Basic Design Of Reactor

Introduction Biological Treatment For Recovery Biological treatment forms the basis of many treatment processes as a first step in the recovery of water, while often presenting the possibility of recovery of other materials. It is therefore important to first address the principles of biological treatment. This module addresses very briefly some of the underlying microbiology as relevant to water and wastewater treatment before embarking on some treatment principles, growth kinetics and recovery applications.

Microbiological Principles

Plants, animals, and fungi comprise three kingdoms of more complex eucaryotic organisms, most of which are multicellular. The main distinguishing factor among these three kingdoms is the nutritional mode; another important difference is cellular differentiation. Fungi are eucaryotic organisms that include the unicellular yeasts, multi-cellular moulds, and macroscopic varieties such as mushrooms. To obtain raw materials for vital functions, a fungus absorbs dissolved organic matter through the membranes of cells in rootlike structures called hyphae. Plantae are multi-cellular eucaryotes, and they include some algae and all mosses, ferns, conifers and flowering plants. To obtain energy, a plant photosynthesizes, a process involving the conversion of carbon dioxide from the air to organic molecules for use by the cell.

The science of classification, especially of living forms, is called taxonomy. The object of taxonomy is to establish the relationship between one group of organisms and another, and to be able to differentiate between them. Until the end of the Animalia are multi-cellular animals with eucaryotic cells. These nineteenth century, all organisms were divided into two include sponges, various worms, insects, and animals with kingdoms: plant and animal. Then, when microscopes were backbones (vertebrates). Animals obtain carbon and energy by developed and biologists came to understand the structure and ingesting organic matter through a mouth of some kind. physiological characteristics of microorganisms, it became In 1978, Whittaker and Margulis proposed a revised classificaapparent that micro-organisms did not really belong to either tion scheme, organizing the five kingdoms according to the plant or animal kingdom, although many microbes have procaryote or eucaryote as overleaf. either plant or animal characteristics. In 1866, a third kingdom, Superkingdom Procaryotae Superkingdom Eucaryotae the Protista, was proposed by Ernst Haeckel to include all microorganisms - bacteria, fungi, protozoans and algae. Kingdom Monera Kingdom Protista Subsequent research indicated that among the Protista, two different basic cell types could be distinguished: procaryotic and Branch Protophyta, plantlike eucaryotic.

Five-kingdom System of Classification Higher organisms, structurally much more complicated, are based on the eucaryotic cell, in which a membrane separates the nucleus from the cytoplasm. This type of cell apparently evolved more recently, a little more than a billion years ago. Simpler eucaryotic organisms, mostly unicellular, are grouped as the Protista and include fungus-like slime moulds, animal-like protozoans, and some algae. Some algae are more complex multicellular types. Algae are assigned to kingdoms based on evolutionary relationships. The more primitive algae (actually cyanobacteria) are placed in the Protista, and other algae are included with the plant kingdom.

Figure 1 Five kingdom system of classification 2.203

protists Branch Protomycota, funguslike protists Branch Protozoa, animallike protists

Kingdom Fungi Kingdom Plantae Kingdom Animali

The division of all living organisms into procaryote and eucaryote has been challenged recently by a number of biologists who claim that there is a third basic category. The organisms belonging to this new category are bacteria, that is, procaryotes. Now it is becoming clear that these bacteria are no more closely related to other bacteria (called eubacteria or true bacteria by these biologists) than they are to eucaryotic organisms such as animals and plants. The bacteria belonging to this new category look like typical bacteria and lack a nucleus and other membrane-bounded organelles. For years, however, it has been known that they are unusual in a number of ways. For example, their cell walls never contain peptidoglycan, they live in extreme conditions and their metabolism is unusual. Their genetic make-up is complete different from that of eubacteria. A classification allowing the archeabacteria is shown in Figure 2.

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LESSON 10 BASIC DESIGN AND CONSTRUCTION OF FERMENTOR AND ANCILLARIES

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The dotted arrows also explains another long held view that chloroplasts really originate from cyanobacteria, except that they are not free-living but really almost live independently, producing photosynthetic products for its host. Similarly, mitochondria are really bacteria-like separate entities within the eucariotic cell.

Figure 2 The 3 Superkingdom classification of living organisms Classification According To Nutritional Requirements

Organisms can also be classified according to their nutritional requirements. This form of classification is most appropriate to wastewater treatment as it describes the opportunities for application of these organisms. It also describes, in general terms, the conditions to be designed for if a certain organism is to be put to use. Classification according to nutritional requirements is shown in Table 1. Microorganism metabolism (principally a process of energy conversion) is sustained by redox reactions, providing the ultimate source of energy. The three major classes of these energy-yielding processes are: •

Respiration (aerobic), constituting the class of biological oxidation processes in which molecular oxygen is the electron acceptor;

•

Respiration (anaerobic), constitutes the class of biological oxidation processes in which inorganic compounds other than oxygen are electron acceptors;

•

Fermentation, constituting the class of energy yielding biologica redox reactions in which organic compounds serve as the final electron acceptors .

Table 1 Classification of microorganisms according to nutritional needs Class

Nutritional Requirements

Autotrophic

The organisms depend entirely on inorganic compounds for energy. These are divided into:

Phototrophic

Use radiant energy for growth.

Chemotrophic

Use oxidation of inorganic compounds as energy source. e.g. nitrifying bacteria.

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Heterotrophic

Organic compounds are required as nutrient.

Lithotrophic

Use inorganic electron donors (e.g. hydrogen gas, ammonium ions, hydrogen suIphate and sulphur) .

Organotrophic

Require organic compounds as electron donors.

Strictly Aerobic

Cannot grow without molecular oxygen, which is used as oxidant.

Strictly Anaerobic

Use compounds other than oxygen for chemical oxidation. Sensitive to the presence of traces of molecular oxygen.

Facultative Anaerobic

Can grow either in the presence or absence of oxygen. Can use nitrate or sulfate as electron acceptor.

The presence or absence of oxygen in water influences the products likely to be present and is of considerable importance as it limits the type of microorganism that can be active. Most biological systems used to treat organic waste, depend upon heterotrophic organisms, which utilize organic carbon as their energy source. Heterotrophic bacteria also play a role in water treatment and distribution. Chemotrophic bacteria are important in nitrogen removal and in corrosion processes during water distribution. Classification According To Identifiable Characteristics

Microbiologists like to classify organisms according to characteristics identifiable under a microscope, according to chemical reactions they may cause and/or their reaction to certain stains. All of these make routine identification easier to accomplish. Bergey’s Manual is the authoritative guide on this. Another very important group of organisms in the field of water and wastewater treatment are the pathogens. Pathogens are parasitic organisms that cause disease and include viruses, bacteria, yeasts, fungi, protozoa and even animalia, such as worms. Many of these are water-borne or involve vectors, such as mosquitoes, that breed in water.

Microbiological Aspects of Water Pollution Control Biological processes are used extensively to treat municipal and industrial wastewater. In advanced countries, the requirement of industry to implement biological treatment (secondary treatment) was the result of governmental regulations enacted over 30 years ago. Prior to that time, many industries used only sedimentation (primary treatment), which removes solids but is rather ineffective in removing dissolved organic substances. In the 1970s, the primary treatment facilities were supplemented with biological systems such as activated sludge and trickling filters. These processes have become sophisticated as a result of advances in process control and microbiology and are capable of removing over 90% of the dissolved organics from the primary effluent.

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Both procaryotic, or unicellular organisms that lack a true nucleus, and eucaryotic, multicellular organisms that have a membrane enclosed nucleus, are active in biological treatment systems. The bacteria are procaryotes. They are typically 0.5 mm wide and 1 mm long and exist as coccus, rod, and spiral shapes. Fungi are filamentous eucaryotes that have rigid cell walls that, unlike eucaryotic algae, lack chlorophyll. Microorganisms require both a carbon and energy source for growth. The pollutant serves as both the carbon and energy source for heterotrophic microorganisms. Autotrophic organisms use either light (photoautotrophs) or an inorganic compound (chemautotrophs) as the energy source and CO2 as the carbon source. Examples of the former are the algae and of the latter are the nitrifying bacteria that convert ammonia to nitrate: 2 NH4+ + 3 O2 è 2 NO2- + 4 H+ + 2 H2O 2 NO2-+ O2 è 2 NO3The bacteria converting ammonia to nitrite are usually Nitrosomonas. Nitrobacter further oxidizes the nitrite to nitrate. Temperature and pH are important parameters influencing growth rate. Each organism has a minimum, optimum, and a maximum temperature for growth. The three temperatures are referred to as cardinal temperatures. Psychrophilic organisms grow at low temperatures (0-20oC), mesophiles at higher (15 45oC), and thermophiles at the highest temperatures (40-70oC). Most microorganisms grow best at pH near neutrality (pH 7) but some are acid tolerant or acidophiles. Thiobacillus thiooxidans, a sulfur-oxidizing bacterium, is capable of growth at a pH of 1. In anaerobic environments byproduct formation is common. Incomplete dechlorination of pesticides has been found to occur both in the laboratory and in the natural environment. However, some chemicals, such as 3-chlorobenzoic acid, are capable of being completely mineralized to methane under anaerobic conditions. The effect of growth rate inhibition at substrate concentrations exceeding a toxicity threshold is shown in Figure 2. Such behavior has been observed with growth of bacteria on dichloromethane and pentachlorophenol (PCP), for instance.

From: Davis, M.I. and Cornwell, D.A. 1985 Introduction to Environmental Engineering. 2nd ed. McGraw-Hill Fig 5-6 p.323

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Microorganisms

Figure 2 Growth Rate Inhibition From: La Grega, MD., Buckingham, P.L., and Evans, J.C. 1994. Hazardous Waste Management. McGraw-Hill, Inc. Fig 10-13 p.578 Upsets or shock loads that result in abrupt increases in pollutant feed concentration commonly occur in industrial wastewater treatment. During such episodes, the increase in pollutant concentration in the biological treatment tank may result in growth rate inhibition and a further increase in concentration. When this occurs, flow to the system must be stopped until residual pollutant concentrations have decreased to below toxic levels. Reseeding of the system with microorganisms may also be necessary. The cell yield is the mass of microorganisms formed per nutrient, usually organics: dX/dt = -YdS/dt where Y is the cell yield, mg dry wt. cells/ mg pollutant and S is the substrate (pollutant) concentration. Values of the cell yield depend upon the specific type of organism, oxygen status of the environment, and carbon content of the pollutant. Under anaerobic conditions, less ATP is produced than under aerobic conditions resulting in a lower cell yield. Aerobic growth on compounds containing a relatively small fraction of carbon, such as pentachlorophenol, results in the production of fewer cells. The cell yield for PCP-degrading bacteria has been reported to be 0.15 mg dry wt/ mg PCB. By contrast, highly carbonaceous materials are metabolized with the production of high cell numbers. The growth yield on naphthalene is 0.7 mg dry wt/mg naphthalene. Pollutant Biodegradability

The public health threat associated with pollutants entering the environment will be greater if they are persistent. The half-life or time required for 50% disappearance of a chemical, is an indicator of susceptibility of the chemical to biodegradation. Half-lives may range from one day to several months depending on the environment and pollutant. Compounds that resist biodegradation are refractory or recalcitrant. Examples of recalcitrant compounds include DDT and PCBs. The factors that influence persistence are related to the indigenous microbiology of the environment and chemical structures of the pollutants. The following chemical structural parameters are normally involved:

Figure 1 Monod Equation

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1.

number of substituents

2.

type of substituents

3.

position of substituents

4.

degree of branching

5.

low solubility in water

6.

atomic charge difference

Chlorine is a common substituent, which imparts both toxicity and persistence. The incorporation of chlorine into an organic compound increases its longevity. In the examples given below the compound on the right is more persistent than the one on the left. Less persistent

More persistent

propionic acid

3-chloropropionic acid

monochloroacetic acid

dichloroacetic acid

phenol

mono-, di-, tri-, and pentachlo rophenol

benzoic acid

3-chlorobenzoic acid

monochlorobiphenyl

polychlorinated biphenyls (PCBs)

The type of substituent will influence pollutant residence time in the environment. Di-substituted benzenes with a carboxyl or hydroxyl replaced with nitro, sulfonate, or chlorine degrade more slowly. The position of the substituent also influences half-life of the compound in the environment as illustrated below.

0.5 mg alkene x (1 mmol cells/mmol alkene)(14 mg N/mmol cells) 96 mg/mmol)

= 0.073 mg N/ mg alkene The cell yield is: (0.5 mg) x (5x12+7x1+2x16+1x14) = (7x12 + 12x1alkene)

Less persistent

More persistent

o,p-chlorophenol

m-chlorophenol

o,p-chlorophenoxyacetic acid

m-chlorophenoxyacetic acid

o,p-amino,nitro,methoxy benzoic acid

Nutrient Requirements for Aerobic Growth Hazardous waste sites are often contaminated in the subsurface with petroleum hydrocarbons. Such contamination arises from petroleum spills on the surface or from underground tank leaks. Effective biological treatment of the contamination (bioremediation) requires the presence of oxygen. The oxygen requirements for degradation of an organic compound may be determined if assumptions are made regarding cellular composition and the fraction of organic compound oxidized for energy. Consider the oxidation of a branched alkene C7H12. The complete oxidation reaction is: C7H12 + 10 O2 → 7 CO2 + 6H2O If 1 mg alkene reacts, the O2 required is: 10(2 x 16)/[(7+12) + (12 x1)] = 3.33 mg O2/mg alkene Oxygen will also be required for the production of biomass C7H12 + 5O2 + NH3 → C5H7O2N + 2CO2 + 4 H2O 5(2 x 16)/((7x12)+(12 x1)) = 1.67 mg O2/mg alkene We assume that 50% of the hydrocarbon is oxidized and the remainder is converted into cell mass, thus 1 mg alkene requires (0.5)(3.33 mg O2) + (0.5)(1.67 mg O2) = 2.5 mg O2 The nitrogen required to produce cells:

mamino,nitro,methoxybenzoic acid

0.59 mg cells/mg

The cell formula containing P, C60H87O23N12P, is used to obtain the phosphorus requirement: (0.59 mg cells /mg alkene)x31 mg P = 0.0133 mg P/mg alkene (60x12+87+23x16+12x14+31 mg cells)

m-nitrophenol

p-nitrophenol

The yield on oxygen Yxo of the mass of microorganisms produced per mass oxygen consumed:

p-methylaniline

o-methylaniline

Yx/o = 0.59/2.5 = 0.24 mg cells/mg O2

Highly branched compounds are more resistant to biodegradation than straight chain compounds. The branched aliphatic acid, b-methylbutyric acid, is degraded more slowly than butyric acid. Water solubility may affect the rate at which pollutants are degraded. Many microorganisms, which grow on compounds of low solubility, utilize only the dissolved form of the pollutant. The growth rate then becomes controlled by the rate of dissolution of the pollutant. Crushing crystals of naphthalene, for instance, results in an enhanced growth rate of microorganisms, due to the higher available surface area at which naphthalene dissolves, making the naphthalene more available as substrate.

Methods of Anaerobic Biological Treatment

Anaerobic processes in industrial wastewater treatment are used mainly when high concentrations of organic material have to be removed or converted or for the further treatment of the organic sludges removed from other biological wastewater treatment. Simple forms of anaerobic treatment, such as anaerobic ponds and septic tanks, however, are used for treating wastewater (rather than sludge) although, even in these cases, the most intense anaerobic action takes place in the layer of concentrated sludge which settles to the bottom. Although the poor level of mixing, especially in the simpler processes, makes classification a little difficult, most conventional anaerobic processes are essentially suspended growth systems. The reason for choosing anaerobic over aerobic treatment should be based on financial considerations. Anaerobic processes usually have a higher capital cost than anaerobic processes due to the longer retention times required, but the cost of treatment is not significantly influenced by the organic

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Anaerobic Ponds.

Anaerobic lagoons are designed with a low surface area in relation to volume. The combination of this ratio and the high oxygen demand of the waste effectively prevents any part of the lagoon becoming aerobic except for a very thin layer at the top of the water during start up. A scum layer then forms above the wastewater which prevents algae from growing due to the absence of sunlight and further oxygen diffusion from the air. The by-products of anaerobic degradation include hydrogen sulfide and methane gas. The organic loading in anaerobic lagoons is controlled to minimise the evolution of malodorous gases. Anaerobic digestion is much slower than aerobic processes. However, in anaerobic ponds, a large portion of the colloidal particles undergo coalescence to form the scum and sludge which hastens the removal of the suspended BOD.

Figure 4.3 Simple anaerobic treatment systems: (a) anaerobic-aerobic lagoon system, (b) septic tank, (c) Imhoff tank Aerobic Treatment

The main objective of conventional treatment is to remove the dissolved and colloidal degradable organic matter that remains after primary treatment, so that the effluent can be rendered suitable for discharge. In many cases, reduction of the BOD to below 20 mg/L and SS to below 30mg/L is sufficient, and conventional secondary treatment can achieve this quality. Very often in industrial systems the objective is to pretreat the wastewater before sewer discharge to reduce municipal charges or to meet disposal restrictions. A second objective in some cases is the reduction of ammonia toxicity and nitrification oxygen demand in the receiving water body. This is achieved by oxidation of most of the ammonia to nitrate during treatment (nitrification). Nitrification is possible with aerobic biological processes if they are operated at long retention times - hence the units must be larger than those that would be required for oxidation of carbonaceous matter alone. These bacterial processes are commonly carried out on either aerated lagoons or in activated sludge plants. The design approach is similar, except that activated sludge plants incorporate a sludge recycle, which allows independent control of the solids retention time or sludge age. The mean cell residence time in aerated lagoons is therefore equal to the hydraulic detention time. To prevent washout of active bacteria, the detention time should not be too short - two to five days’ detention is the general order of the size of aerated lagoons. Activated sludge processes are commonly used industrial treatment systems and their design requires a closer study. Activated sludge processes are now the most widely used in newly installed municipal treatment systems and their design requires a closer study. Disadvantage of this approach is that the other product, which is formed out of the organic substrate, is bacterial biomass, with virtually no reuse value other than as a fertilizer and soil conditioner as the bacterial protein is not of particularly good quality. Design of Activated Sludge Systems

The activated sludge process design involves details of sizing and operation of the following main elements: a

aeration tank (reactor) - capacity and dimensions

b

aeration system - oxygen requirements and oxygen transfer system

c

final sedimentation tank (clarifier)

d

return activated sludge system

e

excess activated sludge withdrawal system and subsequent treatment and disposal of the waste sludge.

Since the whole process takes place in a liquid medium, the hydraulic regime, especially in the aeration tank and the final sedimentation tank, needs to be carefully considered. The main system elements are discussed below.

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loading. Aerobic processes have a higher operating cost, but this operating cost is mainly a function of the oxygen requirements and sludge production, ie aerobic treatment becomes progressively more expensive with increasing organic loading. The total cost usually overtakes that of anaerobic treatment at a BOD or COD of a few thousand mg/L.

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Porous Diffusers

These must be operated continuously, with an airflow less than that which could damage the diffuser but always more than the minimum recommended flow, so that clogging will not occur too rapidly. This requires the provision of standby air compressors, and diesel motors. Most diffused air systems require a supply of air at a pressure of 50 to 80 kPa. Porous diffusers have a theoretical oxygen transfer rate of about 2.8 kg/kWh. Porous diffusers may be made of ceramic material, and they are available in the shape of plates, domes or tubes. They may also be made of plastic material, either fabric or moulded. Fabric diffusers are generally lightweight and they can be readily cleaned. Extensive air filtering before porous diffusers is required to prevent clogging of diffusers. Baghouse systems, sometimes 2 or 3 consecutive filters, make the pre-treatment an expensive part of the system. Non-porous Diffusers

These have been developed with the objectives of a.

Elimination of diffuser clogging problems

b.

Reduction of equipment capital cost (loss in efficiency compared with fine bubble porous diffusers).

Many types of jet, valued orifice and turbulence disc are now available. A system which uses slotted pipes with a shallow immersion of about 0.9 m and a large volume of air at low pressure has been developed. Under these conditions, compressors can be replaced by blowers and the holes in the pipes are so large that they seldom block. Power efficiency is in the range of 1.0 to 2.0 kg/kWh. Vertical Shaft Mechanical Aerators

These have a rotor at the surface of the water, where the oxygen transfer takes place through surface turbulence, splashing and air bubble entrainment. The oxygen transfer capacity depends on the design of the equipment, the depth of immersion, the shape and size of the aeration tank, the energy input per unit volume of tank and the rate of rotation; some of these factors are interrelated. The oxygen transfer rates under average operation conditions are of the order of 2 to 3 kg/kWh. Horizontal Shaft Surface Aerators

Various types of brush and cage rotor have been designed. They provide not only aeration but also a horizontal pumping effect to maintain circulation and mixing. They may be used in tanks of moderate depth to provide a rolling circulation, but the most common use is in oxidation ditches and for supplementing oxygen supply in maturation ponds or oxidation ponds. A typical cage rotor of 700 mm diameter could be operated at a speed of 75 rev / min, to give oxygen transfer of 2.9 to 3.5 kg/ kWh, depending on depth of immersion. These rotors may be mounted on floats that slide in guide-boxes at each end. U-tube Aeration

In U-tube aeration, the partial pressure of the oxygen can be doubled or trebled by passing the water, with diffused bubbles entrained, through a U-tube with its lower portion 10 to 20 meters or more below the hydraulic gradeline. This increases

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the partial pressure 2 or 3x and hence the instantaneous saturation concentration, and speeds up the transfer of oxygen. For ease of construction, the downward tube may be inside the upward flow tube. Further increase of partial pressure of oxygen can be obtained by using pure oxygen. Turbine Aerators

These are submerged turbines with air released from spargers immediately below them. They have been used where the required oxygen input per unit of volume is greater than can be obtained with diffused air. Their oxygen transfer capacity is in the range of 1.5 to 2.0 kg/ kWh. There are also submerged selfaspirating aerators that draw the air through a hollow drive shaft (Kerag aerators). Oxygen Enriched Systems

Molecular oxygen can be used instead of air to increase greatly the partial pressure of the oxygen. If the atmosphere in contact with the sewage under treatment is oxygen, the partial pressure of the oxygen is 5x as large as in air and creates a much greater driving force to transfer it into the liquid. In this way, the saturation concentration at atmospheric pressure changes from 8 - 10 mg/L up to 40 or 50 mg/L. This makes it practicable to maintain higher concentrations of DO in the liquor. Where ‘pure’ oxygen is used for wastewater treatment, it has to be obtained from such a source as cylinders, bulk tankers or reticulated oxygen from suppliers, a cryogenic oxygen distillation plant at the works, or enriching air with ‘molecular sieves’.

Protein Production from Organic Wastes A major problem facing the world, in particular the developing countries, is the explosive rate of population growth. The world population now is annually increasing by approximately 94 million, and could well exceed 10 billion by 2050 if left uncontrolled. Conventional agriculture may be unable to supply sufficient food, in particular protein, to satisfy these demands. The Food and Agriculture Organization (FAO) predicts a widening of the protein gap between developed and developing countries. Currently, at least 25% of the world’s population suffers from hunger and malnutrition, a disproportionate number living in the developing countries. In view of the insufficient world food supply and the high protein content of microbial biomass, the use of biomass produced in fermenters would be an ideal supplement for animal feed. This would release plant proteins for human consumption and increase the animal protein supply into the human food chain. Development of methods to produce microbial biomass protein (MBP) has become an important field of study. MBP has a great nutritional value because of its high protein, vitamin, and lipid content and the general presence of a complete array of all essential amino acids. There are many advantages in biotechnological processing for MBP production. Microorganisms produce protein much more efficiently than any farm animal could, as indicated in Table 1 with the major advantages being: • Microorganisms can grow at remarkably high rates under optimum conditions; some microbes can double their mass every 0.5 to 1 hour;

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Microorganisms are more easily modified genetically than plants and animals, and more amenable to larger scale screening programs to select for high growth rate and improve nutrient content;

•

Microorganisms have a relatively high protein content with a good nutritional value;

•

Microorganisms can be grown, independent of climate conditions, in vast numbers, in relatively small continuous fermentation processes, using relatively small land areas;

•

Microorganisms can grow on a wide range of raw materials, in particular low value wastes, while some can also use plant derived cellulose.

Table 1 Time required to double the mass of various organisms Organism Bacteria and yeasts Moulds and algae Grass and some plants Chickens Pigs Cattle (young) Humans (young)

Time 20-120 minutes 2-6 hours 1-2 weeks 2-4 weeks 4-6 weeks 1-2 months 3-6 months

Many developed and developing countries, particularly Asian nations, have long recognized the nutritional value of some traditional foods produced by fermentation. During the last four decades, there has been a growing interest in the use of microbes for food production, in particular for feeding animals such as poultry. It has been argued that the use of MBP derived from low value waste materials for animal feed would remove some protein-rich vegetable foods from animal diets to become available for human consumption. MBP can serve as a replacement for traditional protein supplements such as fishand soy meal.

Suitable Substrates For Microbial Biomass Protein Production One of the most important factors for MBP production is the choice of substrate. If MBP is to be used to also treat wastes, the substrates should be suitable to sustain growth. We could grow autotrophs such as algae on wastewaters containing plant nutrients (such as from fertilizer wastewater) or heterotrophs on organic rich wastewaters. The latter could easily be classified into synthetic and renewable feedstocks and waste products from these. Chemical Feedstocks

Materials with a high commercial value such as energy sources or derivatives of these chemicals, e.g. gas oil, methanol, ethanol, methane and n-alkane, have attracted wide interest in the early stages. Many scientists have questioned the use of such compounds with a high energy potential for food production, including MBP, as health aspects. A few decade ago, many of the large-scale processes in operation were forced to close down because of health regulations and the high costs of materials used.

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n-Alkane

Alkanes, in particular those of intermediate size (C10 - C20) are the most rapidly metabolised hydrocarbons and were chosen as the substrate for the MBP projects of the 1960s. Alkanes can be catabolised by many yeasts and by some fungi (for instance, fungi of the orders Mucorales and Moniliales) as well as some bacteria (partially through cooxidation). The following yeast species have been intensively studied for MBP production: Candida tropicalis, C. oleophia, and Saccharomycopsis lipolytica (Fukui and Tanaka, 1981). Methane

In some parts of the world, there is an excess of methane, the chief compound of natural gas, making this a desirable energy source for MBP production. Methane can be obtained as a very pure gas. However, in contrast to higher hydrocarbons, methane cannot be liquefied commercially, making long-distance transport difficult and expensive. Considerable security measures must also be taken when handling methane, due to the risk of explosion. Methane as an MBP source has been extensively researched but is now considered to present too many technical difficulties to warrant exploitation. No methane based MBP production process has been developed on a commercial scale, to date. Methanol

Methanol was, for some time, the most important substrate for MBP production and extensive research on methanol utilizing organisms was carried out. Methanol, as a carbon source for MBP, has many inherent advantages over n-paraffins, methane gas and even carbohydrates; composition is independent of seasonal fluctuations. There are no possible sources of toxicity in methanol, it dissolves easily in the aqueous phase in all concentrations and no residue of carbon source remains in the harvested biomass fermentation for the commercial production of MBP. The “ICI Pressure Cycle Fermenter”, a combination of air lift and loop reactor, was installed in a continuous culture system with a capacity of 50,000 - 70,000 tons/y. Operation began in 1980, but proved to be uneconomical at the present high methanol prices and production has ceased (Hiotzman, 1986). Renewable, Biological Feedstocks Cellulose and lignocellulose

Cellulose is the most widely occurring organic material in nature, and the principal source of biomass and therefore of a renewable resource; it is a complex of three classes of polymer, which consists of repeating glucose units largely in crystalline fibers. This material, however, is generally difficult to hydrolyze by microorganisms. At the present time, cellulose from natural sources and waste wood is still an attractive starting material for MBP production as well as a potential source for the fermentation of ethanol. Although there is an abundance of cellulose on earth, cellulose is usually mixed with substances such as lignin, hemicellulose, starch, protein, and salts. Therefore, the cellulose source must be pretreated physically and chemically in order to break down the cellulose into fermentable sugars. Pretreatment may either be enzymatic (cellulose) or chemical (acid) hydrolysis. Production of MBP directly on cellulose

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material has been very rare. There are some major difficulties and disadvantages associated with direct use of cellulose: •

Low solubility of the raw material dictates use of very dilute culture media;

•

Low utilization rates lead to low growth rates, low productivity, and low dilution rates in continuous cultivation;

•

high concentration of solids in the culture medium requires high energy input for mixing and reduces the relative protein content in the biomass.

Starch is a glucose polymer and is one of the most widely available plant polysaccharides. In the biosphere, carbohydrate matter, including starch and cellulose, exceeds the combined amount of all other organic compounds. Starches are manufactured from corn (maize), wheat and potato. The proportions of these ingredients not only vary greatly between raw materials, but also between the different grades of raw materials. A typical analysis of three starch materials is shown in Table 2 (McNicol et al., 1972). Table 2 Typical analysis of selected starches containing raw materials Corn 60.0 9.0 15.0 16.0

Wheat 55.3 13.2 18.7 12.0

Potato 15.0 2.0 4.0 79.0

Wastes from Renewable Feedstock Processes

The definition of “waste material” is changing continuously since more and more re-use is replacing simple disposal. Byproducts that have generally been considered as waste are now in routine use. A suitable definition should take into consideration alternative economical utilization, local pollution control regulations and legislation, and the cost of waste treatment. A major factor in the cost of MBP production is the raw materials and the efficiency of their conversion into MBP production. Waste materials should normally be recycled back into the ecosystem, e.g. straw, bagasse, citric acid, olive and date wastes, whey, molasses, animal manures and sewage. The amount of these wastes can be very high in specific locations and may contribute a significant level of pollution to water courses. Thus, the utilization of such materials in MBP production serve two functions - reduction in pollution and production of edible protein. For MBP production to be economical during waste effluent treatment, certain requirements must be met: 1)

Utilization of all the available nutrients in the treated effluent The primary goal is BOD and COD reduction, and a suitable microorganism with high density growth and productivity rate must therefore be selected.

2)

Sterilization of the waste should be avoided Decontamination, if required, should be carried out using low-cost methods. Methods applying high inoculum, cell recycling, low pH values, and low concentrations of carbon source can be

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3)

Inexpensive preparation of the end product (commercial biomass protein) Recovery should avoid the use of expensive techniques such as centrifugation. Sedimentation, flocculation/flotation, and simple filtration, are usually useful in the first step of product recovery.

4)

Reduction in cooling or heating costs A microorganism that grows at a similar temperature as the treated waste is of great advantage.

Whey

Starch and Starch Wastes

Composition(%) Starch Protein Fat and other Moisture

used for achieving a high disappearance rate of nutrients from the growth system.

Whey is a by-product of cheese-making and contains both protein and lactose. The waste from large scale cheese plants can become a serious environmental problem if it is discharged without treatment. Utilization in the production of MBP was the subject of extensive study (Moulin & Galzy, 1984). One original solution concerned yeast production from crude sweet whey using Candida kefyr LY 496 in batch or continuous culture processes (Hitzman, 1986). Many cheese companies in Europe have successfully used Kluyveromyces fragilis as the organism to produce MBP. This process, however, has to compete with fractionation of the whey as its component lactose and protein (Pellon and Sinseky, 1983). The process also depends on large scale plants giving steady effluent as a source of raw material. The effluent is generally dilute and high transportation costs may be involved in the collection of the whey. This situation has been one of the major reasons why whey is still regarded as an unattractive substrate for MBP production (Moulin and Galzy, 1984). Spent Sulfite Liquor

Spent sulfite liquor (SSL) is an effluent from pulp and paper mills, a sugar-containing waste product. These effluents have been identified as a possible source for the production of MBP. Romantschuk (1975) cultivated Candida utiliz for MBP production using SSL as the substrate. A reduction in sulfite processing and its replacement by the sulfate process has tended to reduce the available effluent and closed some USA facilities for MBP production (Vasey and Powell, 1984). Pretorius et al. (1993) investigated a continuous system of selective cultivation of the thermotolerant Aspergillus sp. on SSL for MBP production.

Selection of Suitable Microorganisms for Microbial Biomass Protein Production Most MBP processes are designed to take advantage of an available, readily degradable substrate using a microorganism. The choice of the MBP microorganism is usually limited to that particular process, and a change in substrate often necessitates a change in the type of microorganisms used. Several factors are involved in the choice of a microorganism for the MBP production. Hamphrey (1974) summarized five criteria for microorganism selection: safety, use, digestibility, growth rate, and maintenance. Apart from substrate cost, operating expenses, and capital investment for process equipment, other important factors in MBP production are the protein content and the quality of the MBP. The chemical composition of bacteria, yeast, fungi and

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not maintained during the large scale MBP production. The potential contamination of algae cultures grown in sewage oxidation ponds by enteric pathogenic bacteria and viruses, therefore, must be given serious consideration (Cooper, 1962).

Table 3 Protein and Nucleic Acid Content of Various Microorganisms

Filamentous fungi play an important role in the food industry, for example in the addition of flavor to certain mould cheeses and acting as a major protein source as food additives and extenders. These fungi are also used in the production of Oriental foods and to improve the protein contents of animal feeds. Wainwright (1992) summarized various fungi that are directly used in food processing, shown in Table 4.

Microorganism Yeast Algae Bacteria Fungi

Protein (%) 45 - 55 47 - 63 50 - 83 31 - 55

Nucleic Acid ( % ) 6 - 10 4-6 10 - 16 2.5 - 6

Protein quality and quantity are the goals of MBP production, although the microorganisms also contain carbohydrates, fats, vitamins and minerals. The most important factor in microorganism selection is safety. The organism must be known to be non-pathogenic and must not produce toxins. Various groups of microorganisms, including bacteria, yeasts, algae and fungi, have been considered for use as a source of protein, the dried cells of these microorganisms being referred to as MBP. Bacteria

Various species of bacteria can utilize a wide range of carbon and energy sources, including sugars, starch, cellulose (either in pure form or as agricultural or forest product waste) hydrocarbons and petrochemicals. Rapid growth rates and high protein contents make bacteria prospective candidates in the MBP production (Bhattacharjee, 1970). The generation time of bacteria is only 20 to 30 minutes compared with 2 to 3 hours for yeast and 16 hours or more for algae, and fungi (Litchfield, 1979). Semi - batch and continuous MBP processes using bacteria are suitable for industrial waste recovery. The conversion efficiency of substrates into MBP by bacteria is very high (0.8 - 1.2 g /g substrate) (Litchfield, 1979). Yeasts

Yeasts can be utilize many substrates for MBP production. These substrates include n-alkanes, methanol, ethanol, diesel oil, gas oil, brewery waste, sulfite waste liquor, starch, anaerobic digester supernatant, molasses, cheese whey and domestic sewage. The species of yeasts most commonly used as MBP include Candida, Hansenula, Kluyveromyces, Rhodotorula and Torulopsis, and particularly Saccharomyces cerevisiae. Because of high MBP yields, wide utilization of substrates, high quality of protein, easier and inexpensive harvesting, yeasts have become the most favourable characteristics for use as a major MBP food.

Cultivating fungi as a high protein food is especially attractive. The major advantages of fungal cultures are summarized as the following: •

Fungal cells contain reasonably high levels of protein;

•

Fungi contain less nucleic acid than yeasts and bacteria;

•

The filamentous nature of fungal mycelia facilitates recovery of fungal MBP from fermentation broths and its physical properties make it acceptable for human consumption;

•

Food produced from fungi is traditionally eaten in many parts of the world.

Table 4 Direct food uses of fungi Application Edible macrofungi Common edible mushroom Shii - take Chinese or straw mushroom Winter mushroom Oyster mushroom Truffle Cheeses Roquefort, Stilton, ‘blue’ Camembert, Brie, soft ripened cheese Oriental food fermentation Ang-kak Hamanatto Miso Ontjom Shoyu ( soy sauce ) Tempeh

Species Agaricus bisporus Lentinus edodes Volvariella volvacea Flammulina velutipes Pleurous sp. Tuber melanosporum Penicillium roquefortii Penicillium camembertii Monascus purpurea Aspergillus oryzae Aspergillus oryzae / A.sojae Neurospora intermedia Aspergillus oryzae / A.sojae Rhizopus oligosporus R. arrhizus, R. oryzae

Yeast Culture

Algae

Algae are photosynthetic microorganisms and thus require either sunlight or artificial illumination. They can be grown either photosynthetically or heterotrophically. Autotrophic growth involves in using carbon dioxide, whereas heterotrophic growth occurs in the dark with organic carbon and energy source. Since illumination is the limiting factor in photosynthetic algal growth, outdoor cultivation is restricted to the use of shallow pond site between latitude 35ºN and 35ºS (Litchfield, 1979). Aseptic conditions with algae in ponds are

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Fungi

Since yeasts can grow rapidly on a wide range of substrates including waste products, they have the potential to provide an extremely versatile protein. A large number of amylolytic yeast species have been screened for MBP production from starch materials to obtain strains that can convert starch directly in a monoculture or improve the performance of Sacchromycopsis fibuligera in a symbiotic system. About 90 species of a total 400 yeast species currently recognised are capable of utilizing starch as a sole source of carbon and energy (Robyt, 1984).

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BIOPROCESS ENGINEERING

algae vary, depending upon the genera and growth condition. The gross chemical composition of various microorganisms is presented in Table 3 (Oura, 1983). Although the protein content can be varied by growth conditions, genetic manipulation can also be employed to alter the amino acid spectrum.

BIOPROCESS ENGINEERING

Table 2-5 Production of MBP by yeast cultivation from starch materials species

a

strain

substrate

process

µ (h

C. utiliz C. tropicalis L. kononenkoae S. cerevisiae Sa. fibuligera

Sc. occidentalis

Sp. holsaticus

NCYC 707 NRRL1048 CBS6948 IGC4052 ICG4052B BRG 530 NRRL Y76 NRRL1062 IMAT3812 ATCC2607 IGC 2829 UCD54-83 IMAT2196 CBS 2863 FRI Y-5

potato waste soluble starch cassava corn soluble starch soluble starch soluble starch corn starch soluble starch potato peeled potato soluble starch soluble starch soluble starch potato peels cassava flour soluble starch

batch batch continuous continuous batch continuous batch batch batch continuous batch batch batch batch batch batch batch

T −1

0.50 0.14 0.26 0.10 0.12 0.04 0.35 0.35 0.21 0.25 0.23 0.14

)

o

( C) 30 23 35 32 25 28 25 30 30 32 28 32 30 30 28 28 23

yield (g/g ) 0.55 0.61 0.42 0.74 0.58 0.48 0.50 0.54 0.50 0.84 0.40 0.51 0.59 0.62 0.54 0.44 0.43

Abbreviations for genera: C. = Candida; L. = Lipomyces; S. = Saccharomyces; Sa. = Saccharomycopsi; Sc. = Schwanniomyces; Sp. = Sporobolomyces. Fungal Culture

Because of the properties of easy harvesting, low nucleic acid content and acceptability as traditional food, filamentous fungi have become more and more attractive in MBP production and biotechnological waste treatment processes. Mixed Cultures

In many practical fermentation processes, the cheapest substrate is always used, and this will not be of the highest purity and will often be a mixture of several materials in large quantities. It would be useful, for example, a mixed culture can be found that not only degrades cellulose but also starches and sugars. Conclusion

Experimental results indicated that the design of wastewater treatment aimed at microbial biomass protein production should be different from the design of conventional biological wastewater treatment systems. Conventional systems are typically designed to achieve a certain effluent quality, but the major design criterion for systems using yeast cultures should be the prevention of bacterial contamination. Review Question •

What are the different suitable substrates for microbial biomass protein production?

Notes:

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