Microbes as test organisms, sensor and tool for future applications of energy production in food and non food industrial products.

1. RAMYA M
M.Tech Food Technology
CFDT, TANUVAS, Chennai - 52.
ramya2798@gmail.com
Microbes as test organisms,
sensor and tool for energy
production

2. Introduction
• Microbes- singlecellular organisms or multicellular- small size
• Found everywhere in natural environment (water, soil, air etc) -
present in foods or on the surface of foods.
• Foods contaminated with microorganisms- bacteria, yeasts, molds or
viruses may pose a risk to the consumer.

3. Microbes
As a test organism
Types of indicator
and detection
methods
As a sensor Biosensor
As a tool
Non-food –
microbial fuel cell
(mediator &
mediator less)
Food - bio
processing,
bio-preservation,
probiotics

4. Microbes as a test organism
• In food industry, it can be distinguished between different
categories of microbiological contamination:
1. Indicator organisms
2. Spoilage organisms
3. Pathogenic organisms
4. Viruses and Bacterial toxins
• Pathogenic microorganisms - either not allowed at all or limited
to a specified number of cells per gram food.

5. • Microbiological tests - cleaning and hygienic control - detect general residues of
foods on insufficient cleaned surfaces.
• Compact dry plates-cultivation of microorganisms on standard nutrient media.
• The test principle is based on the specific chromogenic substrates - converted into
colored products by the metabolism of microorganisms.
• It is also suited for swab samples from surfaces.
• Residues of foods on surfaces - protein tests or ATP measurements.
• Common test formats for microbial food testing - ELISA assays, real-time PCR
tests, nutrient plates and agar plates.

6. Types of indicator
• Total coliforms
• Fecal coliforms
• Fecal Streptococci
• Anaerobic bacteria
• Bacteriophage

7. Indicators of food safety
• A food safety indicator should meet certain important criteria. It should be
1. Be easily and rapidly detectable
2. Be easily distinguishable from other members of the food biota
3. Have a history of constant association with the pathogen whose presence it
is to indicate
4. Always be present when the pathogen of concern is present
5. Be absent for foods that are free of the pathogen.
• Sanitary indicators - to detect fecal contamination of waters.
• The first fecal indicator was Escherichia coli.

8. Coliform
• Coliforms are Gram-negative asporogeneous rods that ferment
lactose within 48hours.
• Coliforms are represented by four genera of the family
Enterobacteriaceae: Citrobacter, Enterobacter, Escherichia,and
Klebsiella.
• E. coli is more indicative of fecal pollution - it could be isolated
and identified more readily.

9. IMViC test
• I = indole production
• M = methyl red reaction
• V = Voges–Proskauer reaction
(production of acetoin)
• C = citrate utilization.
I M V C
E. coli + + − −
E. aerogenes − − + +

10. • Fecal coliforms - the production of acid and gas in EC broth
between 44◦C and 46◦C, usually 44.5◦C or 45.5◦C
• Coliforms - growth in the presence of bile salts, which inhibit the
growth of Gram+ bacteria - have the capacity to ferment lactose
with the production of gas.
• Limitation (not as a sanitary indicator) – poultry (Salmonellae),
frozen blanched vegetable (Enterobacter)

11. Enterococci
• Enterococci - more fastidious in their nutritional requirements.
• They are microaerophiles - grow well in low oxidation - reduction
potential (Eh).
• Coliforms were more efficient indicators of sanitation than
enterococci prior to freezing, whereas enterococci were superior
indicators after freezing and storage.

12. Detection methods
Most Probable Number(MPN)
• Used to detect coliforms
• This test consists of three steps:
• Presumptive test
• Confirming test
• Completed test
Presumptive test:
• Dilute water sample and Inoculate 3 or 5 tubes of lauryl sulfate-tryptose-lactose broth
containing upside-down Durham tubes with water dilutions
• Incubate at 35°C for 48 hours
• Determine number of tubes at each dilution that are positive for gas production (contain
bubble in Durham tube)
- gas production
+ gas production

13. 2. Confirming test – select a positive tube and inoculate a Levines EMB agar and
Endo Agar plate
3. Completed test – inoculate a colony back into MPN media and confirm acid and
gas production.

14. Membrane filter method
• Filter 100 mL water through a 0.45 m filter
• Incubate filter on pad soaked with a differential medium
(Endo medium; contains lactose and Basic Fuchsin dye)
at 35°C for 18-24 hours
• Count colonies that grow on filter
– coliforms will be dark red with metallic gold sheen
• To enumerate Fecal Streptococci, grow on Streptococcus agar at 37°C for 24 hours.
Fecal streptococci reduce 2,4,5-triphenyltetrazolium chloride to formazan, which
makes colonies appear red

15. Heterotrophic Plate Counts
(HPC)
• Enumeration of all aerobic and facultative anaerobic chemoheterotrophs in water
– includes Pseudomonas, Aeromonas, Klebsiella, Flavobacterium, Enterobacter,
Citrobacter, Acinetobacter, Proteus.
• Varies from 1 to 104 CFU/mL, and depends on temperature, residual chlorine
concentration, and availability of organic nutrients
• Indicates general quality of water (particularly levels of organic matter in water)
• HPC > 500 CFU/mL indicates poor water quality

16. Microbes as sensor
• Biosensor - analytical device - biological sensing element with
a transducer - signal proportional to the analyte concentration.
• Biological sensing elements - enzymes, antibodies, receptors,
organelles and microorganisms as well as animal and plant
cells or tissues.
• Transducers - Amperometric, potentiometric, calorimetric,
conductimetric, colorimetric, luminescence and fluorescence.

17. Mechanism of biosensor
SIGNAL
• Proton Concentration, Light Emission, Absorption.
TRANSD
UCER
• Biological Signal To Current Through Electrochemical/Optical
System
SIGNAL
• Amplified, Processed, Stored

18. Microorganisms as
biosensing elements
• Enzymes - widely used biological sensing element -
tedious, time-consuming and costly enzyme
purification.
• The microorganisms - improve the activity of an
existing enzyme.

19. Immobilization of microbes
• Microorganisms can be immobilized on transducer by
chemical or physical methods
• Chemical methods - covalent bonding and cross-
linking.
• Physical methods - Adsorption and entrapment.

20. Biosensor
Electrochemical
Amperometric
Potentiometric
Conductometric
Optical
Bioiluminence
Flourescence

21. Electrochemical Biosensor
Amperometric
biosensor
• current generated
by the oxidation or
reduction reaction
at the surface of
the electrode
• determination of
BOD
• Pseudomonas
putida, Bacillus
subtilis,
Thermophilic
bacteria and yeast.
Potentiometric
biosensor
• change in potential
from ion
accumulation or
depletion
• ionselective
electrodes (ph,
ammonia, carbon
dioxide) and gas
sensing electrode
coated with
immobilized
microbe layer.
Conduct metric
biosensor
• change in the ionic
species
• a net change in the
conductivity of the
reaction solution.
• Eg - urea
biosensor.

22. Optical Biosensor
Bioluminescence biosensor
• emission of light by living
microorganisms
• Quantitative detection of
components their intensity
• pollutant and toxicity test
Fluorescence biosensor
• emission intensity is
directly proportional to the
concentration.
UV absorption, bio- and chemi-luminescence,
reflectance and fluorescence by the interaction of the
biocatalyst with the target analyte.

23. Application
• Microorganisms - low cost, long lifetime, wide range
of suitable pH and temperature - as the biosensing
element.
• Microbial biosensors - the application in extreme
conditions, such as highly acidic, alkaline, saline,
extreme temperature and organic solvent environment.

24. Microbes as a tool (non-food)
Microbial fuel cell (MFC) - bio-electrochemical system - by using bacteria and
mimicking bacterial interactions found in nature.
• Mediator microbial fuel cell - electrochemically inactive - the electron transfer from
microbial cells to the electrode by mediators (thionine, methyl viologen, methyl
blue, humic acid, and neutral red) - expensive and toxic.
• Mediator-free microbial fuel cell - do not require a mediator - use electrochemically
active bacteria to transfer electrons to the electrode (electrons are carried directly
from the bacterial respiratory enzyme to the electrode) - shewanella putrefaciens,
aeromonas hydrophila - some via their pili.

25. Microbial fuel cell
• Anode and cathode separated by cathode specific membrane.
• Microbes at anode oxidize organic fuel generates electrons
and protons.
• Protons move to the cathode compartment through the
membrane.
• Electrons transferred to the cathode compartment through
external circuit to generate current
• Electrons and protons are consumed in cathode chamber,
combining with O2 to form water.
• Anodic reaction:
CH3COO- + H2O → 2CO2 + 2H+ +8e- acetate
• Cathodic reaction:
O2 + 4e- + 4 H+ → 2 H2O.

26. • Microbes - Metal reducing bacteria, Enterococcus
faecium, Pseudomonas aeruginosa, Proteobacteria,
Clostridium butricum, Bacteroides and Aeromonas
species, nitrogen fixing bacteria (e.g. Azospirillum)
• Application - Waste water treatment, Power
generation, Secondary fuel production, Bio-Sensors,
Desalination

27. Microbial electrolysis cell
• MFCS produce electric current by the bacterial
decomposition of organic compounds in water
• MECS partially reverse the process to generate
hydrogen or methane by applying voltage to bacteria
by the microbial decomposition of organics leading to
the production of methane.

28. Microbes as a tool (Food)
Food bioprocessing
• Microorganism are
used to produce
fermented food using
plant and animal
sources (starter
culture).
• Starter culture - A
concentrated
preparation of live
cells which initiates
fermentation.
Food bio-
preservation
• Use anti-microbial
metabolites (lactic
acid, acetic acid,
hydrogen peroxide
and peptide
bacteriocins)
• To control
pathogenic and
spoilage micro
organism and inhibit
the growth of
potentially harmful
microrganism
Probiotics
• A concentrated
supplement of
beneficial live
bacteria
• To improve our
health by promoting
body’s immunity and
improving digestive
system.

29. Enzymes in food waste
treatment
• Food industries generate large volumes of both solid and liquid
wastes. Waste disposal methods - physical, chemical, and some
biological methods (anaerobic digestion).
• Enzymes - to reduce wastes and convert the wastes to value-
added products.
• In food waste treatments - polysaccharidases (cellulase,
pectinase, hemicellulase, chitinase, and amylase) lactase and
proteinases

30. • Treatment of fruits - cellulase and pectinase - increased juice
yield and improved separation of solids from the juice - animal
feed.
• Chitinases - to depolymerize the shells of shellfish - to produce
SCPs.
• Amylases to treat starch-containing wastewater to produce
glucose syrup - alcohol production by yeasts.
• Proteases - to treat wastewater from fish and meat-processing
operations - fish food.

31. Microbial genomics
• a new tool to increase food quality and safety
• offer a new alternative to the classic approach.
• quick identification of microorganisms present in the product
• help to directly measure the total response of the target
spoilage microorganisms to the applied preservation methods.
• to reduce the number of experiments needed to measure all
relevant responses.

32. • Small chips - the information of thousands of genes of food
spoilage micro-organisms - predict the preservation treatment -
gives additional preservation steps if necessary.
• As a result, process control could be improved.
• Improved sensory properties - energy savings - product losses.

33. Conclusion
• Microorganism are not only produce negative effects – spoilage &
harmful disease.
• Microorganism are used in synthesis of industrial chemical products
(vitamins, organic acids, enzymes, alcohol)
• Microbes are used in producing products like vinegar, cheese, yogurt,
bread, buttermilk.
• Enzymes from microbes can be manipulated to cause the microbes to
produce substances like cellulase and insulin (which can’t be
synthesized)

34. Thank You….