EXERCISE 13:

METABOLISM OF
CARBOHYDRATES AND
ORGANIC ACIDS
BAUTISTA, TYRONE D.
ITANG, LORDY GRACE C.
Metabolism of Carbohydrates

Carbohydrates
 major class of naturally occurring organic compounds which have
approximately the general formula (CH2O)n.

Three Major Classes of Carbohydrates

1. Monosaccharide – glucose as an example
2. Oligosaccharide – short chains of monosaccharide units;
sucrose
3. Polysaccharide – starch, glycogen
Three Major Pathways for Utilization of Carbohydrates
(ATP-generating)
1. Fermentation
 Carbohydrates serve as both electron donor and electron acceptor;
oxidation occurs in the absence of electron transport chain; does
not require oxygen.

2. Aerobic Respiration
 Energy from electron transport is used to generate ATP; involves
electron transport system; oxygen is the final electron acceptor.

3. Anaerobic Respiration
 Involves essentially the biochemical pathways as aerobic respiration
except for the terminal electron acceptor.
OBJECTIVES
▪ Identify the type of carbohydrate metabolism
(oxidative or fermentative) using Hugh and Leifson
medium
▪ Determine the glucose fermentation pathway through
methyl red and Voges-Proskauer tests
▪ Determine the ability of a bacterial isolate to utilize
citric acid and malonate
▪ Determine the ability of a bacterial isolate to
hydrolyze starch
▪ Determine the ability of a bacterial isolate to
produce 2-ketogluconate from potassium gluconate
MATERIALS
 Culture Media:
 Hayne’s Medium
 Hugh and Leifson with different sugars (glucose, lactose,
mannitol, sucrose)
 Malonate Broth
 MRVP broth
 Simmon’s Citrate Agar (SCA) slant
 Starch Agar (SA) plate
 Reagents:
 Benedict’s reagent
 Lugol’s/Gram’s iodine
 Methyl red indicator
 VP reagents (freshly prepared)
A. Oxidation and/or Fermentation of
Sugars
UNKNOWN BACTERIUM

Stab inoculate into Hugh and Leifson Medium

Seal 1st set of tubes (glucose, 2nd set of tubes (glucose,

lactose, mannitol, sucrose) lactose, mannitol, sucrose)
with water agar without water agar

Incubate at 30°C for 24 - 48 h

Observe results.
B. Methyl Red (MR) and Voges-Proskauer
(VP) Test UNKNOWN BACTERIUM

Inoculate to 2 MRVP tubes

Incubate at 33°C for 48-60 h
MR VP

MRVP tube #1 MRVP tube #2

Add 1-2 drops of methyl red Add 5 drops of 5% alpha-naphthol and mix
indicator

Add 7-10 drops of 40% KOH

Observe results.

Mix and stand for 15-20 min

Observe results.
C. Citric Acid Utilization

UNKNOWN BACTERIUM

Inoculate to Simmon’s Citrate Agar (SCA) slants

-stabbing the butt and streaking on the surface

Incubate at 33°C for 48 h

Observe results.
D. Malonate Utilization

UNKNOWN BACTERIUM

Inoculate to Malonate Broth (MB) tubes

Incubate at 33°C for 48 h

Observe results.
E. Starch Hydrolysis
UNKNOWN BACTERIUM

Streak to Starch Agar (SA) plates

Incubate at 33°C for 24 -48 h

Flood with Iodine (Lugol’s or Gram’s)

Observe
F. 2-Ketogluconate Production
UNKNOWN BACTERIUM

Inoculate to Hayne’s Medium (HM) tubes

Incubate at 33°C for 48 h

Add 1.0 mL of Benedict’s reagent to 1.0 mL of the liquid culture in a tube

Heat tube in boiling water for 10 min. and cool rapidly. Observe.
RESULTS AND DISCUSSION
A. Oxidation and/or Fermentation of Sugars
(Principle)
 Hugh and Leifson with different sugars (glucose,
lactose, mannitol, sucrose)
 Determine the oxidative or fermentative metabolism of
a carbohydrate or its non utilization
 Employs a semi-solid medium (agar is decreased to 2%
from 3%) in tubes containing the carbohydrate under
test and a pH indicator (bromothymol blue)
 One tube is overlaid with water agar to help achieve
anaerobic environment
A. Oxidation and/or Fermentation of
Sugars (Principle)
 Concentration of peptone ↓ from 11% to 2%
 Decreasing amount of alkaline product prod.by the
metabolism of peptone; thus reducing the neutralizing
effect of these products
 Carbohydrate concentration ↑ by 0.5% to 1.0%
 Inc. concentrations of glucose enhances prod. of weak acids
to a level that can be detected by bromothymol blue
 Dipotassium phosphate promotes fermentation and
acts as pH controlling buffer
 Agar concentration enables the determination of
motility and aids in distribution of acid throughout the
tube
A. Oxidation and/or Fermentation of
Sugars (Principle)

▪ Carbohydrate is added to the medium

▪ Degradation of carbohydrate to
acid=bromothymol blue changes from
green to yellow
▪ Degradation take place while the medium is
exposed to air (oxidative/fermentative) or
under exclusion of air (fermentative only)
A. Oxidation and/or Fermentation of
Sugars (Principle)

Aerogenic fermentation: with gas production (with gas

pockets/bubbles)
Anaerogenic fermentation: without gas production
 A. Oxidation and/or Fermentation of
Sugars

Glucose Glucose Lactose Lactose Mannitol Sucrose Sucrose

w/ WA w/out w/ WA w/out w/ WA w/ WA w/out
WA WA WA

Fig. 1. Aerogenic fermentation of E. coli.

 A. Oxidation and/or Fermentation of
Sugars
Glucose Glucose Lactose Sucrose
Lactose Mannitol Sucrose
w/out w/ WA w/out w/out
w/ WA w/ WA w/ WA
WA WA WA

Fig. 2. P. fluorescens on H & L medium.

UNKNOWN #11

UNKNOWN #4
B.1 Methyl Red (MR) Test (Principle)

 Media: MRVP media (Glucose Phosphate Broth)

 Reagent: Methyl Red Indicator
 All enteric bacteria catabolize glucose for their
energy needs; however, end products vary
depending on the enzyme pathways present in the
bacteria
 Used to distinguish between E. coli (a mixed acid
fermenter) and E. aerogenes (a butanediol
fermenter)
B.1 Methyl Red (MR) Test (Principle)
 Mixed acid fermenters – produce a mixture of
fermentation acids and thus acidify the medium
 Butanediol fermenters – form butanediol, acetoin, and
fewer organic acids; pH of the medium does not fall as
low as during mixed acid fermentation
 Methyl red – detects a pH change to the acid range as a
result of acidic end products (e.g. lactic, acetic, and
formic acids)
 Positive methyl red test – indicator turns red (pH of 4)
 Negative methyl red test – indicator turns yellow (pH
of 6)
 B.1 Methyl Red (MR) Test (Principle)

Fig. 3. Glucose fermentation reaction with methyl red pH reagent.

B.1 Methyl Red (MR) Test
UNKNOWN #4
B.2 Voges-Proskauer (VP) Test (Principle)

 Media: MRVP media (Glucose Phosphate Broth)(pH 6.9)

 Reagent:
A. Alpha-naphthol
B. 40% KOH

 Used to identify bacteria that ferment glucose

leading to 2,3-butanediol accumulation
 Addition of 40% KOH and a 5% sol’n of alpha-
naphthol in absolute ethanol (Barritt’s reagent →
detect acetoin
 acetoin – precursor in 2,3-butanediol synthesis
B.2 Voges-Proskauer (VP) Test (Principle)

 Barritt’s (VP) reagent + acetoin →

cherry-red color

▪ Positive VP test – development of a red

color (after 15 mins)
▪ Negative VP test – absence of red color
 B.2 Voges-Proskauer (VP) Test (Principle)

Fig. 4. Glucose fermentation by E. aerogenes.

Fig. 5. Acetyl methyl carbinol (acetoin) reaction with

Barritt’s (VP) reagent.
B.2 Voges-Proskauer (VP) Test
C. Citric Acid Utilization

 Media: Simmon’s Citrate Agar (SCA)

Sodium citrate – carbon source
NH4+ – nitrogen source
Bromothymol blue – pH indicator
▪ Used to determine ability of bacteria to
use citrate as a sole carbon source.
C. Citric Acid Utilization (Principle)
▪ Presence of citrate permease facilitates transport of
citrate into the bacterium; citrate is converted to
pyruvic acid and CO2
▪ When citrate is oxidized, CO2 is liberated from the
medium. CO2 combines with sodium (from sodium
citrate) and water to form sodium carbonate – an
alkaline product.

 Positive citrate test – raises the pH, turns pH indicator

to blue; growth in medium
 Negative citrate test – absence of a color change; no
growth in medium
 C. Citric Acid Utilization

Fig. 6. Enzymatic degradation of citrate

C. Citric Acid Utilization

S. marcescens E. coli UNKNOWN #11

Fig. 7. S.
marcescens (+)
showing positive
utilization of
citrate indicated by
a shift of color to
Prussian blue. E.
coli (-) and UK #11
showing negative
utilization of
citrate.
D. Malonate Utilization (Principle)

 Determines the ability of an organism to utilize

sodium malonate as its sole source of carbon,
ammonium sulfate as the sole nitrogen source, and
bromothymol blue as the pH indicator.
 Organisms which simultaneously utilize malonate
and ammonium sulfate produce sodium hydroxide
which thereby results in an alkaline reaction and
changes the indicator from its original green color
to light blue or Prussian blue
 This test is used to differentiate Enterobacter from
Escherichia spp.
A B

Figure 8: A. Pseudomonas fluorescens exhibiting a positive result

for the utilization of malonate, B. Escherichia coli exhibited no
change in color, indicating that it does not utilize malonate
E. Starch Hydrolysis (Principle)

 Starch is a large molecule that is consists of: (1) amylose, a

straight chain polymer glucose molecules and (2) amylopectin,
a larger branched polymer of glucose.
 Bacteria that hydrolyze starch produce amylases that degrade
the starch molecule into molecules of maltose, glucose, and
dextrins.

 Gram’s Iodine is the one that will indicate the hydrolytic

activity.
 • If a clear zone is observed it
indicates the presence of α-
amylase and the hydrolysis of
starch

• Blue black coloration indicates

the absence of hydrolytic
enzyme

Figure 9: Unknown bacteria exhibiting

zone of clearing after dropping of gram’s
iodine. Indicating the presence of α-
amylase and the hydrolysis of starch
F. 2-Ketogluconate Production (Principle)

 Used in testing the capacity of an organism to oxidize

gluconate to 2-ketogluconate. The basis of the test is
the change from gluconate (a non-reducing
compound) to 2-ketogluconate (a reducing compound)
when tested with a Benedict’s reagent.

 2-ketogluconate reduces CuSO4 in the Benedict’s

reagent to an insoluble cuprous oxide, when heated.
This cuprous oxide are precipitated out as yellow to
orange-orange red precipitates.
 Figure 10: The change of color from blue-green to
yellow of this unknown bacteria indicates the reduction
of copper sulfate to cuprous oxide with 2-
ketogluconate.
ACTIVITY QUESTIONS
Why are some microorganisms capable of
utilizing certain carbohydrates and some are
not?

The ability of microorganisms to utilize certain

carbohydrate depends on the presence of an
intracellular transport system which allows them to
take up the carbohydrate and secondly is the
presence of the enzyme that will break down the
carbohydrate into a molecule that could be
metabolized to produce energy.
What is the importance of adding vaspar or
water agar to one of the inoculated tube of
Hugh and Liefson medium?

 Adding vaspar/2% water agar to one of the

inoculated tube of H and L medium will serve as a
barrier to oxygen thus helping achieve an
anaerobic environment.
Explain the principle of the MRVP test.

 Methyl Red (MR) Test

 Some bacteria utilize glucose to form large amounts of acid (e.g. acetic,
formic, succinic, etc.) with the result that the pH value of the medium falls
to below 4.4. Other species produce less acid so that the pH is not as great.
This difference can be visualized by using methyl red (as pH indicator)
which is yellow above pH 5.1 and red at pH 4.4 or below.
 Voges-Proskauer (VP) Test
 Many microorganisms metabolize glucose to produce acetoin (acetyl
methyl carbinol), 2,3-butanediol or diacetyl. Oxygen, KOH, and alpha-
naphthol (Barritt’s reagent) react with acetoin to produce diacetyl, which
in turn reacts with creatine or free NH2 of guanidine moiety to form a red
complex seen in the interface of the medium. Oxygen is required for the
formation of diacetyl such that the tube must be shaken vigorously.
Does a bacterium use the same metabolic
pathway for utilization of carbohydrate (like
glucose) and organic acids? Explain your
answer.
 The metabolic pathway for the utilization of
carbohydrate is the same with the pathway it
takes to produce an organic acid. The utilization
of carbohydrates lead to the production of
organic acid. Such example is the utilization of
glucose or lactose to produce lactic acid (Unden
and Zaunmüller, 2009).
Where will a bacterium grow best: glucose
malonate or gluconate? Explain your answer.

 The preferred carbon source for E. coli, as for many

other bacteria, is glucose, supporting faster growth
rate compared to other sugars.
 The best known example of preferential glucose
utilization comes from the work of Monod on the
glucose-lactose diauxic shift: E. coli first grows
rapidly on glucose, and when glucose runs out shifts
to grow more slowly on lactose or other sugars (Bren
et al., 2016).
References

 Benson, Harold J.Brown, Alfred E. (2015) Benson's Microbiological

Applications: Laboratory Manual in General Microbiology, Short Version,
Thirteenth Edition. New York: McGraw-Hill Higher Education.
 Bren A, Park JO, Towbin BD, Dekel E, Rabinowitz JD and Alon U. (2016).
Glucose becomes one of the worst carbon sources for E.coli on poor nitrogen
sources due to suboptimal levels of cAMP. Scientific reports, 6, 24834.
 Cappuccino JG and Welsh CT. (2017). Microbiology: A Laboratory Manual,
Global Edition, Eleventh Edition. USA: Pearson Education Limited.
 https://www.bio-kult.com/research/36-lactase-activity-of-live-bacteria
 Unden G and Zaunmüller T. (2009) Metabolism of Sugars and Organic Acids by
Lactic Acid Bacteria from Wine and Must. In: König H., Unden G., Fröhlich J.
(eds) Biology of Microorganisms on Grapes, in Must and in Wine. Springer,
Berlin, Heidelberg