Biotechnology and bioremediation

• cotton moth - Spodoptera littoralis

  • one of the most destructive pests in subtropical range
  • affects many economically important crops all year round
  • in Europe significant outbreaks in Spain, Italy, Greece and Crete
  • cotton plants - feed on leaves, fruiting points, flower buds and bolls
  • cotton moth damage native to Africa, the Middle East and the Mediterranean countries of Europe
  • greenhouse pest of tomato, pepper, and melon in Spain, Italy and Greece
• biological control of cotton moth

  • pre 1968 control using methyl pyrethroids
  • resistance emerged, synthetic same problem
  • biological control strategies have included use of parasites and predators
  • slow release of pheromone formulations for mating disruption used with limited success
  • nuclear polyhedrosis virus
• nuclear polyhedrosis virus and cotton moth

  • S. littoralis is affected by a naturally occuring nuclear polyhedrosis disease first reported in 1937
  • the NPV was isolated, purified and characterised by Harrap et. al 1977
  • the virus is specific to S. littoralis larvae without affecting beneficial insects
  • in 1978 the UK Overseas Develoment Administration funded a project in collaboration with the Egyptian Academy of Sciences one of the projects was to investigate the potential of the purified NPV as a practical control agent
  • Bacillus thuringiensis as alternative control agent
• lactic fermentation of grass/corn to produce silage

  • glucose and sucrose in grass is converted by fermentation to lactic acid
  • lowers pH and reduces spoilage, better milk yields
  • nutritional advantages and reduce food stock losses
  • isolation and characterisation of inoculant strains
  • product development (fermentation production and other additives including enzymes)
• what is bioremediation

  the use of biodegradative processes to detoxify or to remove pollutants
• what is biodegradation

  naturally occurring degrading properties of microorganisms are used to break down compounds in the environment
• petroleum and oil in the environment

  • 3.4 million T of petroleum and petroleum fractions PA introduced into marine environment
  • main routes of entry include highly publicised accidents eg tanker spills
  • most common mode of entry is via natural oil and tar seeps
  • hydrocarbon contaminated drill cuttings removed from oil platforms and buried in landfills
  • polycyclic aromatic hydrocarbons have received a lot of attention in recent years, however other persistent, toxic fractions also need to be addressed
• oily drill cuttings

  • approx. 700 tonnes during the life of a well
  • previously left on the sea bed
  • due to the anoxic nature of drill cutting piles degradation is severely reduced
  • legislation now requires the oil industry to dispose of this waste
  • when brought to shore, waste remains highly saline
• drilling fluid

  • oil-based drilling fluids in use since the 1930s
  • originally formulated from crude oil
  • currently use low toxicity mineral oil
  • C12 - C16 alkanes
  • these drilling fluids do not readily degrade on the sea floor
  • drilling fluids consist of two phases
  • oil phase
  • brine phase
• extremophiles

  • drill cuttings are highly saline
  • dilution of this waste would be expensive
  • conventional microbial treatments are unsuitable due to high salinity of waste, halophiles tolerate and grow in high salinities
  • halophiles withstand the metabolic burden which the drill cuttings place on them
  • certain halophiles can degrade oil
• hypersaline environments

  • halobacteria refers to the red-pigmented extremely halophilic Archaea
  • halobacteria are the most predominant organisms found in hypersaline environments
  • Gyda oil field is highly saline (290 g l-1)
  • original sample contained entrained brine droplets
  • brine droplets originated from formation water in the well
  • after continuous sub-culturing isolates were obtained from this enrichment
• aromatic compounds

  • discovered in the 19th century
  • they smell sweet
  • often very toxic, carcinogenic
  • huge range of chemical reactions and compounds possible
• bioremediation of pesticides

  • percolation through soild, sediments, into ground water and the subsurface
  • accumulation exceeding government limits and are toxic
  • half life approx. 10 days, but can be much longer depending on microbial degredation
  • auxin mimic
  • usage in the US in 2017 - 18 million tonnes
  • toxicity within the environment and associated human health implications, including reproductive, tetratogenic, mutagenic and carcinogenic effects
  • genetic basis of 2,4-D degradation
  • identification of genetic elements envolved in degradation has been a goal for researchers over past decades
• soil contamination with explosives

  • massive build up of TNT and other production intermediates at former production sites since WWI and II
  • production of TNT increased exponentially between 1940 and 1980
  • highly enriched surfaces, sediments and seeping of hydrophilic intermediates into ground water
  • approx 15 million acres in the US are contaminated, 3000 sites in Germany
  • bacteria can partially degrase TNT - Pseudomonas strains can use it as a nitrogen source
  • consequence of that are often highly recalcitrant and even more toxic intermediates
  • one possible solution is to design bacterial consortia that consist of species that cross-feed each other TNT intermediares for further degradation and mineralisation
• microbial cell factories

  • onset of recombinant DNA technology in the 1980s was followed by a massive increase in biotechnological progress, harnessing microbial metabolism
  • design of microbes to produce or degrade complex organic chemicals
  • growth-yield trade off is the main factor reducing yields of biotechnological processes
  • microbes are tuned for growth, not production of recombinant products such as medicines
  • overproduction of product leads to a growth detriment and selection for variants that have lost production abilities
  • synthetic tools allows to decouple growth and production through dynamic metabolic control
  • genetic engineering
  • delete genes to alter endogenous metabolic fluxes
  • add new or external metabolic pathways to enable production of new metabolic intermediates
• Pseudomonas putida as microbial cell factories

  • soil dwelling, non-pathogenic bacterium
  • was found to have the ability to naturally degrade a host of aromatic compounds such as toluene by using them as sole carbon source
  • large genome with rich secondary metabolism
  • very resistant to oxidative stress - important for many aerobic biochemical reactions
  • high NADH turnover rates
  • EDEMP cycle
  • ED: Entner-Doudoroff EMP: Embden-Meyerhof-Parnas 3 ATP, 4 NAD(P)H
  • instead of going towards pyruvate, a part of GA3P is recycles back into hexoses