome P450 enzymes capable of degrading toxins using versatile in oxidation pathways and catalyze a variety of reactions, including epoxidation. 32  Biostimulation Biostimulation is the release of nutrients, oxidants, or electron donors into the environment to stimulate naturally occurring microorganisms to degrade a contaminant. methyl tert- butyl ether (MTBE) oxygenates gasoline, so that it burns more efficiently, but it can contaminate ground water. The United States Environmental Protection Agency has classified MTBE as a possible human carcinogen, and drinking water must contain less than 20 MTBE to 40 µ g/L. 33  Biostimulation A site in South Carolina, USA, had a large plume of MTBE- contaminated gasoline leaking from an underground storage tank at a gas station. The plume ended at a drainage ditch. The concentration of MTBE in the water was low, and in the 2-meter gap between the anaerobic and aerobic zones, the MTBE was metabolized by naturally occurring microorganisms.  Biostimulation This led to studies that determined that MTBE could be degraded by bacteria such as Methylobium petroleophilum PM1 in areas that transition from anaerobic (anoxic) to aerobic (oxic). If anaerobic regions of the MTBE plume in South Carolina were injected with a compound that released oxygen, the concentration of MTBE decreased from 20 mg/L to 2 mg/L, suggesting that biostimulation may be a good approach to clean up contamination. 36 37 Phytoremediation ─ GM plants are used which are capable of direct uptake of pollutants from the environment. ─ can be applied to both inorganic and organic pollutants present in solid and liquid substrate 38 Phytoremediation ─ Biotechnology offers the opportunity to transfer hyper accumulator phenotypes into fast growing, high biomass plants that could be highly effective in Phytoremediation 39 Phytoremediators An ideal phytoremediator would have: 1. high tolerance to the pollutant; 2. the ability to either degrade or concentrate the contaminant at high levels in the biomass; 3. extensive root systems; 4. the capacity to absorb large amounts of water from the soil; 5. and fast growth rates and high levels of biomass. 40 Phytoremediators Introduction of novel traits for the uptake and accumulation of pollutants into high biomass plants is a successful strategy for improving phytoremediators Genes targeted include metal transporter genes, as well as genes that facilitate chelator production and genes that facilitate conversion to volatile forms of molecules. 41 BTEX compounds Benzene, Toluene, Ethylbenzene, and Xylene (volatile organic compounds- VOCs) Naturally found in gasoline emissions sources of exposure to BTEX compounds include breathing contaminated air, particularly in areas of heavy motor vehicle traffic and petrol stations. 42  BTEX compounds To increase phytoremediation of BTEX chemicals, the genes for degrading the BTEX component, e.g., toluene, can be transferred to an endophyte and inoculated onto a flowering plant. The inoculated plants are able to tolerate 10X the levels of toluene. When the original toluene-degrading strain was inoculated into another plant e.g., poplar, the strain conjugatively transferred the plasmid to the native endophytes, resulting in increased tolerance to toluene 43 BTEX compounds The endophyte also reduced the phyto- transpiration of the chemical. Furthermore, transgenic plants expressing mammalian cytochrome P450 2E1 had greatly increased rates of removal of toluene and benzene. Toluene was removed from the hydroponic solution within two days, at a rate ten times faster than the vector-control plants. Benzene was nearly completely removed within three days by CYP2E1 transgenic tobacco, while the vector-control plants removed benzene 44 Bioethics Ethics and laws are not equal The law will often incorporate ethical standards, to which most citizens subscribe. Ethics is a set of well-founded standards of right and wrong that prescribe what humans ought to do, usually in terms of rights, obligations, benefits to society, fairness, or specific virtues. A general philosophy of "Do no harm" while maximizing benefits for the research project and minimizing risks to the research subjects is a standard followed by scientists involving human subjects. 45  Biotechnology especially modern biotechnology can produce unanticipated consequences that cause harm to people or affect in a negative fashion the problem it was made to improve or solve. Biotechnology Bioethics includes assessment of the rights and wrongs of specific technologies and applications and Bioethics (e.g. cloning). Bioethics also examines the broader goals and aims of activities such as biotechnology. E.g. The relief of sickness or reduction in starvation is are examples. 46  Ethics are guided by rules based on moral norms or principles which may differ based on Biotechnology culture and vary in different communities and Bioethics The question is “What principles and factors are needed for guiding decision-making in the use and applications of modern biotechnologies?” 47  Who should control technology? -control What should be banned or permitted and who should decide? control Bioethics questions Who should profit? - control Should access to novel and expensive technology be provided to those who cannot afford it? -access If so, who should pay, the government or private individuals? Access /control 48 ̶ Access to expensive health care, whether drugs, surgery, or simply high-quality Access nursing is a question of individual access to money. ̶ Treatment with botulinum toxin (Botox) used to remove wrinkles from the skin is expensive. Botox injections range from $300 to $500, with each treatment lasting approx. 5 months. ̶ Botulinum toxin type A is a protein toxin Taken from Biotechnology by Clark made by the bacterium Clostridium botulinum, which causes food poisoning ̶ Access to biotechnology and new ̶ For example, individuals may want treatments can be defended on the basis. reproductive cloning, but the concerns of of individual rights. Yet it is not easy to future generations and society as a whole defend concerns about people seeking need to be considered treatments that are ethically questionable or of uncertain benefit. 49  Control Transgenic crops able to grow in poor soils and give higher yields without fertilizers help rural communities and poor nations worldwide. Large strides in traditional plant breeding increased crop yields drastically between the 1940s and 1980s eg.in India “Green Revolution”. However improved crop yields and freedom from starvation may cause population expansion which in turn, causes overcrowding, thus promoting the spread of infections. ( unintended consequences) Distribution o f seedless improved varieties s controlled by agricultural biotechnology companies e.g. terminator seeds. http://www.academicjournals.org/article/article1380729637_Yusuf.pdf 50 IFBA – International Federation of Biosafety Associations » IFBA Welcomes Biosafety Associations in Caribbean, Turkey, and Morocco (internationalbiosafety.org) 51  References: Clark, D. and Pazdernik, N. J. 2016. Biotechnology. Elsevier https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2267612/ https://microbiomejournal.biomedcentral.com/track/pdf/10.1186/ s401 68-019-0648-z 52