TEKNIK BIOPROSES

Pertemuan II

REVIEW : DASAR-DASAR BIOLOGI DAN BIOKIMIA

Kinerja Bioproses

ditentukan oleh :• daya guna (efiseinsi) • tingkat keterulangan

(reproduksibilitasnya)

Prinsip Rekayasa Biproses:

• Identifikasi Produk, Substrat dan produk antara

• Stoikiometri Proses• Kinetika Laju Bioproses• Rancangan Bioreaktor

a. Identifikasi Produk, Substrat dan produk antara

• Tujuan memaksimalkan rendemen (produk)• Substrat adalah sumber karbon terasimilasi:

hasil pertanian (pati, sukrosa, laktosa, whey…)petrokimia (metanol, etanol, asam asetat, ….)

• Faktor : kendala teknik (rendemen, pemurnian, kosentrasi)kendala ekonomi (harga, tingkat keamanan)

b. Stoikiometri Proses

• prinsip kekekalan masa…..• Rasio antara substrat (sumber karbon) yang

digunakan, dengan produk atau hasil antara yang diperoleh

c. Kinetika Laju Bioproses• Reaksi bioproses sangat tergantung pada waktu

(skala waktu)• Perubahan substrat dan pembentukan

produk/produk antara melibatkan waktu proses• Kinetika bioproses harus dipahami untuk

memaksimalkan produksi dan kosentrasi produk• Berhubungan dengan purifikasi dan pemisahan

metabolik

d. Rancangan Bioreaktor

• Skala laboratorium (tahapan seleksi mikroba)• Skala pilot plant (menerapkan kondisi

optimum/ optimation process)• Skala industri (pertimbangan ekonomi dan

finansial)

Kinetika Pertumbuhan Mikroba• Fase awal (lag) …masa penyesuaian dengan substrat

(belum terjadi penambahan sel mikroba) X =Xo• Fase Penyesuaian , mulai terjadi reproduksi seluler

tapi belum maksimal• Fase Eksponensial/Logaritmik, laju pertumbuhan

mencapai titik maksimum• Fase Perlambatan, substrat mulai habis dan

penumpukan penghambat/inhibitor• Fase Stasioner, mulai berkurang jumlah sel akibat

kematian• Fase Kematian/penurunan, jumlah sel makin

menurun tidak adalagi substrat

An Overview of Biological Basic

• Microbial Diversity– Temperature to grow

• < 20 oC : Psychrophilic• 20 – 50 oC : Mesophilic• > 50 oC : Thermophilic

– pH– Oxygen

• Aerob• Anaerob• Anaerob facultative

– Nutrients• Some cyanobacteria (formerly called as blue green

algae) can grow on environment with only a little moisture and a few dissolved minerals

– Photosynthetic and convert C02 from atmosphere into organic compound necessarily for life.

– Convert N2 into NH3 for use in making the essential building block of life.

• Size and Shape– Spherical, cylindrical, ellipsoidal, spiral and pleomorphic.

• Cell Nutrient– All organism except viruses contain :

• Water (+ 80 %)• Dry weight (+ 20 %)

– 50 % of Dry weight = protein– 10-20% of dry weight = nucleic acid

– The intracellular composition of cell varies depending on the type and age of the cell, and the composition of the nutrient media

– Nutrients• Macro nutrients

– Needed in concentration larger than 10-4 M– Those are : C, N, O, H, S, P, Mg2+ and K+.

• Micro nutrients– Needed in concentration less than 10-4 M.– Those are: Mo2+, Cu2+, Fe2+, Ca2+, Na+, Vitamins, growth

hormones and metabolic precursors.

• Macro Nutrients– Carbon

• Major source of cellular carbon and energy.• Microorganism classified into categories based on the

source of C:– Heterotrophs : Carbohydrate, fat, and hydrocarbon.– Autotrophs: CO2– Mixotrophs– Others

» Chemoauatotrophs: CO2, inorganic energy sources» Photoautotrophs: CO2 and Light.

– Nitrogen• Ammonia, ammonium salts, protein, peptide, amino

acids and urea.• Nitrogen→ Cell Mass (protein and nucleic acid).

– Oxygen• Introduced into the growth media by spraying air or by

surface aeration.– Hydrogen

• Source : Carbohydrate• Some bacteria such as methanogens can utilize

Hydrogen as an energy source.

– Phosphorous• Source : Inorganic phosphate salts (KH2PO4 and

K2HPO4), organic phosphate source (Glyceride phosphate).

– Sulfur• Source: sulphate salt (NH4)2SO4, sulfur containing

amino acid• Certain autotrophs utilize S2- and So as energy source.

– Potasium• Source : K2HPO4, KH2PO4 and K3PO4

– Magnesium• Source : MgSO4.7H2O.; MgCl2.

• Micronutrients– Lack of essential trace elements

• Increase the lag phase ( the time from inoculation to active cell replication in batch culture).

• May decrease the specific growth rate and the yield.

– Categories• Most widely needed trace elements: Fe, Zn and Mn.

• Trace elements needed under specific growth condition:Cu, Co, M0, Ca, Na, Cl, Ni and Se.

• Trace elements that are rarely required: B, Al, Si, Cr, V, Sn, Be, F, Ti, Ga, Ge, Br, Zr, W, Li and I, chelating agents, growth factors, vitamins (vitamin B1, B2, B6, B12, folic acid, lipoic acid, p-amino benzoic acid and vitamin K).

• Growth Media– Defined Media– Complex Media.

• Major Metabolic Pathway– The three of the most important pathways in the

cell are”• EMP• TCA• HMP → Plays a key role in supplying carbon skeleton

and reducing power for direct use in biosynthesis.

Primary mean for energy and generation

– The difference between aerob and fermentation.• If O2 is the final electron acceptor for the reducing

power → aerob respiration.• If another electron acceptor is used in conjunction with

the electron transport chain → anaerobic respiration

– The original meaning of the term fermentation = “ The organism grow without the generation energy by electron transport chain”.

Terminology (“trophs”) in microbial metabolism

Energy sourceChemo= chem bonds

Organo = organicLitho= inorganic

Photo= sunlight

e- acceptorAerobic = oxygenAnaerobic= not O2

CarbonHetero- = organicAuto- = inorganic

Nomenclature based on energy sources

Examples

• Anaerobic chemo-organo heterotroph– e.g. denitrifying Pseudomonas

• NO3- as an e- acceptor

• breaks chemical bonds for energy• Organic C as energy and C source

• Aerobic chemolithoautotroph– e.g. ammonia oxidizing bacteria Nitrosomonas

• O2 as terminal electron acceptor• Break chemical bonds for energy• Energy comes from breaking down NH4

+

• CO2 as C cource

Concept: energy uses and orginsHow is the energy used?

1) transport2) biosynthesis (anabolism)3) polymerization4) maintenance (regulation of pH, osmotic pressure,

motility)All biochemical reactions = metabolism; anabolic are biosynthetic; catabolic

are energy generating

Where does energy come from?• Breaking chemical bonds (inorganic or organic substrates) = chemotrophs• Sunlight= phototrophs

Enzymes: key concepts• proteins that catalyze biological reactions• have quarternary structure • can chemically / physically denature and renature • specific to specific reactions; except those involved in co-

metabolic reactions • typically act unidirectionally • re-available for catalysis after reaction • naming indicates type of reaction and substrate • intracellular or extracellular • constituitive or induced

MICROBIAL METABOLISM

OVERVIEW : MICROBIAL METABOLISM

• Anabolism– The assimilation of nutrients and the construction of new cell.– The cell assembles small molecules into more complex ones.– Need energy.

• Catabolism– All the chemical reaction of the cell that result in the breakdown of organic

molecules into simpler one.– Produce energy (some energy is conserved in nucleotide such as ATP and

GTP).• The digestion is accomplished by the secretion of exoenzymes.• Then it is followed by the transport of small molecules into the cell.• The third step, the degradation of small organic molecules into sampler

molecules. Then…..• The complete oxidation of pyruvate or acetate to carbon dioxide

NUTRIENT TRANSPORT

• A few of the nutrient such as water and glycerol pass readily into the cell by diffusion, but the plasma membrane is impermeable to most ions and molecules.

• Mode of transport:– Passive diffusion (no energy, high conc → low conc (cell))– Facilitated diffusion (no energy, carrier protein, high conc → low conc (cell))– Active transport (energy used, permeases, high conc ↔ low conc (cell))

BIOENERGETICS• Not all chemical reaction result in products with less energy than the

reactants.• The term : exorgenic (product have lower energy) and energonic (product

have higher energy).• Bioenegetics is a study that is concerned with the production and use of

energy by living organisms.• The energy available to these organism may be in the form of

electromagnetic radiation or stored in chemical bonds of nutrient molecules.

• Chemotrophic organisms obtain their energy from the oxidation of molecules.– Chemoorganotrophs : organic molecules (carbohydrate) as the electron donor

in energy-yielding metabolism.– Chemolithotrophs : inorganic molecules (ammonia, molecular hidrogen and

hydrogen sulfide) as the electron donor in energy-yielding metabolims.

• Among microorganisms there are three basic mechanisms of ATP synthesis:– Substrate level phosphorylation– Oxidative phosphorylation– Photophosphoryalation

• The fate of pyruvate depend upon a number of factors.– In the absence of an external electron acceptor (oxygen or nitrate ions), or it

the cell is deficient in some enzymes of the krebs cycle, the energy metabolism is restricted to fermententation.

– When an organism synthesizes its ATP by substrate level phosphorylation and disposes of its electron by giving them to organic molecules, the organism has carried out a fermentation.

– In facultative anaerobs, when the oxygen concentration is high, oxygen rather than an organic molecule is the final electron and proton acceptor.

CHEMOLITHOTROPHIC METABOLISM

• Obtaining their energy from the oxidation of reduced inorganic compounds (H2S, S, Fe2+, NH3, No2-, H2) and do not carry out fermetations.

• For example Nitrosomonas is an obligately aerobic chemolithotroph that obtains its energy from the oxidation of ammonia.– FAD as the electron transport system (not NAD).– The final electron acceptor is oxygen, which is reduced to water.

PHOTOTROPHIC METABOLISM• The phototrophs, utilize light energy to oxidize chlorophyll.• The electrons obtained during this oxidation are passed along various

electron carrier in an electron transport system to produce ATP and or NADPH.

• Both of these molecules are employed by phototrophic organism to power anabolic reactions such as the fixation of carbon dioxide.

• Autotrophic microorganism can build their organic compounds by fixing carbon dioxide in process called the calvin cycle (dark reaction of photosynthesis).

• Heterotrophic microorganisms, on the other hand, must have a ready source of organic molecules for their biosynthesis. These molecules can be used by the cell not only to suply its carbon but also as a source of energy.