Microbial physiology

Microbial physiology 楊倍昌 Colonies, Turbid suspension, Biofilm

略 Where to find microbe? Everywhere! Cell Phone Bacteria: http://www.youtube.com/watch?v=4lmwbBzClAc

Learning Objectives After reading this section, students will be able to... • Explain how to make microbes accountable. • Describe the pattern and requirement for bacterial growth. • Explain growth methods used to synchronize cells. • Describe how microbes do catabolism to get energy and metabolism to build structure.

略 Microbial physiology All before doing anything: If you do not separate an individual from a mix population, you can not really known who is it, not to say how it works. By making photomicrographs, I can reveal the bacteria true to nature and free of subjective misinterpretation ---- Robert Koch

Counting the viable cells: Dilution (兩種物理特性) 科學的第一步在於數字化: 定量

Finding a solid medium What can be a replacement? Agar's first use discovered by Frau Franny Eilshemius (a physician's wife) whose husband told Robert Koch, who is credited with the discovery! Dr. Walther and Fanny Hess Agar is derived from Gracilaria (Gelidium species) a bright red sea vegetable with the botanical name of Gleidium purpurascens. Agar due to its high gelling properties is considered the queen of gelling agents.

Growth measurement • Cell count: microscopic observation; flow cytometer (direct) • Colony formation: Measure the living cell (direct) • Biomass determination: dry weight; essential cell component (indirect) • Turbidity (indirect)

Doubling time 1, 2, 4, 8, 16, 32, 64, 128, 256, …. • Escherichia coli:12.5 min • Vibrio cholerae : 13 min (can kill a man within 12 h) • Mycobacterium tuberculosis : 24 h (develop symptom after months)

Growth and survival • Lag phase: adaptation to the environment • Exponential logarithmic growth: machine in full rum • Stationary phase: nutrition exhausted, toxin increased • Decline: cell die (steady biomass) or lysis (decrease biomass) • Dormant as spore, non-viable state

略 in 37oC, pH 5.1 ; in 45oC, pH 6.2 In bioreactors

Factors affecting Growth • The orderly increase in the sum of all the components of an organism Affected by: • Nutrients • pH: neutrophils, acidophils, alkalophis • Temperature: psychrophils; mesophils; themophils • Aeration • Pressure • Ionic strength and osmotic pressure: halophils, osmophils

The ideal temperature range for maximum reproduction rate of most bacteria is between 80° F (27oC) and 105°F (40.5oC). The ideal pH range for maximum bacterial growth of most strains is 6.5 to 8.5.

Thermophiles 70o-110o Mesophiles 10o-50o

Thermophils • visible in hot springs when single-celled photosynthetic bacteria (cyanobacteria) form dense layers of biomass called bacterial mats. • contained "environmentally friendly" enzymes that were stable under high temperatures and could be used in place of more dangerous chemicals that have been concocted by modern industry. • Bacteria and Archaea live in hot springs heated by geothermal reactions deep in the Earth. Some springs reach temperatures of 80 C (177F). Taq polymerase Distinct membrane

There is no O2 in the earth in ancient times. What is the advantage of growth in O2-containing environment? Aerobic respiration produce free radicals. To detoxify oxygen, you need: Catalase: H2O2 --- H20 and 02 Superoxide dismutase (SOD): oxygen radical --- H20 and O2

Growth pattern • Obligate aerobe • Obligate anaerobe • Microaerophile • Aerotolerant anaerobe • Facultative anaerobe/aerobe

略 Air requirement:O2 • Aerobe: A microorganism whose growth requires the presence of air or free oxygen • Anaerobe: A microorganism that grows only or best in the absence of free oxygen. Organisms utilize bound oxygen • Microaerophile: A microorganism that grows best in the presence of low concentrations of oxygen • Facultative anaerobe/aerobe: A microbe that adjusts its metabolism to depending on the oxygen concentration in which it is growing • Aerotolerant anaerobe: an organism that always grows in an anaerobic mode -- it ignors the presence of oxygen. • Capneic microbe: An organism that requires 3 to 10% CO2 for growth

Create an environment to grow bacteria Capneic microbe: An organism that requires 3% to 10% CO2 for growth Obligate anaerobes. These will not grow in presence of O2 Some find oxygen very toxic, even at short exposures. Example: Bacteroides spp. Q: How they grow in nature?

略 Growth/Culture condition Plating technique of Robert Koch for single colony isolation Microbiol. Mol. Biol. Rev. 64:847-867 (2000) The majority of microbes persist attached to surfaces within a structured biofilm ecosystem and not as free floating organisms.

Differences in culture: when cells grow as biofilm 1. On metabolism 2. Drug resistance

Symbioses Termite, ruminant digestion Sewage treatment bioreactors Water pipes Dental units Contact lens cases Dental plaque Endocarditis Cystic Fibrosis Otitis media Urinary catheter Implants 略 Two types of biofilms Environmental Disease-associated

略 Technique: continuous cultivation

Synchronization What for? • Starvation • Temperature shock • Refresh from stationary phase • Filter binding/release

Viable But Non-culturable (VBNC) Bacteria Bacteria in the VBNC state fail to grow on the routine bacteriological media on which they would normally grow and develop into colonies, but are alive and capable of renewed metabolic activity (Oliver, 2000b).

http://nihroadmap.nih.gov/hmp/ This initiative will begin with the sequencing of up to 600 genomes from both cultured and uncultured bacteria, plus several non-bacterial microbes. Combined with existing and other currently planned efforts, the total reference collection should reach 1000 genomes.

Run a store for life: 6 major tasksPlease refer to biochemistry course • Nutrition uptake • Metabolism: synthesis and catabolism • Energy generation • Discard garbage/toxin • Reproduction • Adaptation

Metabolism: key of physiologya very short summary • Bacteria feed by absorption through their cell membranes • In the first stage, they secrete enzymes (extra-cellular) which break down the large particulates and solids. • In the second stage, the reduced particles are absorbed through the membrane where cellular enzymes break down the extra cellular enzymes. • This process will produce CO2 and H2O and seed bacteria since cell division occurs when sufficient food is processed.

Energy and building block generation

Glycolysis • Two molecules of ATP are use to phosphorylate glucose and start glycolysis. • The phosphorylated molecule is then broken down in a series of reactions into two three carbon molecules (lysis).

Pentose phosphate shunt • When reducing power are needed • Sensitive to the level of NADP+ • Did not generate ATP

Metabolism & Bacterial Identification Some microbes can metabolize certain molecules while others can’t. When molecules are metabolized, specific waste products are created such as acids. Those waste products can be labeled by color.

http://www.genome.jp/kegg/pathway.html 這個網頁中有很完善的整理資料 (KEGG PATHWAY Database) Current knowledge on molecular interaction networks,including metabolic pathways, regulatory pathways,and molecular complexes • 你如果要把所有的生理代謝路徑背起來, 保證你一整年都還背不完全。

Life is a kind of chemical reactionSwedish chemist Arrhenius discovered how temperature affects the rate of chemical reaction and it can apply to cell growth Arrhenius plot of growth rate of E. coli. Individual data points are marked with corresponding degrees Celsius. (Herenden et al 1979)

Key messages • Cell counting is not always as obvious. There are 4 surrogate ways: Cell counting (direct), Colony formation (direct) , Biomass determination (indirect), Turbidity (indirect) • Environmental factors affecting growth include: Nutrients, pH, Temperature, Aeration, Pressure, Ionic strength and osmotic pressure. • Cell growth can be divided into 4 phases: Lag phase (adaptation to the environment), Exponential logarithmic growth (machine in full run), Stationary phase (nutrition exhausted, toxin increased), Decline (cell or lysis) • Microbes are useful tools in research because of their rapid life cycle, their simple growth requirements, and their small size. Due to this simplicity, microbes have been essential in understanding core questions in biology.

Microbial physiology