Respiration (Bioremediation) Flashcards
What is respiration?
the oxidation of reduced carbon with oxygen
production of energy (ATP)
What is the net reaction of respiration?
CnH2nOn + O2 + ADP + Pi → nCO2 + nH2O + ATP
What are the major processes involved in respiration?
glycolysis
Citric acid cycle or tricarboxylic (3-COOH) acid cycle
oxidative phosphorylation
T or F: most cells use respiration as a key metabolic process
true
T or F: respiration is key for OM decomposition
true
Describe the major steps of the CAC
- condensation
oxaloacetate + acetyl CoA + H2O to regenerate CoA and produce citrate
2a. dehydration
citrate loses H2O = intermediate
2b. hydration
+ H2O = isocitrate
- oxidative decarboxylation
CO2 released = ketoglutarate
NAD+ = NADH - oxidative decarboxylation
CoA-SH + ketoglutarate, CO2 released = succinyl-CoA
NAD+ = NADH - substrate level phosphorylation
GDP + Pi + succinyl CoA = GTP and CoA-SH regenerated = succinate - dehydrogenation
FADH2 released = fumarate - hydration
+ H2O = malate - dehydrogenation
NADH released
= oxaloacetate
What stages of CAC is NAD+ reduced to NADH?
the 2 oxidative decarboxylase steps and dehydrogenation of malate
What stage(s) of CAC release CO2?
the 2 oxidative decarboxylation stages
What stage of the CAC is ATP produced?
substrate level phosphorylation of succinyl-coa
When is FADH reduced to FADH2 in the CAC?
dehydrogenation of succinate
What is the major coenzyme for the CAC of respiration?
coenzyme A
What is the Principle of Microbial Infallibility?
given optimal environmental conditions, no natural compound is resistant to degradation
Explain how even synthetic compounds are not resistant to degradation?
they have natural counterparts that can be broken down by microbial metabolism
What are xenobiotics?
compounds with molecular structures and chemical bond that are not recognized by existing degradative enzymes
What 8 characteristics of xenobiotics can lead to them being recalcitrant?
- unusual substitutions (Cl, F)
- unusual bonds (tert or quaternary carbons)
- highly condensed aromatic rings
- very large molecular size
- inability to synthesize degradative enzymes
- inappropriate permease
- insoluble
- toxic parent compounds
What is biodegradation?
the transformation of a molecule into another molecule
**NOT complete degradation
T or F: biodegradation = complete degradation
false!! it’s just the conversion of one molecule into another
T or F: a toxic molecule cannot be toxic in its biodegraded state
false, it can still be toxic
ex. DDT biodegrades to DDE which is still toxic
What is mineralization?
the COMPLETE degradation and recycling of an organic molecule to its mineral constituents
What is DDT mineralized into? What is it biodegraded into?
Cl and CO2 - both non-toxic
DDE - toxic
Does DDT normally decompose naturally? why/why not?
no, it consists of Cl (inorganic, unusual substitutions) and benzene rings (aromatic)
requires specific bacteria
Most aromatic compounds have ____ pathways that lead to the same intermediate - which intermediate?
convergent pathways that lead to catochol (oxygenated 6C benzene ring + 2 OH)
What happens to the catechol intermediate?
central pathways with enzymes to break carbon rings and produce
acetyl CoA and succinate > TCA cycle
and
acetaldehyde and pyruvate which either are
- converted into acetyl CoA and succinate then TCA/CAC
- or directly into TCA
What are petroleum hydrocarbons?
natural products from the anaerobic conversion of biomass under high temperature and pressure
How does the rate of degradation of petroleum compare to the force of recovery? why is this problematic?
degradation MUCH slower than force of recovery
problematic because there’s a high risk of spills = very difficult to get rid of and mitigate damage
How are most xenobiotics classified?
as substituted or modified hydrocarbons
What is crude oil?
the main component of an oil spill
what are the components of crude oil?
cycloparaffins
aromatics and cycloparaffinic aromatics
straight chain alkane
branched alkane
What is aliphatic hydrocarbon?
non-aromatic hydrocarbons
ex. cycloparaffins, straight and branched alkanes
What co-proteins initiate oxidation of aliphatic hydrocarbons?
Rubredoxin (Fe based protein), a cosubstrate (aka a coenzyme) that is required for initiating the attack by monooxygenase
Which major enzyme is involved in the oxidation of aliphatic hydrocarbons?
monooxygenase
What is monooxygenase?
an enzyme that catalyzes the addition of a single O2 to an organic compound
Which state (oxidized/reduced) does rubredoxin need to be in to be active and assist monooxygenase?
the Fe of rubredoxin needs to be reduced by rubredoxin reductase
What are the major steps of oxidizing aliphatic hydrocarbon?
ex. n-Octane
n-Octane + NADH
monooxygenase catalyzes addition of O from O2 = octanOL + NAD+ (oxidized) + H2O
NAD+ reduced to NADH
= octanAL (aldehyde)
H2O oxidized + NAD+ reduced to NADH
= octanoic acid (Oxidized)
+ ATP (to AMP + PPi) + CoA
= beta oxidation to acetyl CoA (central pathway for fatty acid metabolism)
What is the model organism for the genetic biodegradation of aliphatic hydrocarbon?
Pseudomonas putida
What is the other name for monooxygenase?
GPo1 alkane hydroxylase
What are the 3 components required for oxidation of aliphatic hydrocarbon?
alkane hydroxylase (monooxygenase)
rubredoxin
rubredoxin reductase
What are the major genes that code for the 3 components required for oxidation of aliphatic hydrocarbon?
alkB
alkG
alkT
What does alkB code for?
alkane hydroxylase (aka monooxygenase)
What does alkG code for?
rubredoxin
What does alkT code for?
rubredoxin reductase
Which major organisms mineralize aliphatic hydrocarbons?
Pseudomonas putida
Alcanivorax borkumensis
Bacillus cepacia
Rhodococcus erythropolis
gram+ and gram-
what enzymes catalyze the oxidation of aromatic hydrocarbons?
monooxygenases and dioxygenases
which gene codes for oxygenases (XO) for aromatic oxidation?
xylMA
T or F: genes for aromatic oxidation are highly regulated
true
Which gene represses the aromatic hydrocarbon oxidation (degradation) pathway? how?
xylR blocks the expression of xylS which leads to the production of toluene = blocks pathway
which hydrocarbon intermediate is key to produce for the ongoing oxidation of an aromatic carbon? which genes code for it?
Toluene
coded for by xylS and repressed by xylR
which plasmids encode for catabolic functions (degradation/respiration)?
TOL encodes for toluene and p- and m-xylene
What are some major genera of organisms that can degrade aromatic hydrocarbons?
Micrococcus in soil
Vibrio (gammaproteobacteria) in aquatic
pseudomonas (gammaproteo)
Achromobacter (betaproteo)
Which 2 betaproteobacteria conduct anoxic degradation of aromatic hydrocarbons?
Thauera aromatica (named for Rolf Thauera from Marburg, Germany)
Azoarcus tolulyticus
Would the oxygenic or anoxygenic degradation of aromatic hydrocarbons be slower/faster?
oxygenic faster
anoxygenic slower = less energy is produced
What are 3 major ways to measure potential for biodegradation + biomineralization of hydrocarbons?
- ecology: study abundance
- measure activity
- determine diversity - use molecular DNA techniques to identify species
How can the ecology (abundance) of organisms which biodegrade or biomineralize hydrocarbons be studied?
viable count on specific medium that only has toluene, benzene or octane as carbon source
or
isolating degrading bacteria with colony hybridization for confirmation
Describe the steps of aromatic hydrocarbon oxidation for benzene
monooxygenase steps:
benzene monooxygenase uses energy from NADH > NAD+ to split H2O and adds an O to the benzene ring to produce an epoxide
another O is added to the epoxide > diol
NAD+ takes H+ from diol to produce a catechol (intermediate) and regeneate NADH
Which cofactor is key for the oxidation of hydrocarbons and must be regenerated by the process?
NADH
Describe the steps of aromatic hydrocarbon oxidation for toluene
toluene DIoxygenase adds 2 O2 with energy from NADH to toluene
= diol
NAD+ removes H+ and regenerates as NADH to produce a methyl catechol
methyl catechol dioxygenase breaks the benzene ring and opens it up for the central pathway
How do dioxygenases help prepare an aromatic hydrocarbon such as toluene for the central pathway of degradation?
by breaking the benzene ring open
What type of genes would be useful for identifying to determine whether an organism can oxidize aromatic hydrocarbons?
ones that code for oxygenases or the production of the catechol intermediate
What form do aromatic hydrocarbons need to be in before they can enter the central degradation pathway?
catechol intermediate
Which genes code for the degradation of xylene into benzoic acid?
(Pu)xylCMABN
Which genes code for the degradation of benzoic acid into benzoate?
(Pm)xylXYZLEGFJKIH
Which genes code for the degradation of benzoate into toluene?
xylS ?
What are the major sources of carbon for organisms conducting biodegradation of hydrocarbons?
toluene, benzene, octane, etc.
Describe how you would count abundance from a specific medium
using a petri dish where the only C source is xylene
only bacteria containing XO for xylene would be able to survive
isolate the bacteria that have grown transfer them to another petri dish
using xylMA as a probe, identify which have it = these can degrade xylene
Describe a method for measuring activity of biodegradation
using a 14C probe to determine rate:
- add 14C-hexadecane or napthalene to soil or sediment microcosms
- add test tube with KOH = traps CO2
3/ sample KOH levels at different times
- measure radioactivity using liquid scintillation
radioactivity = degradation
Would microorganisms at Mt Doug be better at mineralizing xylene than in Sooke?
mt doug
What other methods of assessing activity of biodegradation are there?
analyzing xenobiotic compounds and/or biodegradation product by gas chromatography
or
RNA extraction and analysis by RT-PCR of gene product (alk B, nahA, xylE)
How can diversity of biodegraders be measured?
molecular DNA techniques
extraction of DNA from samples
PCR amplification of alkB
clone amplicons
dot blot
What is Dot blotting?
similar to colony hybridization - a step of molecular DNA sequencing
Which gene would you use to determine whether an organism can oxidize alkane hydrocarbons? aromatic HCS?
alkB - the gene that codes for alkane hydroxylase (monooxygenase for alkane hydrocarbons)
xylMA for aromatic
T or F: identification genes (ex. alkB) need to be turned on in order to determine organism
false
why does it matter which carbon you tag with 14C label to determine rate of mineralization?
carbons on the ring need to be tagged because they take much longer to degrade than a carbon on a chain
= accuracy of rate of degradation
What 4 environmental factors limit presence or activity of hydrocarbon degradation?
presence of HC-degrading bacteria
presence of O2 for monooxygenase and dioxygenase
nitrogen source (NO3-, NH4+) for protein and nucleic acid synthesis
phosphorous source (PO43-) for nucleic acid synthesis
Why is O2 required for the presence of biodegraders?
O2 is required for monooxygenases and dioxygenases = the enzymes that catalyze the breakdown of hydrocarbons
What environments might there be less biodegraders?
subsurface environments like fine soil or sediment because limited O2 diffusion
Why is nitrogen source for protein and nucleic acid synthesis and P limiting environmental factors? How can this be mitigated?
generally because biodegraders’ carbon source is low in N and P (hydrocarbons low in N and P)
addition of fertilizer can help increase N and P
T or F: hydrocarbons are very water soluble
false, poor solubility, especially with increasing molecular weight
What other chemical compound may be useful in degrading oil compounds in water?
surfactants (which some bacteria can produce) and emulsifiers
How is petroleum absorbed in soil?
to particulate matter with decreased toxicity but persistent and long lasting
What do petroleum spills cause?
increased populations of HC-degrading bacteria or bacteria encoding hydrocarbon catabolic genes
Describe the steps and set up of bioremediation of petroleum hydrocarbon
in a gas station:
H2O2 and nutrient tanks (NO3-, PO43-) drain into an injection well with pores that leads into the underground contamination zones
H2O2 will have an influence in zone closest to injection
O2 will have influence
nutrients will have influence and the zone of contamination should decrease
How can we measure the elimination of contamination underground?
map underground contaminations and sample petroleum concentrations using gas chromatography
Describe how PCB-contaminated sediment can be bioremediated
Steel Caissons over time
using gas chromatography to measure anaerobic dechlorination and aerobic degradation to break apart carbon rings
