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Nutrient Cycling + Prokaryotes > Respiration (Bioremediation) > Flashcards

Respiration (Bioremediation) Flashcards

1
Q

What is respiration?

A

the oxidation of reduced carbon with oxygen

production of energy (ATP)

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2
Q

What is the net reaction of respiration?

A

CnH2nOn + O2 + ADP + Pi → nCO2 + nH2O + ATP

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3
Q

What are the major processes involved in respiration?

A

glycolysis

Citric acid cycle or tricarboxylic (3-COOH) acid cycle

oxidative phosphorylation

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4
Q

T or F: most cells use respiration as a key metabolic process

A

true

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5
Q

T or F: respiration is key for OM decomposition

A

true

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6
Q

Describe the major steps of the CAC

A
  1. condensation
    oxaloacetate + acetyl CoA + H2O to regenerate CoA and produce citrate

2a. dehydration
citrate loses H2O = intermediate

2b. hydration
+ H2O = isocitrate

  1. oxidative decarboxylation
    CO2 released = ketoglutarate
    NAD+ = NADH
  2. oxidative decarboxylation
    CoA-SH + ketoglutarate, CO2 released = succinyl-CoA
    NAD+ = NADH
  3. substrate level phosphorylation
    GDP + Pi + succinyl CoA = GTP and CoA-SH regenerated = succinate
  4. dehydrogenation
    FADH2 released = fumarate
  5. hydration
    + H2O = malate
  6. dehydrogenation
    NADH released
    = oxaloacetate
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7
Q

What stages of CAC is NAD+ reduced to NADH?

A

the 2 oxidative decarboxylase steps and dehydrogenation of malate

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8
Q

What stage(s) of CAC release CO2?

A

the 2 oxidative decarboxylation stages

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9
Q

What stage of the CAC is ATP produced?

A

substrate level phosphorylation of succinyl-coa

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10
Q

When is FADH reduced to FADH2 in the CAC?

A

dehydrogenation of succinate

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11
Q

What is the major coenzyme for the CAC of respiration?

A

coenzyme A

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12
Q

What is the Principle of Microbial Infallibility?

A

given optimal environmental conditions, no natural compound is resistant to degradation

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13
Q

Explain how even synthetic compounds are not resistant to degradation?

A

they have natural counterparts that can be broken down by microbial metabolism

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14
Q

What are xenobiotics?

A

compounds with molecular structures and chemical bond that are not recognized by existing degradative enzymes

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15
Q

What 8 characteristics of xenobiotics can lead to them being recalcitrant?

A
  1. unusual substitutions (Cl, F)
  2. unusual bonds (tert or quaternary carbons)
  3. highly condensed aromatic rings
  4. very large molecular size
  5. inability to synthesize degradative enzymes
  6. inappropriate permease
  7. insoluble
  8. toxic parent compounds
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16
Q

What is biodegradation?

A

the transformation of a molecule into another molecule

**NOT complete degradation

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17
Q

T or F: biodegradation = complete degradation

A

false!! it’s just the conversion of one molecule into another

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18
Q

T or F: a toxic molecule cannot be toxic in its biodegraded state

A

false, it can still be toxic

ex. DDT biodegrades to DDE which is still toxic

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19
Q

What is mineralization?

A

the COMPLETE degradation and recycling of an organic molecule to its mineral constituents

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20
Q

What is DDT mineralized into? What is it biodegraded into?

A

Cl and CO2 - both non-toxic

DDE - toxic

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21
Q

Does DDT normally decompose naturally? why/why not?

A

no, it consists of Cl (inorganic, unusual substitutions) and benzene rings (aromatic)

requires specific bacteria

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22
Q

Most aromatic compounds have ____ pathways that lead to the same intermediate - which intermediate?

A

convergent pathways that lead to catochol (oxygenated 6C benzene ring + 2 OH)

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23
Q

What happens to the catechol intermediate?

A

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

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24
Q

What are petroleum hydrocarbons?

A

natural products from the anaerobic conversion of biomass under high temperature and pressure

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25
Q

How does the rate of degradation of petroleum compare to the force of recovery? why is this problematic?

A

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

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26
Q

How are most xenobiotics classified?

A

as substituted or modified hydrocarbons

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27
Q

What is crude oil?

A

the main component of an oil spill

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28
Q

what are the components of crude oil?

A

cycloparaffins

aromatics and cycloparaffinic aromatics

straight chain alkane

branched alkane

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29
Q

What is aliphatic hydrocarbon?

A

non-aromatic hydrocarbons

ex. cycloparaffins, straight and branched alkanes

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30
Q

What co-proteins initiate oxidation of aliphatic hydrocarbons?

A

Rubredoxin (Fe based protein), a cosubstrate (aka a coenzyme) that is required for initiating the attack by monooxygenase

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31
Q

Which major enzyme is involved in the oxidation of aliphatic hydrocarbons?

A

monooxygenase

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32
Q

What is monooxygenase?

A

an enzyme that catalyzes the addition of a single O2 to an organic compound

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33
Q

Which state (oxidized/reduced) does rubredoxin need to be in to be active and assist monooxygenase?

A

the Fe of rubredoxin needs to be reduced by rubredoxin reductase

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34
Q

What are the major steps of oxidizing aliphatic hydrocarbon?

A

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)

35
Q

What is the model organism for the genetic biodegradation of aliphatic hydrocarbon?

A

Pseudomonas putida

36
Q

What is the other name for monooxygenase?

A

GPo1 alkane hydroxylase

37
Q

What are the 3 components required for oxidation of aliphatic hydrocarbon?

A

alkane hydroxylase (monooxygenase)
rubredoxin
rubredoxin reductase

38
Q

What are the major genes that code for the 3 components required for oxidation of aliphatic hydrocarbon?

A

alkB
alkG
alkT

39
Q

What does alkB code for?

A

alkane hydroxylase (aka monooxygenase)

40
Q

What does alkG code for?

A

rubredoxin

41
Q

What does alkT code for?

A

rubredoxin reductase

42
Q

Which major organisms mineralize aliphatic hydrocarbons?

A

Pseudomonas putida
Alcanivorax borkumensis
Bacillus cepacia
Rhodococcus erythropolis

gram+ and gram-

43
Q

what enzymes catalyze the oxidation of aromatic hydrocarbons?

A

monooxygenases and dioxygenases

44
Q

which gene codes for oxygenases (XO) for aromatic oxidation?

A

xylMA

45
Q

T or F: genes for aromatic oxidation are highly regulated

A

true

46
Q

Which gene represses the aromatic hydrocarbon oxidation (degradation) pathway? how?

A

xylR blocks the expression of xylS which leads to the production of toluene = blocks pathway

47
Q

which hydrocarbon intermediate is key to produce for the ongoing oxidation of an aromatic carbon? which genes code for it?

A

Toluene

coded for by xylS and repressed by xylR

48
Q

which plasmids encode for catabolic functions (degradation/respiration)?

A

TOL encodes for toluene and p- and m-xylene

49
Q

What are some major genera of organisms that can degrade aromatic hydrocarbons?

A

Micrococcus in soil

Vibrio (gammaproteobacteria) in aquatic

pseudomonas (gammaproteo)

Achromobacter (betaproteo)

50
Q

Which 2 betaproteobacteria conduct anoxic degradation of aromatic hydrocarbons?

A

Thauera aromatica (named for Rolf Thauera from Marburg, Germany)

Azoarcus tolulyticus

51
Q

Would the oxygenic or anoxygenic degradation of aromatic hydrocarbons be slower/faster?

A

oxygenic faster
anoxygenic slower = less energy is produced

52
Q

What are 3 major ways to measure potential for biodegradation + biomineralization of hydrocarbons?

A
  1. ecology: study abundance
  2. measure activity
  3. determine diversity - use molecular DNA techniques to identify species
53
Q

How can the ecology (abundance) of organisms which biodegrade or biomineralize hydrocarbons be studied?

A

viable count on specific medium that only has toluene, benzene or octane as carbon source

or

isolating degrading bacteria with colony hybridization for confirmation

54
Q

Describe the steps of aromatic hydrocarbon oxidation for benzene

A

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

55
Q

Which cofactor is key for the oxidation of hydrocarbons and must be regenerated by the process?

A

NADH

56
Q

Describe the steps of aromatic hydrocarbon oxidation for toluene

A

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

57
Q

How do dioxygenases help prepare an aromatic hydrocarbon such as toluene for the central pathway of degradation?

A

by breaking the benzene ring open

58
Q

What type of genes would be useful for identifying to determine whether an organism can oxidize aromatic hydrocarbons?

A

ones that code for oxygenases or the production of the catechol intermediate

59
Q

What form do aromatic hydrocarbons need to be in before they can enter the central degradation pathway?

A

catechol intermediate

60
Q

Which genes code for the degradation of xylene into benzoic acid?

A

(Pu)xylCMABN

61
Q

Which genes code for the degradation of benzoic acid into benzoate?

A

(Pm)xylXYZLEGFJKIH

62
Q

Which genes code for the degradation of benzoate into toluene?

A

xylS ?

63
Q

What are the major sources of carbon for organisms conducting biodegradation of hydrocarbons?

A

toluene, benzene, octane, etc.

64
Q

Describe how you would count abundance from a specific medium

A

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

65
Q

Describe a method for measuring activity of biodegradation

A

using a 14C probe to determine rate:

  1. add 14C-hexadecane or napthalene to soil or sediment microcosms
  2. add test tube with KOH = traps CO2

3/ sample KOH levels at different times

  1. measure radioactivity using liquid scintillation

radioactivity = degradation

66
Q

Would microorganisms at Mt Doug be better at mineralizing xylene than in Sooke?

A

mt doug

67
Q

What other methods of assessing activity of biodegradation are there?

A

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)

68
Q

How can diversity of biodegraders be measured?

A

molecular DNA techniques

extraction of DNA from samples
PCR amplification of alkB
clone amplicons
dot blot

69
Q

What is Dot blotting?

A

similar to colony hybridization - a step of molecular DNA sequencing

70
Q

Which gene would you use to determine whether an organism can oxidize alkane hydrocarbons? aromatic HCS?

A

alkB - the gene that codes for alkane hydroxylase (monooxygenase for alkane hydrocarbons)

xylMA for aromatic

71
Q

T or F: identification genes (ex. alkB) need to be turned on in order to determine organism

A

false

72
Q

why does it matter which carbon you tag with 14C label to determine rate of mineralization?

A

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

73
Q

What 4 environmental factors limit presence or activity of hydrocarbon degradation?

A

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

74
Q

Why is O2 required for the presence of biodegraders?

A

O2 is required for monooxygenases and dioxygenases = the enzymes that catalyze the breakdown of hydrocarbons

75
Q

What environments might there be less biodegraders?

A

subsurface environments like fine soil or sediment because limited O2 diffusion

76
Q

Why is nitrogen source for protein and nucleic acid synthesis and P limiting environmental factors? How can this be mitigated?

A

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

77
Q

T or F: hydrocarbons are very water soluble

A

false, poor solubility, especially with increasing molecular weight

78
Q

What other chemical compound may be useful in degrading oil compounds in water?

A

surfactants (which some bacteria can produce) and emulsifiers

79
Q

How is petroleum absorbed in soil?

A

to particulate matter with decreased toxicity but persistent and long lasting

80
Q

What do petroleum spills cause?

A

increased populations of HC-degrading bacteria or bacteria encoding hydrocarbon catabolic genes

81
Q

Describe the steps and set up of bioremediation of petroleum hydrocarbon

A

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

82
Q

How can we measure the elimination of contamination underground?

A

map underground contaminations and sample petroleum concentrations using gas chromatography

83
Q

Describe how PCB-contaminated sediment can be bioremediated

A

Steel Caissons over time
using gas chromatography to measure anaerobic dechlorination and aerobic degradation to break apart carbon rings

Nutrient Cycling + Prokaryotes (13 decks)

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