INDUSTRIAL MICROBIOLOGY Industrial Microbiology • Use of microbes to obtain a product or service of economic value constitutes industrial microbiology • Any process mediated by or involving microorganisms in which a product of economic value is obtained is called fermentation – The terms industrial microbiology and fermentation are virtually synonymous in their scope, objectives and activities. Microorganisms: importance in industry Microorganisms: importance in industry • Negative or detrimental effects: – Causative agents of diseases – Causing food poisoning and spoilage – Affecting water quality – Deterioration of materials and products • Positive effects: produce valuable products or useful services – Producing therapeutic compounds, foods and beverages, additives – Pollution control – Alternative fuels – Agriculture • Microorganisms can be used 3 ways industrially – Products of microbial metabolism • metabolic end product, pathway intermediates, enzymes, vitamins – Bioconversion • microorganisms as biocatalysts, e.g. bioremediation – Microbial cells or cellular components • foods or food supplements PROPERTIES OF AN INDUSTRIAL MICROORGANISM • Genetically stable • Suitable for scale-up • Easily grown • Rapid growth rate • Grown on relatively cheap media or waste materials – molasses, corn steep liquor, whey, cassava, potatoes, grapes, etc Factors affecting growth of industrial microorganisms • Availability of carbon and energy sources • Ability to replicate DNA • Size and mass of cells • Physical and chemical environment – media (balance between cost and maximum growth and product formation), conditions • Maintenance energy requirements – energy needed to keep the cell alive but not for growth • Culture type – batch cultures (closed culture systems, nutrients are not renewed) – continuous cultures (continuous supply of substrates gives better control over variables) • Growth rates (increase in cells per unit time) – cell population goes through a number of phases when inoculated into fresh medium PRODUCT FORMATION i. Primary metabolites • Product formed during the primary growth phase, e.g. ethanol • Growth dependent ii. Secondary metabolites • Products formed near the end of the growth (or stationary) phase, e.g. antibiotics • Not essential for growth and reproduction • Highly dependent on growth conditions, repression frequently occurs • Usually produced from primary or intermediate metabolites, not the substrate METHODS OF FERMENTATION • The goal of biotechnology is to obtain useful metabolic products from biological material. • Biotechnology encompasses two distinct phases: – Fermentation and Product recovery • Fermentation procedures must be developed for the cultivation of microorganisms under optimal conditions and for the production of desired metabolites or enzymes by the organisms. • Product recovery involves the extraction and purification of biological products PRINCIPLES OF MICROBIAL GROWTH • The growth of organisms may be seen – as the increase of cell material expressed in terms of mass or cell number. • Growth is dependent on – the availability and transport of necessary nutrients to the cell and subsequent uptake and – environmental parameters such as temperature, pH and aeration being optimally maintained. • Cells grow in four typical phases – lag phase, log phase, stationary phase and death phase LAG PHASE • No increase in the number of cells • Time for adaptation to the new environment arising from: – Change in the pH value – Increase in the supply of nutrients – Decrease in growth inhibitors • New transport system must be induced within the cell • Enzymes of primary metabolism must be adjusted to the new conditions • Physical condition of the inoculum is essential to the length of the lag phase LOG PHASE • By the of end of lag phase, the cells have adapted to the new conditions of the growth • Microbial growth proceeds at the maximum possible rate for that organism, with nutrients in excess, ideal environmental parameters and growth inhibitors absent • Growth can be described as: – Doubling of the cell number per unit time (yeasts & bacteria) – Doubling of biomass per unit time (filamentous organisms such as fungi & Actinomycetes) • Cells alter the medium through uptake of substrates and excretion of metabolic products STATIONARY PHASE • As soon as the substrate is metabolized or toxic substances have been formed, growth slows down or is completely stopped. • The biomass increases only gradually or remains constant during this phase • Due to lysis, new substrates are released which may the serve as energy for the slow growth of the survivors • The various metabolites formed in this phase are of great biotechnological interest DEATH PHASE • In this phase the energy reserves of the cells are exhausted • The cells die at an exponential rate • In biotechnological processes microorganisms in a bioreactor can be grown in – batch or continuous fermentation • Within the bioreactor reactions can occur with – static or agitated cultures – in the presence or absence of oxygen – in liquid or low moisture conditions (e.g. on solid substrates) 1. BATCH FERMENTATION – Is considered to be a closed system – A fixed volume of sterilized nutrient solution in the fermenter is inoculated with microorganism • incubation is allowed to proceed under optimal conditions – Catalysts and medium are mixed by a rotating central shaft that carries several impellers. – Metabolically active microorganisms evolve heat • Jackets that surround it provide cooling to maintain an optimum operating temperature. – Equipments to monitor and control the temperature and the pH of the medium are common – If the process is aerobic, oxygen must be supplied continuously. • The cheapest means of sterilization of air is – to pass it through a sterile filter composed of glass, wool, carbon particles, or some finely divided material that will trap microorganism present in the air. – Aeration and agitation of a liquid medium can cause the production of foam. • medium containing high levels of proteins or peptides. – Foam must be controlled if fermentation is to be carried out in a proper manner. • The usual procedure for controlling foam is to add an antifoam agent. – Samples of mixture can be removed for analysis by means of other tubes and pipes. • In the course of the fermentation, nothing is added except: – Oxygen (in the form of air) – An antifoam agent – Acid or base to control the pH • As a result of metabolism of microbial cells, the following change constantly: – The composition of the culture medium – The biomass concentration – The metabolite concentration • In most batch processes, the biocatalyst is discarded with the spent medium, which is potentially disadvantageous. 2. CONTINUOUS FERMENTATION • An open system • Are fermentations that are run continuously – without empting the fermenter at each harvest of microbial cells or biosynthetic products. • Fresh nutrient solution is added to the bioreactor continuously and – an equivalent amount of converted nutrient solution with microorganisms is simultaneously taken out of the system. • Continuous methods of cultivation – will permit organisms to grow under steady state (unchanging) conditions • growth occurs at a constant rate and in a constant environment. • Factors such as pH and the concentrations of nutrients and metabolic products – held near constant in continuous cultivations. • Potential benefit of continuous methods is – reducing losses of catalyst. In most batch processes, the catalyst is discarded with the spent medium. Approaches to control microbial activity in continuous culture Turbidostat: – the total cell population of microbial cells is held constant • by employing a device that measures the culture turbidity – regulate both the nutrient feed rate to the fermenter and the culture withdrawal rate from the fermenter. – If the population numbers rise above a predetermined level • a greater amount of fresh medium is added to the fermenter Chemostat: – In contrast to the turbidostat, a chemostat maintains the nutrient feed and harvest culture withdrawal rates at constant values. – The growth rate is controlled by • supplying only a limiting amount of a critical growth nutrient in the feed solution. • Thus, cell multiplication cannot proceed at a rate greater than that allowed by the availability of this critical nutrient. Advantage of continuous fermentation • The size of the bioreactor and other equipment used in continuous fermentation are – relatively smaller compared to batch fermentation for the production of the same quality of product • They yield of the product is more consistent since the physiological state of the cells is uniform • The time gap between two successive fermentations for cleaning and preparing the bioreactor for reuse is avoided in continuous fermentation • Potential benefit in reducing losses of catalyst Disadvantages of continuous fermentation • Maintaining sterile conditions on an industrial scale over a longer period of time is difficult • It is not to maintain the same quality of the culture medium for all the additions. – Nutrient variations will alter the growth and physiology of the cells and consequently the product yield • While using high-yielding strains, reverse mutants arise, which can overgrow the production strains in continuous culture SOLID SUBSTRATE FERMENTATION • Is a fermentation processes that do not involve liquid medium. – growth of the microorganisms is carried out on solid substrates in the absence or near absence of free water • The presence of moisture (about 15%) is necessary for solid state (solid substrate) fermentation • The most regularly used solid substrates are – cereal grains, legume seed, wheat grain, lignocellulose materials such as straws, sawdust or wood shavings • Most of this compounds are cheap, easily obtainable and represents a concentrated source of nutrients for microbial growth • There is a need to pre-treat the substrate raw material to facilitate the availability of the bound nutrients. Advantages of solid substrate fermentation – Simple cheaper substrate – Low technology, low energy expenses – Low moisture content of materials gives economy of bioreactor space – No need for sterilization – Easier downstream processing – Yield of product can be high • Disadvantages of solid substrate fermentation – Process limited mainly to moulds that tolerate low moisture levels – Slower growth rate of microorganisms – Process monitoring, e.g. moisture levels, oxygen and carbon dioxide levels is difficult to achieve accurately FERMENTATION EQUIPMENT AND ITS USE
