IN THE NAME OF GOD Islamic Azad University Falavarjan Branch Falavarjan Branch School of Biological Sciences Department of Microbiology

MICROBIAL NUTRITION By: Keivan Beheshti Maal

Metabolism Metabolism – coordination of different chemical reactions into specific structures –energy releasing – catabolic rxn – catabolism –energy requiring – anabolic rxn – anabolism We will concentrate on chemoorganotrophs = organic compounds for carbon and energy

Nutrition Nutrition – process in which nutrients acquired from environment and used for metabolism and growth

5 Chemical composition of cytoplasm 70% water proteins 96% of cell is composed of 6 elements –Carbon –Hydrogen –Oxygen –Phosphorous –Sulfur

6 Nitrogen Main reservoir is nitrogen gas (N 2 ) 79% of earth’s atmosphere is N 2 Nitrogen is part of the structure of proteins, DNA, RNA & ATP – these are the primary source of N for heterotrophs Some bacteria & algae use inorganic N nutrients (NO 3 -, NO 2 -, or NH 3 ) Some bacteria can fix N 2 Regardless of how N enters the cell, it must be converted to NH 3, the only form that can be combined with carbon to synthesis amino acids, etc.

7 Oxygen major component of carbohydrates, lipids and proteins plays an important role in structural & enzymatic functions of cell component of inorganic salts (sulfates, phosphates, nitrates) & water O 2 makes up 20% of atmosphere essential to metabolism of many organisms

8 Hydrogen major element in all organic compounds & several inorganic ones (water, salts & gases) gases are produced & used by microbes roles of hydrogen –maintaining pH –forming H bonds between molecules –serving as the source of free energy in oxidation- reduction reactions of respiration

9 Phosphorous main inorganic source is phosphate (PO 4 -3 ) derived from phosphoric acid (H 3 PO 4 ) found in rocks & oceanic mineral deposits key component of nucleic acids, essential to genetics serves in energy transfers (ATP)

10 Sulfur widely distributed in environment, rocks, sediments contain sulfate, sulfides, hydrogen sulfide gas and sulfur essential component of some vitamins and the amino acids: methionine & cysteine contributes to stability of proteins by forming disulfide bonds

Common Nutrient Requirements macroelements (macronutrients) –C, O, H, N, S, P, K, Ca, Mg, and Fe –~10 –required in relatively large amounts micronutrients (trace elements) –Mn, Zn, Co, Mo, Ni, and Cu –trace amounts –often supplied in water or in media components

Requirements for Carbon, Hydrogen, and Oxygen often satisfied together –carbon source - provides H, O and electrons autotrophs –carbon dioxide - sole or principal carbon source –Electrons from other sources heterotrophs –organic molecules - carbon and energy sources

Nutritional Types of Microorganisms

Organic nutrient – C and H molecules –Living matter –Proteins, carbohydrates, lipids, nucleic acids Inorganic nutrient – combination of atoms other than C and H –Non-living matter mixotrophy –chemical energy source (inorganic) –inorganic H/e- donor –organic carbon source

Autotrophs – obtain carbon from inorganic CO 2 –Chemoautotroph: CO 2 = carbon source Inorganic compounds = energy source Nitrifiers oxidise ammonia or nitrite to nitrate. Other bacteria oxidise sulfur, hydrogen gas, etc. –Photoautotroph: CO 2 = carbon source Light = energy source Cyanobacteria, purple and green sulfur bacteria

Heterotroph – require organic compounds as source of carbon –Chemoheterotroph: Organic compounds = carbon and energy source decomposers and most pathogens –Photoheterotroph: Organic compounds = carbon source light = energy source purple and green non-sulfur bacteria.

based on energy source –phototrophs use light –chemotrophs obtain energy from oxidation of chemical compounds based on electron source –lithotrophs use reduced inorganic substances –organotrophs obtain electrons from organic compounds

Four Basic Groups of Organisms Figure 6.1

Requirements for Nitrogen, Phosphorus, and Sulfur needed for synthesis of important molecules (e.g., amino acids, nucleic acids) Nitrogen: supplied in numerous ways –organic molecules –Ammonia (NH 3 ) –nitrate via assimilatory nitrate reduction (NO 3 ) –nitrogen gas via nitrogen fixation (N 2 )

Anabaena sp. Heterocysts -Heterocysts - Special cells - filamentous cyanobacteria - fixing atmospheric nitrogen

Sulfur: usually supplied as sulfate via assimilatory sulfate reduction –H 2 S, FeS, SO 4 2- –Sulfur granules Phosphorus: usually supplied as inorganic phosphate –phosphate - main inorganic source –Limiting nutrient –Stockpile in polyphosphate granules

Growth Factors organic compounds essential cell components (or their precursors) that the cell cannot synthesize must be supplied by environment if cell is to survive and reproduce industrial production of growth factors by microorganisms

Classes of growth factors amino acids –needed for protein synthesis purines and pyrimidines –needed for nucleic acid synthesis vitamins –function as enzyme cofactors

Table 5.3

Uptake of Nutrients by the Cell Some nutrients enter by passive diffusion Most nutrients enter by: –facilitated diffusion –active transport –group translocation

Passive Diffusion Molecules (solutes) move from region of higher concentration to one of lower concentration because of random thermal agitation Concentration gradient No energy used H 2 O, O 2 and CO 2 transport Osmosis – movement of solvents!!

Facilitated Diffusion similar to passive diffusion –movement of molecules is not energy dependent –direction of movement is from high concentration to low concentration –size of concentration gradient impacts rate of uptake

Figure 5.1 rate of facilitated diffusion increases more rapidly and at a lower concentration diffusion rate reaches a plateau when carrier becomes saturated carrier saturation effect

Figure 5.2 note conformational change of carrier

Facilitated diffusion… differs from passive diffusion –uses carrier proteins (permeases) –Undergoes conformation change when bound to substrate –Returns to original shape after transport –specificity –smaller concentration gradient required for uptake of molecules –transports glycerol, sugars, and amino acids –competition

Active Transport energy-dependent process –ATP or proton motive force used moves molecules against the gradient – “uphill” concentrates molecules inside cell involves carrier proteins (permeases) or pumps –carrier saturation effect is observed

ABC transporters ATP-binding cassette transporters observed in bacteria, archaea, and eucaryotes Figure 5.5

Figure 5.6 antiport symport

Group Translocation molecules are modified as they are transported across the membrane energy-dependent process PTS system- phosphoenolpyruv ate: sugar phosphotransferas e system Figure 5.7

Iron Uptake ferric iron (Fe 3+ ) is very insoluble so uptake is difficult microorganisms use siderophores to aid uptake siderophore complexes with ferric ion complex is then transported into cell Figure 5.8

growth factors: organic compounds required in small amounts not every growth factor is required by all cells

STUDYING MICROORGANISMS 5 Is – inoculation, incubation, isolation, inspection and identification Inoculation: –Grow or culture from different specimens –Microorganism introduced to nutrient medium (pl. media) –Aseptic techniques

Isolation: –Individual cell separated from other cells – grows on agar as colony pure culture –population of cells arising from a single cell –3 major techniques – streak plate; pour plate; spread plate

CO 5

Fig. 5.11

The Spread Plate and Streak Plate mixture of cells spread on an agar surface - individual cells are well separated from each other each cell reproduces to form a separate colony (visible growth or cluster of microorganisms)

Streak plate technique

Figure 5.8 inoculating loop

Spread-plate technique

Figure dispense cells onto medium in petri dish sterilize spreader 4. spread cells across surface

Laboratory Culturing Trying to get a pure culture of a single organism Unwanted organisms are called contaminants – try to avoid them –more than one colony type may indicate contamination Use solid medium to isolate bacteria rather than liquid –can tell that colonies are separate –differentiate based on size, color and shape

Different Colony Morphology

Aseptic Technique - Broth

Aseptic Technique - Plate

The Pour Plate sample is diluted several times diluted samples are mixed with liquid agar mixture of cells and agar are poured into sterile culture dishes

Figure 5.9

Culture Media preparations - support the growth (reproduction) of microorganisms 3 properties – physical state; chemical composition; functional type Physical state: –Liquid, semi-solid or solid –Liquid - broth –solid - solidified with agar (1 - 5%) –Semi-solid – ( %) – motility tests

Agar – algal sulfated polysaccharide –Solid at room temperature –Melts at 100 °C –Liquid at 45 – 50 °C –Solidifies at 42 °C –Bacteria cannot break down agar – can use gelatin as nutrient source Cell cultures, host animals

Chemical composition: Synthetic or Defined Media all components and their concentrations are known Pure organic and inorganic compounds

Complex Media contain some ingredients of unknown composition and/or concentration Blood, meat extracts, yeast, peptone Rich nutrient source

Functional type: General purpose media –Supports growth of many microorganisms –e.g., tryptic soy agar Enriched media –general purpose media supplemented by blood or growth factors –e.g., blood agar –Fastidious bacteria

Selective media –favour the growth of some microorganisms and inhibit growth of others –Dyes, antibiotics, bile salts –Suppress growth of Gram-positive organisms –e.g., MacConkey agar selects for gram-negative bacteria

Differential media –distinguish between different groups of microorganisms based on their biological characteristics –Dyes (ph indicators), specific nutrients –Colony size, colour, media colour changes, formation of gas bubbles or preciptates –e.g., Blood agar haemolytic versus nonhaemolytic bacteria –e.g., MacConkey agar – lactose + neutral red lactose fermenters versus nonfermenters

Table 5.7

Incubation – –Optimal temperature, gaseous conditions – visible colonies –Days to weeks Inspection – –Macroscopic colony characteristics –individual species form characteristic colonies –Microscopic analysis

Figure 5.10a

Colony growth most rapid at edge of colony –oxygen and nutrients are more available at edge slowest at center of colony in nature, many microorganisms form biofilms on surfaces

Identification: –correlate morphological, physiological, and genetic traits –Species or strain