L9 - Improving Bioremediation

Question 1

How can you introduce genes to bacteria?

Answer 1

Plasmids and mating or donor and recipient microbes

Can occur naturally

Question 2

What are the issues of GM bacteria?

Answer 2

Stability of gene within pop

Risk of transfer to other bacteria (may be better to incorporate gene into host chromosome)

Question 3

What is an example of a gene introduction into bacteria?

Answer 3

Wantanabe (2002)
Took phenol hydroxylase from Comamonas R5 (very good phenol degrader, bad surviving out of lab)
Put in another Comamonas (rN7) that’s dominant in sewage
Result - survive and degrade phenol 3x faster

Question 4

How can a gene be altered to make it optimal in field?

Answer 4

Alter txnal promoter and terminator
Increase copies
Improve stability of protein

Question 5

What is an example of gene alteration in bacteria?

Answer 5

Ramos et al (1986)
Took pWWO from Pseudomonas putida (degrades toluene and zylene but no ethylbenzoate (EB) because pathway not induced)
Cloned xylS gene (regulator of pathway) and generated e coli mutant that responds to EB
Introduced gene back in putida

Question 6

What is the horizontal gene transfer method?

Answer 6

Introduce remediation genes into indigenous microbes already adapted to that environment

Question 7

What is good about horizontal gene transfer method?

Answer 7

No need for long term survival, can insert conditional suicide genes into donors

Question 8

What is the conditional suicide system devised by Contreras?

Answer 8

In presence of 3-methylbenzoate production of LacI repressor blocks Gef production
When 3-MB all degraded, Gef produces porins and kills cell

Question 9

What are 6 molecular techniques that can be used to measure what microbes are doing?

Answer 9

16S rRNA PCR
RT PCR
Real time PCR
Fluorescent in situ hybrid
Microarrays
Reporter genes

Question 10

How does 16S rDNA PCR help and how is it done?

Answer 10

Allows for detection of specific microbes in soil.

Lyse cells, specific primers, amplified material analysed

Question 11

How does fluorescence in situ hybridisation work?

Answer 11

Fluorecent probes attached to oligonucleotides
Probe binds targets in ribosomes
Targets only viable and active cells
Can be designed to only label specific types of bacteria

Question 12

How are reporter genes used?

Answer 12

Gene codes for easily detected trait (GFP) attached to regulatory sequence of gene of interest in bacteria
Easily detected

Question 13

What are the features of metal working site in Spain contaminated by hydrocarbons?

Answer 13

7000 kg LNAPLs flowing towards river
3000kg hydrocarbons in vadose zone
Fuel oil from heating system, lubricant from metalworking, diesel from underground tanks

Question 14

How did they initially treat the metalworks?

Answer 14

Started physicochemical
Hydraulic barrier to stop groundwater entering river
Pump and treat to remove free hydrocarbons

Question 15

How did the initial treatment go?

Answer 15

Removed 3/4 of hydrocarbons in 1y
2000kg hydrocarbons retained in vadose
500kg in groundwater

Question 16

What did they find in tests after initial strategy?

Answer 16

Nutrients and oxygen at rate limiting levels

Microbial pops capable of degrading detected

Question 17

What 3 treatments did they use to stimulate microbial activity after initial treatment?

Answer 17

Continuous addition of H2O2 at 250mg/l to increase oxygen
Biodegradable surfactant added once a month
Oleophilic fertiliser applied to achieve C:N of 10:1 once a month

Question 18

What had to be carefully controlled with these treatments?

Answer 18

Prevent excess levels of surfactant or nutrients building
pH to remain between 6.5 and 8
Temperate between 22 and 26

Question 19

What type of sites are poor candidates for bioremediation?

Answer 19

High clay content
Water saturation
Pollutant sorbed strongly to soil

Question 20

What are the limitations of bioremediation?

Answer 20

Incomplete metabolism can lead to build up of intermediates

Physico-chemical methods often chosen

Question 21

What are the advantages of bioremediation?

Answer 21

Efficient, rapid, versatile and inexpensive alternative
No major disruption in situ
Can improve soil fertility and structure
No harsh chemicals
Acceptable to public