Production of Biofuels – Biosafety Issues, Bioreactors, Biopiracy and Patents

Microbiology is one of the Biology Topics that involves the study of microorganisms, including bacteria, viruses, and fungi.

Production of Biofuels Options by Contribution of Effective and Suitable Enzymes

A biofuel is a product of biological organisms that can substitute or enhance existing fuels. The production of biofuels has the potential to provide an alternative energy source and reduce global warming resulting from the burning of fossil fuels.

Today there are two commonly produced biofuels: ethanol from corn or sugarcane and biodiesel from oil crops such as soybeans or oil palm. Ethanol is added to gasoline to improve the emission of carbon dioxide and other greenhouse gasses from the combustion of gas. Because ethanol is produced from corn or sugarcane means that the resource is easily replaced year after year. The other major biofuel in use is biodiesel, which is made by taking oil either vegetable oil from soya beans or animal-based fats, and converting it into fuel by adding alcohol. This fuel is not an additive but replaces regular diesel fuel completely. Recombinant DNA technology is being used to produce E. coli with an increased ability to produce ethanol. The creation of biorefineries is in progress in which leftover biomass from conventional crops such as corn husks and stalks, wood waste such as chips, sawdust, and yard clippings would incorporate microbial enzymes to process sugars with great efficiency from biomass into fuels.

Biosafety

The purpose of biosafety is to give protection to humans and the environment from any harmful effects generated by biotechnological procedures. Zaid (2001) tried to give a definition of biosafety and according to him, biosafety is the avoidance of risk to human health and safety and of degradation of the environment due to the use of biotechnological procedures and products in research and commerce.

During the last part of the 20th century, people considered the destruction of habitats and the environment, a great concern, and biosafety has gained prime importance in the scenario of human development. Countries all over the world give much importance to the conservation of biodiversity and the protection of human health. Convention on Biological Diversity (CBD) held in 1992 discussed on conservation of biodiversity and its judicious use for sustainability. The convention also discussed research, the use of GMOs, and their commercialization.

In 2003 Cartagena Biosafety Protocol was recognized. About 138 countries of the world signed the protocol upto 2007. The objectives of this protocol were to give protection to human health and to conserve biodiversity making it enjoyable equally to all humans of the world. The Indian government started adoption of guidelines of this protocol in January 2003 and the Government has taken two issues into important consideration in order to protect human health-

• GMOs should be protected from their uncontrolled use.
• The indigenous concerns of an Indian country must not make storage of seriously harmful GMOs or toxic materials of GMO origin.
• It is to be pointed out that GMOs and their products are used sometimes as weapons against men and countries.

Some regulatory bodies have been framed to look after these matters. These statutory bodies control research on genetically modified organisms and their use. The Recombinant DNA Advisory Committee (RDAC) is a statutory body like this. Institutional Biosafety Committee (IBSC), Review Committee on Genetic Manipulation (RCGM), and Genetic Engineering Approval Committee (GEAC) are three other committees that give approval to research programmes related to recombinant DNA technology. State Biotechnology Coordination Committee (SBCC) and District Level Committee look after (DLC) the use of biotechnological products. In this regard, the name of several guidelines for biotechnology may be mentioned as under-

• Recombinant DNA safety Guidelines, 1990.
• Revised Biosafety Guidelines, 1994.
• Revised Guidelines for Research in Transgenic Plants, 1998.
• Guidelines for Generating Preclinical and Clinical Data for rDNA Vaccines, Diagnostics and Other Biologicals, 1999.
• Guidelines for the Conduct of Confined Field Trials for Regulated, Genetically Engineered Plants in India and Standard Operating Procedures (SOPs), 2008.
• Guidelines for the Safety Assessment of Foods Derived from Genetically Engineered Plants in India, 2008.
• Protocols for Safety Assessment of Genetically Engineered Plants, 2008.

Regulatory Process for Use of Genetically Modified Plants

At the initial stage Genetically Modified plant remain confined to the laboratory and it comes under field trial. For a field trial of a GM plant permission is necessary from the respective authority. The Institutional Biosafety Committee (IBSC) on the basis of reports on laboratory tests in greenhouse experiments issues permission for field trials. On the basis of a recommendation from IBSC, RCGM (Review Committee on Genetic Manipulation) gives permission for a first-level field trial (For Biosafety Research Level-1). A person may carry out a field trial for the plant for about one year. After this first level trial, a trial for Biosafety Research Level II may be carried out. To carry out a trial for Biosafety Research Level II permission from GEAC (Genetic Engineering Approval Committee) is required. To complete investigations on BRL-I and BRL-II about 3 years time period is required. However, prior to BRL-I testing some information should be necessary and these are-

• Description of the GE plant.
• Description of its natural counterparts.
• Description of genetic modifications.
• Information regarding possible toxicity or allergic reactions.
• The pattern of inheritance of the new character in the GE plant.

Field trial of any GE plant gives information about risks and hazards in cultivating the plant. The tolerance of the GE plants to live in the environment and their quality in comparison to the normal counterpart can also be assessed in field trial. Besides these, whether the GE plant has any detrimental impact on other plants and whether the protein produced in the plant has any toxic effects on the domestic animals as well as its utility etc., may be known from field trials. Having known all this information the consistency between the information obtained from laboratory investigation as well as greenhouse test and that obtained from field trial may be ascertained. To know the safety aspects of GE plants as following issues must be taken into consideration.

• Immediate oral response.
• The efficiency of being digested by pepsin.
• Toxicity on laboratory animals like rats.
• Testing on domestic animals.
• Information on the molecular organization of the component.
• Information on the evolutionary aspect of new traits.
• Stability of the imposed character.
• The structure of the protein is produced as a result of the expressed character.
• The efficiency of the GE plant to survive in the environment and its reproduction.
• Impact on other plants and animals.

The data obtained on the above aspects are considered to assess the fitness of the GE plant towards open and wide cultivation. In India, the GE plants that have got approval for field trial or commercial cultivation may be shown in the following table.

GM plants are obtained for cultivation and field trial:

Stage of Approval of GE Plant	GM Crop Plant
1. Commercial Cultivation	1. Bt Cotton
2. Open cultivation in the environment	2. Bt brinjal
3. Field trial	3. Brinjal, cauliflower, cabbage, ground nut, maize, mustard, pea, potato, tomato, cotton, okra.

Several GE Plants in the Light of Biosafety

A. Bt brinjal:
Bt brinjal plant has been produced by inserting the cry IAC gene of Bacillus thuringiensis in the natural brinjal plant. This genetically modified brin¬jal plant may produce protein in the plant body that is toxic to the insect pest. The fruit and stem borers can not attack the plants because of this toxic protein. This GE plant was developed by the year 2000 and its greenhouse testing was also completed by this time, it was found that the toxic protein has no detrimental effect on other animals. As per the recommendation of RCGM field trials of this plant continued for six years and based on reports of field trials RCGM sent its recommendation to GEAC in 2006. In 2009 GEAC gave approval for open cultivation of GE brinjal plant. However, Government approval for its cultivation was obtained by February 2010.

Biosafety-related information as obtained on Bt brinjal are the following.

• Cry IAC gene product Bt protein acts against only lepidopteran insects.
• The gene produces the protein throughout the whole life of the plant.
• The protein and GE plant have no harmful effect on soil micro-organisms and other plants.
• Bt protein has no adverse effect on man and other animals. Only by 30 seconds Bt protein gets digested in the intestine by digestive juice.
• The cry IAC gene remains in the GE plant with stability and it cannot be transferred to other plants.

B. Bt cotton:
Bt cotton also contains the cry IAC gene of Bacillus thuringiensis. As a result, the Bt cotton plant may produce Bt protein that is toxic to insect pests. Such GE cotton is pest-resistant and profitable in cultivating the plant. Bt cotton has got approval for open cultivation at the Government level.

Biopiracy and Patent

Biopiracy and patents are two interrelated terms. Biopi¬racy means an unlawful claim of a substance of others. On the other hand, the patent is a legal procedure through which the claim on a substance may be established. Due to such legal aid, the substance of another’s possession may be shown as the property of a particular individual. In the case of biopiracy, the matter claimed by some person is related to some living organism. The matter may be an organism (plant, animal, or microorganism), a product of any of these organisms, a gene from such organism, an engineered organism, or a process used by an organism. Considering all these aspects a definition of biopiracy may be given.

Biopiracy
Biopiracy refers to the unlawful patenting of biological matters; some organism or bio-chemical product of the organism, the gene of the organism, or a biological process that is used by some other person or country without proper permission. As a result, the organism or its product in possession of another country or person becomes the right of another concern. The native country or local people lose the legal right to use them.

Some Examples of Biopiracy
Many examples of biopiracy may be obtained from poor and developing countries full of biodiversity. India is a country with great biodiversity. By population, it is the second-largest country in the world. It contains about 1.2 billion people with four hundred languages and thirty religions. In India, there are 45500 plant species and about 91200 known animal species. Among the four main biodiversity hot spots of the world as East Himalayas, Indo-Burma, the Western Ghats, and Ceylone, two hotspots are present in India. For this reason, except the rich countries of the world, most of the multinational concerns in the world have a greedy look upon India. Probably because of this, many cases of biopiracy may be observed in India.

1. Basmati is a type of good-quality scented rice produced in the foothill areas of the Himalayas. The common farmers of Northern India not only cultivate this rice but they also know details of its qualities and cultivation. A company in Texas named Rice-Tec. Inc. in the United States produced text through the hybridization of Basmati and ordinary dwarf variety rice. The concern claims Texmoti as their own property and they also patented this rice. Therefore, patenting of this rice is not only illegal but also a deprivation of the Indian people economically.

2. A company in America patented Atta formed from wheat.

3. Neem (Azadirachta indica) is a known beneficial Indian plant from the very old days. The people of India are known for the useful property of this plant. However, some multinational companies produce extracts from neem and have patented the products in their name. W. R. Grace, an American company patented an oil of neem in 1994. The company claims that the patented neem oil controls fungi.

4. In various parts of India regional medicinal plants are protected and cultured by the local people. But pharmaceutical companies use these plants for the preparation of medicines and they use the experience and knowledge of the native people in marketing the medicine. As a result, the rich organization may earn huge amounts of money with sufficient profit, but the local people in return get nothing. This incidence is equivalent to biopiracy.

5. Two Indians based at the University of Mississippi Medical Centre obtained a patent against the wound-healing property of curcumin in 1995. But so to say it is not a new discovery because this property of curcumin is known to the Indian people from the very remote past. Therefore, this patent for curcumin is a biopiracy.

Alarming Sides of Biopiracy

Biopiracy is a type of theft and therefore, it appears to be alarming for many reasons as

• A person or a concern in the process of biopiracy focuses on the national property and cultural knowledge of local people as their own property.
• Because of biopiracy local people are deprived of random and easy use of the genetic wealth of the locality with their acquired knowledge.
• Biopirates or thieves by obtaining patents obtain gain and unlawfully deprive common people of the benefit from the medicinal plants.
• Because of biopiracy, the livelihood of local people is affected.

Measures to Prevent Biopiracy in India

Biopiracy has a potential negative impact on the national economy and therefore, to protect against economic degradation of the country, the Indian Government has been alert with various measures. Council of Scientific and Industrial Research (CSIR) and Ayurveda, Yoga, Unani, Siddha and Homeopathy (AYUSH) have been very conscious of preventing Biopiracy. These two organizations of India have come to an agreement with the European Patent Office, the American Patent and Trademark Office, the Intellectual Property Office of Canada, the German Patent Office, and the United Kingdom Patent Office in order to prevent biopiracy. CSIR has formed the Traditional Knowledge Digital Library (TKDL) which has described all the traditional knowledge on medicinal plants with a listing of all medicinal plants in India. When a person or any concern applies for a patent on herbal products, the same may be scrutinized with the help of TKDL and may be considered in the light of biopiracy.

If any indication of biopiracy is detected, the application for a patent will be rejected then and there. The traditional knowledge is recorded in many languages in India and TKDL has recorded them in English in this digital library. All the countries that issue patents may enter this TKDL and judge the appeal of a patent for biopiracy. This measure may control biopiracy substantially. Besides this globally an agreement has been prepared which is named the Convention of Biological Diversity (CBD) which provides protection to the Biodiversity and genomic property of nature. About 190 countries have signed this CBD agreement with the condition that all the countries will protect biodiversity conjointly. World Trade Organization (WTO) is an international organization that is also committed to giving protection to biodiversity throughout the world.

Unlawfully patenting of traditional knowledge and genetic property of India has now come under judgment. When a case of biopiracy and patenting of genetic wealth comes to the knowledge of the Government, the issuing country of the patent is informed about the case of biopiracy through TKDL, and the concerned country is requested for reconsideration of the patent. With such measures, several patents such as biopiracy have been null and void. Basmati, Neem, and Curcumin patents are some of the invalid patents. Besides, very recently some patent applications have been rejected. Several such examples are-

• The patent application of Babul (which acts as a remedy for constipation, diabetes, and indigestion) was filed in 2006 by Jaffe Russel M.
• Patent application for Tulsi (used as a remedy to skin and heart disease as well as diabetes) filed by Helen Schweitz.
• Patent application for Ashwagandha (used in the treatment of anxiety, insomnia, and fatigue) filed by Natreon Inc. USA.

Patent

A patent is a right granted to a person (inventor) by the Government for a new useful invention. New inventions such as tools, a product of the tool or machinery, or a composition formed by mixing different substances may appear for obtaining a patent. In the case of biotechnology, engineered plants, animals, or microbes may obtain patents. Any matter which was present before cannot be eligible for patenting. Naturally available plants or animals and scientific principles always remain outside patenting.

The term ‘patent’ is derived from ‘patere’ which means to expose i.e., to make a matter exposed to the common man. It denotes a type of royal recognition by which a person achieves the legal right to enjoy the benefit of the matter. Through registration, a person achieves the legal right of a bond and it becomes his own property, so a new invention may be the property of a person through patenting. Such property is called intellectual property. Except for the person obtaining the patent on a matter nobody will be able to use the matter without permission of that person.

In 500 BC patenting was first introduced in Greece. However, in the present form patent came into force in 1474 in Venice. In England, patenting was introduced during the time of King Gems-I. Usually for some new invention patent is given to any person or concern. In North America patent was given to Samuel Winslow in 1641 for the invention of a novel method of salt preparation. Subsequently, the system of patenting has been introduced in other countries of the world.

Patent and Stability of Right on it
In most cases, the right to a patent lasts for 20 years. Within this period another person cannot use the patent. However, on proper permission, the patented matter may be used by others. After 20 years the patent becomes the property of general people and the right of any person on the patent will be invalid. In some countries, the validity of a patent may be less than 20 years. For example, in the United States of America, the duration of a patent right is 17 years.

The Items or Subjects Having Patentability
Usually, a new invention that has some industrial application gets eligibility for obtaining a patent. But if the invention occurs based on some previous or existing knowledge or an invention becomes a part of biodiversity, that is unsuitable to get a patent. However, for obtaining a patent the conditions that should be fulfilled are-

• The invention should not be a scientific principle.
• The invention should have some industrial application.
• The matter should not be part of traditional knowledge and should be new without previous existence.
• Usually, if the matter is a part of biodiversity it is not eligible to obtain a patent. However, an organism that appears as a product of biotechnology is eligible for patenting.
• A new product or procedure developed for preparing a substance, tools invented and any conversion of the existing tool or method are eligible to obtain a patent.

Procedure for Obtaining a Patent
By patenting the right to a matter and invention is developed as per approval of the Government. Therefore, to obtain a patent the inventor has to apply for a patent to the concerned office. The patent examiner reviews the patent application and determines the eligibility of the matter for obtaining a patent. Only after that, the patent is issued to the inventor applicant. The step in obtaining a patent may be shown.

Legal Protection to a Patent
A patent is always related to new inventions and therefore, the matter of a patent is the result of the thinking of a person. For this reason, it is considered intellectual property. This intellectual property is protected by law in every country and in every state it is protected by National Patent Laws. On the basis of the patent as the invention, a person gets legal recognition due to which the inventor may use it as his own property as applicable for immovable properties like land, houses, ponds, hotels, cinemas, etc. Like immovable property intellectual property may be sold. Therefore, with permission from the inventor, intellectual property may be used by other people. Throughout the world, the patent is regulated by common regulatory principles. For this purpose, WTO (World Trade Organization) is alert about the issue of patents. WTO countries are bound by Trade Related Intellectual Property Rights (TRIPS) and the member countries have to inform WTO about the patents issued by the country.

Advantages and Disadvantages of Patent
On the basis of patent, as the patent holder gets some advantage, also it has some disadvantageous aspects. The advantages and disadvantages of a patent may be indicated in the following manner.

Advantages	Disadvantages
1. Possession right for a patented matter is valid for a limited period.	1. After a stipulated time, the patented matter becomes the property of the general people.
2. Administrative control on a patent is easy to regulate.	2. Disputed right on a patent is expensive to give protection to a patent.
3. Patented matter or object is much protected from misuse.	3. Non-patented matter very often faces misuse.
4. The owner of a patent may be benefited economically.	4. Patented matter sometimes goes beyond the reach of general people.

Bioreactors

Bioreactors are large chambers or vats used to grow organisms such as yeast or bacteria so that the biotechnological product (for example, organic or amino acids, antibiotics, enzymes, vaccines, or alcohol) can be harvested on a large scale. Living transgenic animals like genetically engineered cows or goats can also serve as “bioreactors” to produce desired pharmaceutical proteins.

Characteristic Features
In all forms of fermentation, the ultimate aim is to ensure that all parts of the system are subject to the same conditions. Within the bioreactor, the microorganisms are suspended in the aqueous nutrient medium containing the necessary substrates for the growth of the organism and required product formation. All nutrients, including oxygen, must be provided to diffuse into each cell, and waste products such as heat, carbon dioxide, and waste metabolites removed.

Fermenting vessels allow scientists to control and monitor optimal growth conditions such as temperature, pH, substrate, nutrient concentration, and cell density for achieving the desired product. Fermenters do not require oxygen when anaerobic organisms are fermenting nutrients. Bioreactors use non-fermenting organisms that require oxygen. For each biotechnology process, the most suitable containment system must be designed to give the correct environment for optimizing the growth and metabolic activity of the biocatalyst.

Types of Bioreactors
Bioreactors occur in two main types.

• Non-aseptic systems where it is not absolutely essential to operate with entirely pure cultures, e.g., brewing, and effluent disposal systems.
• Aseptic conditions are a prerequisite for successful product formation, e.g., antibiotics, vitamins, wine, beer, and cheese.

Operation of Bioreactors
Small-volume cultures cannot yield large quantities of desirable products, therefore to assist large-scale production, Bioreactors are necessary. Bioreactor operation is either batch-wise or continuous. Batch-wise operation or batch cultures include all the nutrients required for the growth of cells prior to the cultivation of the organisms. After inoculation, cell growth commences and ceases once the organisms have exhausted all the available nutrients in the culture medium. A modification of this type of operation is a fed-batch or semi-batch operation in which the reactants are continuously fed into the bioreactor, and the reaction is allowed to go to completion, after which the products are recovered. Continuously operated bioreactors, use “continuous culture systems” that continuously feed culture medium into the bioreactor and simultaneously remove excess medium at the same rate. Batch-culture bioreactors work best for fast-growing biological organisms. Slow-growing organisms usually require continuous-culture bioreactors.

Factors that Influence the Bioreactor-Based Operations
Several factors influence the success of bioreactor-based operations.

• Choosing the right strain to make the desired product is essential.
• The culture medium and growth conditions must optimize the growth of the chosen organism.
• Supplying the culture with adequate oxygen requires the use of agitators or stirring equipment that must operate at high enough levels to aerate the culture without severely damaging the growing cells.
• The bioreactor must have sensors to measure accurately the physical properties of the culture system, such as temperature, acidity (pH), and ionic strength.
• The bioreactor must be integrated into a network of peripheral equipment that allows automated monitoring and adjustment of the culture’s physical factors.

Fermentation reactions are multiphase, involving a gas phase (containing N2, O2, and CO2), one or more liquid phases (aqueous medium and liquid substrate), and a solid microphase (the microorganisms and possibly solid substrates). All phases must be kept in close contact to achieve rapid mass and heat transfer. In a perfectly mixed bioreactor, all reactants entering the system must be immediately mixed and uniformly distributed to ensure homogeneity inside the reactor.

The performance of any bioreactor depends on many functions, such as those listed below:

• Biomass concentration (in a growth-associated bioprocess, the biomass concentration must be high enough to achieve a high product yield).
• The culture volume should remain constant, i.e., no leakage or evaporation.
• The dissolved oxygen level must be maintained above critical levels of aeration and culture agitation for aerobic organisms.
• Environmental parameters such as temperature, pH, etc., must be controlled and the culture volume must be well mixed.
• Sterile conditions (sterile conditions must be maintained for a pure culture system).
• Effective agitations (effective agitation is required for perfect mass transfer and uniform distribution of substrate and microbes in the working volume of a bioreactor) .
• Heat removal (efficient heat transfer is needed to operate the bioreactor at a constant temperature, as the desired optimal microbial growth temperature).
• Correct shear conditions (high shear rate may be harmful to shear-sensitive organisms and disrupt their cell wall; low shear may also be undesirable because of unwanted flocculation and aggregation of the cells, or even growth of organisms on the reactor wall and stirrer.
• Nutrient supply should be well-controlled and desired product should be removed to keep metabolisms/microbial activities at optimal levels.
• Bioreactors may be of different types. However, the Stirred Tank Bioreactor is more popular.

Stirred Tank Bioreactor

A stirred-tank bioreactor is either cylindrical in shape or has a curved base to help proper mixing of the contents.

The stirring equipment operates to evenly mix the content and makes sure of the availability of oxygen throughout the bioreactor. A stirred-tank bioreactor should have a good agitator system as well as efficient oxygen delivery, foam, temperature, and pH control systems. Sampling ports are also present in a stirred-tank bioreactor so that small samples of the culture can be withdrawn periodically for testing.

Sparged Tank Bioreactor

This type of tank is similar to a stirred tank bioreactor. The additional facility is that there is an arrangement for sparging of sterile air bubbles in the culture medium of the bioreactor.

Downstream Processing

Downstream processing refers to the isolation and purification of a biotechnologically formed product to a state suitable for the intended use. Once a bioreactor makes a product, separating this molecule or group of molecules from the remaining contaminants, byproducts, and other components is an integral part of preparing that molecule for market.

In most, biotechnology processes the desired product(s) will be in a dilute aqueous solution. There are several different separation techniques like evaporation and distillation, precipitation, membrane filtration, adsorption, and chromatographic techniques (affinity, ion exchange, and gel-filtration). After separation, the product is sterilized by using heat, radiation, chemicals, filtration or moist steam. Then the sterilized product is mixed with suitable preservatives. The final products of the downstream purification stages should be stable for commercial distribution.