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Fermentation

Image of plant for aseptic fermentation from Industrial Alcohol by John G McIntosh

Fermentation is a metabolic process during which carbohydrates are converted into alcohol and carbon dioxide or other acids. Fermentation is an anaerobic process in which energy is released from glucose without the need for oxygen. Fermentation occurs in yeast cells, they obtain energy by converting sugar into alcohol. Bacteria are also involved in fermentation; they convert carbohydrates into lactic acid. Yeasts are involved in both bread and alcohol production. During alcohol production, fermentation yields beer, wine, and other spirits. The carbon dioxide produced by yeast activity combines with the carbon dioxide emitted in the Krebs cycle which results in the rise of bread. Another use of fermentation is its ability to preserve foods for example, it produces lactic acid in yogurt, and it is also used in the pickling of foods with vinegar. Fermentation also occurs naturally and has been happening since before human time. However recently fermentation has become the controlled process we know it as today. The study of fermentation is known as zymology. In 1856 French chemist Louis Pasteur became the first known zymologist, when he demonstrated fermentation was caused by living cells. In 1860 he demonstrated that bacteria cause souring in milk, it was previously thought this a simple chemical change. He also successfully identified the role of microorganisms in food spoilage; this resulted in the discovery of pasteurization. While he was investigating the fermentation of sugar to alcohol by yeast, Louis Pasteur found that the fermentation was caused by forces called ferments, which were inside the yeast cells. The metabolising of glucose can occur in yeast cells by cellular respiration. This can also occur in other cells. In the absence of oxygen, glycolysis occurs, which results in the metabolising of glucose into pyruvic acid. This pyruvic acid is then converted first to acetaldehyde and then to ethyl alcohol. The conversion of energy to the yeast cell results in the production of two molecules usually produced in glycolysis. This process is known as the crabtree effect

Submerged Fermentation

SmF utilizes free flowing liquid substrates, such as molasses and broths. The bioactive compounds are secreted into the fermentation broth. The substrates are utilized quite rapidly; hence need to be constantly replaced/supplemented with nutrients. This fermentation technique is best suited for microorganisms such as bacteria that require high moisture content. An additional advantage of this technique is that purification of products is easier. SmF is primarily used in the extraction of secondary metabolites that need to be used in liquid form.

Principle of Submerged Fermentation

Submerged fermentation involves the growth of the microorganism as a suspension in a liquid medium in which various nutrients are either dissolved or suspended as particulate solids in many commercial media. Submerged fermentation is a process involving the development of microorganisms in a liquid broth.This liquid broth contains nutrients and it results in the production of industrial enzymes, antibiotics or other products.The process involves taking a specific microorganism such as fungi and placing it in a small closed flask containing the rich nutrient broth.A high volume of oxygen is also required for the process. The production of enzymes then occurs when the microorganisms interact with the nutrients on the broth resulting in them being broken down. The bioactive compounds are secreted into the fermentation broth.

Methods of Carrying Out Submerged Fermentation

There are two common methods by which submerged fermentation takes place; they are batch-fed fermentation and continuous fermentation.

In batch-fed fermentation sterilized growth nutrients are added to the culture. It is most common in bio-industries as it occurs during the growth of biomass in the fermenter. It helps raise the cell density in the bioreactor and it is typically highly concentrated to stop dilution. The rate of growth in the culture is maintained by adding nutrients, this also reduces the risk of overflow metabolism.

An open system is constructed for continuous fermentation. Then sterilized liquid nutrients are slowly and continuously added to the bioreactor at the same rate at which the converted nutrient solution is being recovered from the system. This results in a steady-rate production of the fermentation broth. In order to maintain a successful fermentation, certain variables must be monitored, for example, temperature, pH, as well as oxygen and carbon dioxide levels.

Submerged Fermentation Substrate

Some common substrates used in submerged fermentation are soluble sugars, molasses, liquid media, fruit and vegetable juices, and sewage/wastewater.

Enzymes

SmF is usually implemented in case of bacterial enzyme production, due to the requirement of higher water potential. More that 75% of the industrial enzymes are produced using SmF, one of the major reasons being that SmF supports the utilization of genetically modified organisms to a greater extent than SSF. Another reason why SmF is widely used is the lack of paraphernalia regarding the production of various enzymes using SSF. This is highly critical due to the fact that the metabolism exhibited by microorganisms is different in SSF and SmF, and the influx of nutrients and efflux of waste materials needs to be carried out based on these metabolic parameters. Any slight deviation from the specified parameters will result in an undesirable product.

Bacterial Enzymes

Bacterial Enzymes

Bacteria have been used to produce various enzymes such as amylase, xylanase,L-asparaginase, and cellulase. It was earlier believed that that the best method of production of enzymes from bacteria is by using submerged fermentation. However, recent studies have shown that SSF is more efficient than SmF for bacterial enzyme production. The main reason can be attributed to the metabolic differences. In the case of SmF, the accumulation of a variety of intermediate metabolites results in lowered enzyme activity and production efficiency.

Hypercholestrolemic Agents

Hypercholestrolemic agents are those substances that block the overproduction of cholesterol in the liver. They are of great medical importance as high blood cholesterol levels (hypercholesterolemia) are believed to be one of the primary causes of arthrosclerosis which leads to coronary heart disease. The first hypercholestrolemic agent to be discovered was compactin. Over the years, research has led to the discovery of other hypercholestrolemic agents such as lovastatin, mevastatin, pravastatin, and simvastatin. Compactin, Lovastatin, and Pravastatin are direct products of fermentation; they are also called natural statins. Natural strains possess a polyketide part and a hydroxyl-hexahydro naphthalene part to which side-chains are attached . Simvastatin is a semi synthetic statin obtained by the bioconversion of Lovastatin. Simavstatin differs from natural statins in the possession of an additional methyl group. The change in human lifestyle has led to the sudden spurt in cases of arthrosclerosis over the years, which has driven the demand for statins. This has resulted in the need to scope out newer and more efficient ways to produce them. Fermentation is a cost-effective way of mass production of statins. Both SSF and SmF have been used for this purpose. Fungi are widely used for producing statins.

Applications of Submerged Fermentation

SmF is primarily used in the extraction of secondary metabolites that need to be used in liquid form. Submerged liquid fermentations are traditionally used for the production of microbially derived enzymes.

Citric Acid Production

An example of submerged fermentation can be seen in the production of citric acid. Every year over a million tones of citric acid is produced by fermentation. In 1893, C. Wehmer became the first person to produce citric acid from sugar by using Penicillum mold. However it was James Currie who first established that strains of the fungus, Aspergillus niger, could be used to produce citric acid. Two years later the first industrial scaled production of citric acid was started by a pharmaceutical company named Pfizer. This fermentation process is still the process used by industries to produce citric acid today. It is the preferred method for production of citric acid as the probability is low and therefore economical constrictions are in place. A. niger is the chosen strain of fungi used at it is used as a substrate and it produces consistently high yields. In the process A. niger cultures are placed onto a sugar containing medium, the mold is then removed by filtration and the citric acid is separated using precipitation with lime. Sulfuric acid is then used on the resulting calcium citrate salt to produce citric acid.

Advantages of Submerged Fermentation

•Submerged fermentation technology has the advantages of short period, low cost and high yield. •Purification of products is easier. •In liquid culture the control of the fermentation is simpler and consequently significant reductions in fermentation times can be achieved. •In the same way, the use of submerged culture can benefit the production of many secondary metabolites and decrease production costs by reducing the labour involved in solid-state methods.

Limitations of Submerged Fermentation

•In recent years, many researchers have demonstrated that SSF has a large impact on productivity, leading to higher yields and improved product characteristics compared to SmF •Low volumetric productivity •Relatively lower concentration of the products •More effluent generation •Complex fermentation equipments