Microbial Process Technology LECTURE 1

Introduction Biobased Economy “The biobased economy produces non-food products from green resources (biomass) through which the generation of greenhouse gasses can be reduced and the dependence on fossil resources is diminished. The use of biomass can lead to economic opportunities. However, knowledge on the technical potential of the biobased economy is still limited. Scientific research to help finding a healthy balance between the production of food, chemicals and bio-energy is required to establish a sustainable biobased economy.”

Introduction Biobased Economy “The development of a Biobased Economy is a necessity as also mentioned by the State of the World 2006 report of the World Watch Institute: “Rising demands for energy, food, and raw materials by 2.5 billion Chinese and Indians creates an urgent need for a new path of industrialization: based on new production and consumption technology with low consumption of resources and low environmental pollution, and the optimal allocation of human resources. The resource-intensive model for economic growth can't work in the 21st century.”

Introduction Biobased Economy “There is a growing demand for energy (in 2050 a worldwide estimate of 2 to 3 times the current consumption) and resources. Triggered by this demand, there is a growing market for products which exploit biomass for pharmaceutical, fine- chemical, bulk chemical, bio-energy and biobased materials.”

Introduction materials chemicals transport fuels energy food cattle feed resource crops biomass rest streams crops biomass rest streams products cattle feed

Bioprocess engineering application of chemical engineering principles to effect desirable chemical conversions using living cells or enzymes in bioreactor containing biological catalyst followed by a product recovery section ancient art = food and beverages modern art due to genetically modified organisms ≠ (petro)chemical processes due to fragility and complexity of the biocatalyst

Biocatalyst – enzyme = protein = polymer of amino (-NH 2 ) acids (COOH) = organic catalysts any substance that speeds up the rate of a chemical reaction but is itself not affected once the reaction is completed highly specific and fairly rapid reaction rates at near ambient temperature typical turn over of 10 2 to 10 6 molecules of product per minute per active site a typical bacterium will make over 1000 distinct enzymes

Biocatalyst – enzyme active site = cavity with specific side groups that reversibly hold a substrate in a particular orientation while a reactive points breaks or forms new bonds (lock and a key mechanism)

Biocatalyst – enzyme important in genetic engineering and bioprocess operation (large scale): – inhibition due to excess substrate, products or byproducts – denaturation due to extreme pH or temperature

Biocatalyst – cell Produced for biomass itself – baker’s yeast – starter cultures (yoghurt) – Single Cell Proteins (SCP) – … Produced for cell product – enzymes – metabolites primary (directly related to growth) secondary (not related to growth)

11 Industrial Biotechnology Industrial bioreactor Reactor model Yield optimization Cell factory Metabolic network model Flux distribution optimization From molecular biology to industrial biotechnology … Complementary optimization of microbial cell factory and bioreactor process conditions Courtesy Kristel Bernaerts

Biocatalyst – cell Also to be put in the right environment – need for the appropriate amount of “food” = C, N, P to build up carbohydrates, proteins, phospholipids, DNA, RNA etc. – need for energy to make the abovementioned new cell building blocks for transport systems: selected molecules in and out the cell – cellular composition ≠ medium composition – pump desirable components into the cell against a concentration gradient – much energy needed (active transport)

Biocatalyst – cell – need for energy (Ctd.) catabolism = breaking down to obtain energy and building blocks energy is captured in ATP - ADP - AMP (breaking P bound releases energy) oxidation of substrates also generates reducing power = NAD/FAD → NADH/FADH 2 (=electron shuttle) NADH → H with O to H 2 O in respiratory chain → yield of 3 ATP no O 2 present → electrons to other substrates = fermentation

Microbial diversity eubacteria and archaebacteria = prokaryotes yeast/molds = eukaryotes: – nucleus and nucleus membrane – more ribosomal RNA, more complex structure that includes – organelles such as mitochondria (sites of respiration) & chloroplasts aerobic / anaerobic / facultative anaerobic wide T range wide pH range

Genetic engineering plasmids = extrachromosomal piece of DNA capable of autonomous replication contains specific target DNA + some antibiotic resistance (AB) transferred to E. coli (or to Streptomyces) AB resistance enables selection

Impact of environment on strain performance Optimization of heterologous protein production by Streptomyces lividans Impact of heterologous protein production on central metabolism Products: industrial enzymes and therapeutic proteins Bioprocess engineering: examples Courtesy Kristel Bernaerts

Bioprocesses profitable? See Figures on next slides

Bioprocesses profitable?

Bioreactor (liquid fermentations) modes of operation: batch, fed-batch, CSTR accessories

Bioreactor – Modes of operation: Batch lag - exponential - stationary - decline – lag: adaptation of the cell – exponential: – stationary: growth ceases because of depletion of Cs build up of toxic (by)products physical crowding → internal machinery (composition) changes if cell senses that it cannot grow rapidly anymore → maintenance energy (from break down, e.g., of excess ribosomes) – decline: no maintenance energy left = dying

dCx/dt= µ Cx Td = ln2/ µ = generation time primary metabolites = growth associated (metabolite essential for growth) secondary metabolites = non growth associated disadvantage: down time, substrate inhibition, catabolite repression, … Bioreactor – Modes of operation: Batch

µ = D = F/V advantage w.r.t batch – higher productivity disadvantages w.r.t batch – mechanical instability – absolute sterility difficult – genetic instability Bioreactor – Modes of operation: CSTR

avoids substrate inhibition, catabolite repression maintains sterility Bioreactor – Modes of operation: Fed-Batch

Still to come Bioreactor configurations/mode of operation: – cell retention systems – solid state fermentations Bioreactor monitoring and control Advanced bioprocess modeling – Immobilized systems – Multispecies models – Structured models Applications – Seminars – Biomedical application

Introduction materials chemicals transport fuels energy food cattle feed resource crops biomass rest streams crops biomass rest streams products

Possible seminar topics Genetically engineered plants/micro-organisms Biofuels (biodiesel, bioethanol, ABE) Bio-energy – from solid waste to energy – from liquid waste to energy Bioplastics, biomaterials Micro-algae Microbial fuel cells Bioremediation Production of biodetergents, enzymes, fine chemicals (citric acid, lycopene, …), prebiotics,... Nutrient recovery from waste streams