klarmaya news about microbes

Microbes: The unseen entities in human economy, environment, food and industry (2)

Thursday, February 26, 2009

Being continuation of the text of inaugural lecture series 101 of the University of Benin delivered by Prof. E. A. Nwanze, professor of microbiology, on Thursday, December 11, 2008.

Microbial problems encountered in drilling for oil

SULPHATE-REDUCING organisms such as Desulfovibrio often occur under anaerobic conditions in boreholes and pipelines where water and minerals are present for its growth. In this type of environment, hydrogen sulphide is produced which with water and the cathodic effect of the iron pipes and minerals result in sulphate acid (H2SO4) production, which causes corrosion of pipes such as drilling pipes. Corrosion may be very rapid and may lead to 30-40 per cent dissolution of steel within a period of 4 years. This type of damage requires replacement of pipe sections at the bottom of oil wells, which is very difficult and expensive.

Problems may also arise due to microbial attack on drilling fluids, which are used to lubricate the drill bit and seal the bore when passing through permeable strata. The drilling fluids are compounded with various clays, supplemented with colloidal starch and cellulose, both of which may be attacked by micro-organisms causing severe damage to the well. The drilling fluid is employed as lubricant to reduce frictional force. The fluid may also cause problems to oil workers, as it has been associated with different skin infections.

Micro-organism may grow to block injection wells. These wells are used to pump water into oil bearing strata and in so doing, help to force oil out. Fungi, bacteria and algae have been implicated as they accumulate in the pipes to block the pores in oil bearing rocks preventing efficient use of the injection well and reducing oil yield. This may be combated by using filtration processes from which contaminating micro-organisms are excluded.

IMPORTANT USES OF MICRO-ORGANISMS IN PETROLEUM INDUSTRY

Soil micro-organism are affected by gas such as methane exuding from underlying oil beds and it has been claimed that such oil deposits may be detected by studying the variation in number of methane utilising micro-organisms. This method of prospecting has been used in Soviet Union and patents have been issued in U.S.A. for the use of bacteria, such as Desulforibrio, to increase the yield from oil wells by introducing them to the oil bearing rocks or strata. In so doing, the viscosity of the oil is decreased and simultaneously the calcareous rocks are dissolved by metabolic acids, produced by the micro-organisms, to increase in rock pore size and gas pressure within the well result in greater oil recovery and yield.

In an attempt to produce and provide more protein rich food and feed various yeasts have been grown on petroleum substrate in fermentors. These substrates include paraffin fractions of petroleum. Protein rich organisms in final powder form have been obtained and the system is partially commercialised. The cost efficiency of conversion in nutritional value of the protein product compares favourably with protein obtained from conventional sources such as animals, fish and plants.

The degradation of crude oil by bacteria, yeasts and moulds is of beneficial effect in connection with pollution clean up, especially in marine pollution during oil spill. The more volatile portions of the oil spill evaporate leaving residual tar-like mass, which destruction is slow. This takes place under alternating aerobic and anaerobic conditions. As the oil sinks with accumulated weight of micro-organisms and then rises again due to gas production such as methane which accumulates in the mass the final result is a hard pebble like piece of utilisable tar residues. The spreading of detergents and artificial chemicals, sometimes believed to be necessary to break up large patches of oil-spill, may ultimately be counter-productive in terms of biological degradation as the detergent may kill bacteria, yeasts and moulds and other marine life beneficial to oil-spill clean-up or degradation. Other activities of micro-organisms in petroleum products involve the degradation of asphalt used in road building and in corrosion of metals employed in tankers and pipelines for petroleum products distribution.

USE OF MICROBES AS FOOD FOR HUMANS

SINGLE CELL PROTEIN (SCP)

• Various groups of micro-organisms have been considered for food or feed use. These include algae, bacteria, yeasts, moulds and higher fungi. The dried cells of these organisms are collectively referred to as single cell proteins (SCP).

• It is known that several people have eaten certain micro-organisms as a portion of their diet since ancient times. The top-fermenting yeasts, Saccharomyces spp., was recovered and employed as a leavening agent for bread as early as 2500 BC. Bread and yeasts leavened baked products contain 1-4 per cent of bakers' yeasts based on the wt of the flour used. The 1-4 per cent is subsequently consumed as food by man regularly.

• Fermented milks and cheeses produced by the use of lactic acid bacteria of the genera Streptococcus and Lactobacillus were consumed by early Egyptians, Greeks and Romans (100-50 BC.). These dairy products contain from 107 to 10 lactic acid bacteria per gram, which are consumed by man with the products. Wild mushrooms were reported to be prized by Pharaohs of Egypt and were also eaten in ancient times in the orient.

• The blue green algae of the genus Spirulina found extensively growing in alkaline lakes formed a useful food source for the inhabitants of Lake Chad region and the Aztecs of Mexico.

• Notwithstanding their usefulness, the purposeful cultivation of micro-organisms for direct use in human foods or animal feeds is a fairly recent development. The Industry started in Germany and its rapid development was based on the two world wars. During the First World War Bakers yeast, Saccharomyces cerevisiae was grown in aerated molasses ammonium salts medium while during World War II the aerobic yeast, Candida utilis (Torula yeast) was also produced in Germany for food and feed.

• More recently there have been large outputs of information on development of processes for utilising raw materials, including simple sugars, starches, cellulose, agricultural and food-processing wastes and hydrocarbons by bacteria, yeasts, mould, higher fungi and algae by means of photosynthetic wastes conversions (Kihlberg, 1972; Litchfield, 1977)

  FERMENTATION AND BIOREACTORS

  Production of microbes/microbial products useful to man usually involves fermentation. Fermentation per se in general terms as employed in Industries refers to large scale production of microbes or single cells to produce a commercially viable substance needed by man under anaerobic situation in classical terms or aerobic condition as now being loosely used. The anaerobic condition is employed in such food fermentations as brewing, wine making and dairy industries. Similar technology is employed but with aeration in the production of industrial products like insulin and human growth hormone.

  Large scale fermentations are carried out in a vessel called FERMENTOR/FERMENTER OR BIOREACTOR. The vessel is designed with close attention to aeration, pH control and temperature control. Different designs of bioreactors are available but the most popular bioreactors are of the continuously stirred types. In a typical bioreactor, air is continuously introduced through a diffuser at the bottom to maximise aeration. A series of stirrer paddles and stationary wall baffles are installed to keep the microbial suspension agitated. Oxygen is not very soluble in water, which makes proper aeration of microbial population a problem. To overcome such problems, highly sophisticated bioreactors have been developed to achieve maximum efficiency in aeration and other growth parameters to pH control and medium formulation. The premium placed on genetically engineered micro-organisms and products therefrom have stimulated development of exotic and newer types of bioreactors with installed computerised controls as obtained in breweries and some other food industries.

  Micro-organisms involved in fermentation produce either primary metabolites or secondary metabolites. A typical primary metabolite is ethanol and its production follows in parallel cell population curve with a minimal lag. Primary metabolites are largely produced at TROPHOPHASE OR GROWTH PHASE. An example of a secondary metabolite is penicillin antibiotic, which is produced at the IDIOPHASE stage when the micro-organism has largely completed its growth phase.

  RANGE OF FERMENTATION PROCESSES

  There are four (4) major groups of commercially important fermentations:

• Those that produce microbial cells (SCP or biomass as the product)

• Those that produce microbial enzymes

• Those that produce microbial metabolites

• Those that modify a compound or substrate as in steroids transformation processes.

  THE COMPONENT PARTS OF A FERMENTATION PROCESS

  Any fermentation process may be subdivided into six basic steps as listed below:

• Preparation formulation of the growth medium to be used in culturing selected organism. During the development of inoculum, the process is usually carried out in stages to the right size in the production fermenter.

• Sterilisation of medium, fermenters and auxiliary equipment.

• Production of an active, pure culture in sufficient quantity to inoculate the medium in the production vessel.

• Growth of the organism in the production fermenter under optimum conditions for product formation.

• Extraction of the product and its purification.

• Disposal of effluents produced by the process, which may serve as substrate for other processes such as Single Cell Protein production.

  USE OF MICROORGANISMS IN ENERGY GENERATION

  Large amount of organic waste materials are produced from agricultural products such as corn cobs, rice bran, sawdust, cassava processing wastes, and animal dung, which abound virtually in all cities and towns and municipal areas in Nigeria and globally. This 'waste' is collectively called BIOMASS.

  Biomass may be acted upon by micro-organisms to produce fuel in a process referred to as BIOCONVERSION. Fuel generated can be used for energy production. Examples of such fuel include Methane Biogas, Ethanol and GASOHOL. Gasohol is 90 per cent GASOLINE + 10 per cent ETHANOL. Ethanol has been generated from agricultural waste products like corn cobs, when acted upon by yeasts to give ethanol. The yeasts harvested from the process can also serve as additive in human snack food or as animal feed. The pollution, which naturally follows these 'wastes', is abated resulting in cleaner environment.

  CONTRIBUTIONS

  Mr. Vice Chancellor Sir, one big challenge encountered in the preparation leading to this Inaugural Lecture is what to INCLUDE OR EXCLUDE out of several contributions. After series of approaches I finally settled for the format of presentation as:

• Initial contribution;

• Contributions involving production of single cell protein (SCP,);

• Fermentation processes involving bio-conversion of inedible Parkia filicoidea Welw or African Locust Bean Seeds to nutritious edible product Dawadawa; and

• Microbiological investigations to add value to local products of fermentation and stability of products obtained from wastes and allied products.

  INITIAL CONTRIBUTION ANTIBIOTICS AND ANTIMICROBIAL EFFECT OF CASSIA ALATA EXTRACT ON SELECTED MICROORGANISMS

  Achievements recorded over disease causing micro-organisms are directly due to man's persistent and relentless struggle against these ubiquitous inhabitants of the microcosm that have plagued and lethally assaulted not only man, but also his domestic animals, plants and aquatic forms upon which his survival depend. .

  Man, against the lopsided advantage of numbers possessed by micro-organisms, has two answers his sciences and ability to think. Such ability and thinking resulted in herbal production of anti-microbial agents in earlier times; two of which have succeeded to the modern times. These are 'CINCHONA' bark for treatment of malaria and 'IPECACUANHA' root for amoebic dysentery.

  Cinchona bark was used by the Indians of Peru for treatment of malaria and was introduced into European medicine by the Spaniards in the early 17" century. The active agent or principle, QUININE, was isolated in 1820. Quinine remained the only treatment for malaria well into twentieth century and still has a place in chemotherapy today.

  Ipecacuanha (IPECA) was known in Brazil and probably in Asia for its curative action in control of diarrhoea dysentery. EMETINE was isolated as the active constituent in 1817 and was shown in 1891 to have a specific action against amoebic dysentery and is still used today to treat this disease.

  As a result of research into different anti-microbial agents contained in some plant extracts, well-documented information have surfaced into the world of chemotherapy. From Impatiens balsamina, Garden blossom, Little et al., 1948 isolated a naturally occurring 2-methoxy-1, 4-naphthoquinone active against several phytopathogenic organisms. In 1968, Japanese Investigators, Kosuge et. al., obtained anti-fungal activity from 'rice bran-ter' which was potent in the treatment of eczema. Neeman et al., 1970 observed anti-microbial activity against 13 different species of bacteria and yeasts from avocado pear.

  In a report by AI-Delamy and Ali (1970), vegetable extracts of garlic, onion, turnip, green peppers and radishes were reported to inhibit growth of Escherichia coli, Salmonella typhosa, Shigella dysenteriae and Staphylococcus aureus all of which are pathogenic bacteria.

  In line with above reports, extracts from fresh leaves of Cassia alata were tested against some selected dermatophytes, phytopathogenic moulds and bacteria. Results indicate that Cassia alata extract was most active against Trichophyton species with minimal effect on Candida albicans. The two phytopathogenic moulds tested Aspergillus and Penicillium were both susceptible to the extract. The extract of Cassia alata also produced inhibitory effect on E. coli, E. freundii and B. Subtilis (lkenebomeh, 1974).

  The extract of Cassia alata leaves, filtered or unfiltered, produced growth inhibition in vitro against dermatophytes and appeared to have broad anti-microbial spectrum (Ikenebomeh, 1974; Ikenebomeh and Metitiri, 1988). Cassia alata extract potency is not affected by the extracting medium as cold water extraction produced the same inhibition with that of Sorensen's buffer. However, Sorensen's buffer at pH 6.8 was used as the extracting medium to ensure long period of Cassia alata extract potency and to avoid deterioration arising from either high acidity or alkalinity. Gugnani (1982), Kubeyinje (2008) have discussed the public health problems in Nigeria and globally with respect to mycoses caused by dermatophyes. The extract of Cassia alata leaves from our investigations could be used to control some of these dermatophytes and their spread. Industrial production of the extract locally would have considerable effect on our scarce foreign exchange expended on importation of fungicidal and fungistatic medicaments (Ikenebomeh and Matitiri, 1988).

  CONTRIBUTIONS INVOLVING SINGLE CELL PROTEIN (SCP)

  Resulting from improved medical knowledge and scientific investigations in the elimination of microbes, surgery for example is no longer a desperate gamble with human life, thanks to Joseph Lister who made use of Louis Pasteur's pasteurisation techniques as a base for the introduction of antisepsis which eliminated surgical sepsis. Side by side with Lister's innovation are the lessened perils of childbirth due to control of puerperal fever, death of children and some adults from meningitis, tuberculosis and septicaemia, all due to micro-organism which have now been put under check MINUS HIV presently. 0ne of the obvious results of improved medical care and scientific knowledge is the attendant increase in human population.

  Communities where endemic diseases and periodic plagues previously kept the death rate in balance with the birth rate are now expanding at an alarming rate as witnessed in developing nations. Nigeria for example in the 1940s had a population of about 30 million but today, the figure officially quoted is 140 million.

  The situation is accentuated by the fact that while population increases geometrically, agricultural output in developing countries follows arithmetic progression. For example, 10 x 10 x 10 = 1,000 Geometrical, compared to 10 + 10 + 10 = 30 Arithmetic is not the same. Above scenario is one of the major reasons for SCP production.

  Although population is one of the major prong for raison d'etre for SCP, yet there are contrary views Uriah (1997) in his Inaugural Lecture Series 51, University of Benin stated in reference to problems caused by affatoxin "The incidence of declining human fertility is presently causing a lot of concern all over the world. The male spermatozoa have" reduced to one half in number between 1942-1992. The spermatozoa abnormalities increased within this period. In Nigeria, about 4 million people have been reported with fertility problem." This may be true, but notwithstanding, our population is still on the increase.

  Other reasons for SCP production are:

• Critical need for protein

• Inexpensive raw materials 'WASTES'

• Economic advantages. Cleaner environment

• Less labour compared to conventional agriculture

• Small space needed Fermentor/Bioreactor

• Independent of seasonal and climatic variations

• Precise yield plan

• Insurance against pest and crop failure

• Dry cells of microbes contain 50-70 per cent protein

  UNCONVENTIONAL PROTEINS AND SOURCES

  Interest in the synthesis of food by micro-organisms arises from three main considerations:

• The critical need for food, and in particular for protein, that exists in many parts of the world.

• The economic advantages of microbial elaboration of foods or vitamins or amino acids from relatively inexpensive raw materials and

• The reduction in cost of disposal of fermentable wastes. Since these proteins are obtained from unconventional sources, they are referred to as unconventional proteins (Han et al., 1971;Worgan, 1973).

  SINGLE CELL PROTEIN

  The term 'Single Cell Protein' and its acronym SCP was coined by a group of scientists at a conference in Massachusetts Institute of Technology (MIT) in 1967 (Tannenbaum, 1976). However, it is believed that the term was first used in 1966 by Prof. Carol Wilson also of MIT as a form of nomenclature to describe a wide range of yeasts, bacteria, fungi and algae which are considered to be of potential food ingredients. At present, the term has become, by definition, a generic name for crude or refined sources of proteins which origin is unicellular.

  The most unique aspect of SCP is that it is a protein resource that can be produced without resorting to an agricultural base. Of equal interest is the possibility of using sources of carbon derived from under-utilised or waste resources, such as cellulose. This possibility led me to Dr. J. T. Worgan's laboratory in Weybridge, University of Reading, who supervised my M.Sc. project work titled 'Cassava Wastes as substrate for Single cell Protein Production from Geotrichum candidum and problem of pigmentation. In addition to SCP production, the process has the advantage of decreasing biological oxygen demand (BOD) levels of effluents or wastes.

  CASSAVA TUBERS AND WASTES AS SUBSTRATE FOR SCP PRODUCTION

  A large percentage of world's cassava (Manihot esculenta, Crantz) production is used as human food either as fresh vegetable oil in some simple processed form (Phillips, 1974). Nigeria produces about 10-20 per cent of world cassava output and about 50 per cent of cassava tubers is used in Garri production. Garri production involves removal of cassava tubers pericarp, grating into pulp and fermenting under pressure for about 48 hours to expel cassava whey. The de-wheyed pulp is passed through a mat-like sieve and dried over large hot hemispherical bowls to give garri. The process generates about 10-30 per cent waste from the tubers as a result of peeling and run-off cassava whey. The whey and other wastes can be harnessed for the production of SCP, which can be re-introduced into the cassava pulp or garri to upgrade their protein content. The use of micro-organisms along this line has been studied and documented (Trevelyan, 1974; Reade and Gregory, 1975) and in some cases covered by patents (Stanton and Wallbridge, 1972).

  In our contribution, cassava starch, dissolved in a liquid medium, was investigated as a carbon source for the production of Single Cell Protein (SCP) from Geotrichum candidum. In the presence of a nitrogen source (ammonium tartrate) inorganic salts, trace elements and growth factors, fermentation at 30C in flasks on a rotary shaker mycelia biomass was cropped. The resultant mycelium weight was maximum at initial medium pH 4.5 and 4 per cent (w/v) cassava starch. The crude protein content (N x 6.25; Kjedahl) of the starch was 0.25 per cent whereas for the SCP, it varied from 25-35 per cent (Ikenebomeh, 1989). It is envisaged that SCP from cassava starch will find application in increasing the protein contents of garri and allied products.

  Other contributions include cassava whey treatment with Aspergillus niger (Ikenebomeh and Chikwendu, 1990); biomass production in cassava whey medium (Ikenebomeh and Chikwendu, 1997). Apart from SCP, the cassava whey has been used in other fermentation processes to produce ethanol (Akpan et al., 1988) and for the production of glutamic acid (Akpan et al., 1998).

  PARKIA SEED AND ITS FERMENTATION PRODUCT DAWADAWA

  The Parkiae are a small but widely spread genus of Leguminosae found in Africa Brazil, Java and Surinam (Henderson, 1890). They are perennial plants which pods contain a sweet, pulpy carbohydrate material and dark Brown seeds rich in proteins, fat and lysine. In West Africa, the seeds are called 'African Locust beans'. In the raw form, they are extremely hard and practically inedible. They are however processed and fermented to a tasty product called Dawadawa in West Africa.

  Early interest in these beans and the fermented product obtained from them was expressed in a report of Imperial Institute of London (Anon, 1922) subsequently, Platt (1964), Oyenuga (1968) and FAO (1970) have all reported on the chemical composition and nutritional significance of African Locust beans. However, at the point of our investigation of these seeds, there were no detailed report of the processing and fermentation of these seeds (Ikenebomeh and Kok, 1984) and the mass balance and yield data of the fermentation process. Dawadawa production as was earlier practical in West Africa was based on small-scale traditional methods. This imposed a need for scientific investigation and quantification to allow for process scale up. The mass balance revealed that 1.0kg of raw beans (6.4 per cent moisture by wt) yielded 1.3kg of processed substrate (63 per cent moisture by wt) which in turn was converted to 1.2kg of Dawadawa (65 per cent moisture by wt). The loss of bean solids during processing was due to the removal of adhering pulp and testa as well as to solids extraction during boiling and washing. The loss of solids during fermentation was presumably due to microbial utilisation of a fraction of the processed substrate (Ikenebomeh and Kok, 1984).

  Normally prepared fermentation substrate (PS) was compared with both sterilised and radappertised beans in terms of conversion to Dawadawa, number of colony forming units present and the development of pH and titratable acidity. The presence of micro-organisms was found to be obligatory for the conversion to proceed (Ikeneb-meh et al., 1986).

  CHANGES IN PARKIA FILICOIDEA WELW. TEXTURE DETERMINATIONS WITH INSTRON MACHINE DURING FERMENTATION

  During Parkia filicoidea Welw. fermentations, textural changes were observed as the substrate becomes progressively softened. Partial solubilisation of the beans was also observed when the substrate was kept for longer than 72 hours period of bioconversion. It was simultaneously observed that the control of radappertised beans (RADB) retained their original hard texture throughout the period of investigation. In order to quantify these observations, a test known as the Magness Taylor Puncture Test or simply puncture test was carried out using the Instron machine (Bourne and Mondy 1967).

  In this test, a probe was forced at constant speed into the material under study. The resulting 'bioyield and peak force' were determined from the output on a strip chart. The processed substrate (PS) had 20.6+1.0N and 26.0+2.0N as bioyield and peak force respectively.

  The fermented product gave 5.9+2.0N for bioyield and 7.4+3.0N for the peak force. The RACB bioyield and peak force remain relatively high at over 300N. On a large production scale, the instron may be used to characterise the African Locust Bean fermentation.

  EFFECT OF SALT (NaCI) ON DAWADAWA FERMENTATION

  Sodium chloride (NaCI) is known to play useful role in fermentation of foods such as sauerkraut, cheese, Japanese shoyu and other indigenous foods (Yokotsuka, 1977; Pederson, 1979; Steinkraus, 1983). An appropriate salt level may select for micro-organism useful to the fermentation by reducing contamination and improving product consistency. Fermentation temperature is also important for good growth of micro-organisms. The influence on microbial growth by different salt contents and temperatures were followed by changes of pH and titratable acidity. A 1 per cent (w/w) salt addition and fermentation at 37C improved the organoleptic quality of the Dawadawa product (Ikenebomeh, 1989a). Salt addition above 3 per cent (w/w) and temperature below 25'C resulted in lower microbial counts, low pH and titratable acid values. Fermentation of processed substrate of African locust bean seeds was inhibited and the product organoleptic quality was poor. The predominant micro-organisms present throughout the fermentation was a Bacillus species with characteristic similar to Bacillus subtilis (Ikenebomeh, 1982, 1989a) indeed more appropriately Bacillus Dawadawa.

  PROTEOLYTIC BACTERIA IN DAWADAWA PRODUCTION

  Spontaneous bacterial fermentation of processed African locust bean seeds, Parkia filicoidea Welw. at 37.C to produce Dawadawa is accompanied by qualitatively proteolytic activity. Unfermented but processed bean seeds called processed substrate (PS) and 11KGY radappertised PS (RADB) also investigated gave quantitatively reduced proteolytic activity and protein content. Aerobic, spore forming proteolytic bacterial rods predominate in the ferment and their numbers increased from log,. 4.30 CFU.g 1 at Oh to log10 9.48 CFU.g' at 72 hours period. Presence of proteolytic organisms during fermentation resulted in increased production of extra-cellular proteolytic enzymes and simultaneous increases in pH and titratable acid values characteristic of African locust bean seeds fermentation (Ikenebomeh, 1982; Ikenebomeh et al. 1998).

  Extra-cellular protease production by micro-organisms is well known and documented as method of classifying, differentiating and identifying them (Kelly and Fogarty, 1976; Deak and Timar, 1988). The release of ammonia during' fermentation as a result of the presence of proteolytic micro-organisms indicates that the PS bio-conversion to the desirable fermented Dawadawa product occurs, at least partly, by proteolysis.

  HYPOTHESIS OF PAR KIA FILICOIDEA WELW BIOCONVERSION TO DAWADAWA

  A hypothesis can be defined in one of several ways as:

• A tentative assumption made in order to draw and test its logical or empirical consequences, or

• An interpretation of a practical situation or condition taken as the ground for action, or

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