Nature's minor gifts

Even as microbes -silent and efficient behind-the-screen contributors to the ecosystem -continue to revel under the spotlight abroad, in India, it has yet to acquire its due importance. Indian germplasm, aided by non-existence of biodiversity conservation laws, thus finds its way into leading research laboratories in different parts of the world

 
By Indira Khurana, Indira Khurana
Last Updated: Sunday 07 June 2015

Nature's minor gifts

An Escherichia coli bacillus d Over 90,000 Indian soil samples have been diligently screened by Hoechst, a German multinational company, for their microbial diversity. The company is now building a new, high efficiency screening system in Frankfurt, where it will further screen their Indian soil booty. Remarks Richard Helmut Rupp, head of Hoechst's research and development wing, on the company's website, "The most important publications for our researchers are not chemistry journals but journals of patent offices around the world." Indian custom authorities are as yet unaware of the importance of microbial diversity and hence do not prevent the export of the same, according to Vinod Vaishya, additional secretary in the ministry of environment andforests (MEF).

In 1995-96, close to Rs 2,500 crore of products involving microbes were produced in India. In fact, India has been identified as one of the 12 megadiversity centres, in the world.

MICROBIAL diversity, an essential part of biological diversity and ecosystem conservation is an unseen national resource of any country. Recent developments using modern technology in microbial diversity research indicate that as much as 99 per cent of naturally occurring microorganisms worldwide are undiscovered, and their ecological role is currently unknown.

Microbes or microorganisms can be defined as tiny living things that include bacteria, viruses, algae, fungi and other self- replicating units like plasmids (strands/fragments of genetic material or DNA capable of existing outside the nucleus) which are ordinarily not visible, except through a microscope. While some efforts have gone into estimating the number of plant and animal species, microbes have been generally ignored till the early nineties.

The problem of identification and conservation is complex. The number of microbes known today represent only the tip of the iceberg. While many microbes are difficult to find and isolate, maintaining such strains in culture collections is the only practical way to ensure access to them for beneficial properties. Conversely, it has now generally been accepted that the microbes capable of growing in the laboratory under artificial conditions are not necessarily those abundant in the environment.

Omnipresent
Estimate of the number of described species and possible existing species of microorganisms
Group known Described species Existing species Percentage
Algae 40,000 350,000 11.0
Bacteria(including cyanobacteria and unculturables) 4,000 3,000,000 0.1
Fungi (inlcuding yeasts, lichens, fungi, slime moulds) 70,000 1,500,000 5.0
Protozoa 40,000 100,000 40.0
Viruses (including phages and plasmids) 5,000 500,000 1.0
Total 159,000 5,0450,000 3.0
Note:
ALGAE: extreme diverse group consisting predominantly of aquatic plants, includes prokaryotes (microbes whose genes are not encapsulated in a nucleus) and eukaryotes (microbes whose genes are encapsulated in a nucleus)
BACTERIA: all prokaryotes except blue-green algae
CYANOBACTERIA: prokaryotic division of blue-green algae, capable of fixing nitrogen
PROTOZOA: group of small, single-called, usually microsopic eukaryotic organisms
VIRUS: very small infectious agent capable of replicating only in its specific host
PHAGE: virus infecting bacteria
The total number of known species is currently estimated at close to 1,60,000 (See table: Omnipresent). The general consensus is that less than five per cent of the world's microorganisms have been described and around 20 per cent of this are preserved.

A high degree of microbial diversity also exists in extreme environments like hot springs, or in induced stress conditions due to alteration by human intervention like polluted rivers. Microbe research and exploitation requires cooperative efforts. Conservation of microbes is complex - both within the original habitat or natural environment as well as outside it. While the former is difficult, given the tremendous ability of these organisms to adapt and gradually change or evolve due to changes in their environment and their seasonal or periodic appearance, the latter is a laborious process requiring finances, trained manpower, storage space, low temperatures and equipment. Often novel characteristics or traits are found at low frequency and the isolated microbes then need to be appropriately maintained and conserved, as these traits may be lost if sufficient care is not taken to preserve them.

Indian efforts at microbial conservation are minimal and are seriously hampered by lack of funds, ideas and trained scientists (taxonomists capable of identifying them). Some collections do not even meet international guidelines. While P K Ghosh, advisor, department of biotechnology (DBT) feels that India is running a handicapped race in this area, Anil Gupta of the Centre for Management in Agriculture, Indian Institute of Management, Gujarat, is more optimistic. He feels that if the importance of microbial research in India is realised even now and efforts initiated, "we can make up with intelligence and diligence what India lacks in technology".

Microbes: tiny but mighty
Microbes not only have commercial applicability in pharmaceuticals, food, agriculture and healthcare, but are also an integral part of ecosystems. They have been involved in the evolution and diversification of higher organisms. They, especially fungi, maintain the health and fertility of the soil. Microbes are also involved in the geochemical cycles of nitrogen, sulphur, carbon and metals.

The extent of diversity in micro-organisms is much larger than that of macroorganisms simply because the former have been around on the earth much longer. Human intervention has been responsible for a tremendous erosion in the microdiversity - in several regions in Europe, diversity of fungal species has dropped by more than 50 per cent during the last 60 years. Microbes, however, have a tremendous capacity to survive difficult conditions, resulting in their greater genetic diversity. Regions of macrodiversity are naturally rich in microdiversity and act as additional habitats for microorganisms. Recent research in the ecology of extreme environments - either natural or induced - which were earlier thought to be populated by few microbial species have revealed a whole new range of microbes.

"Different microbial populations will definitely be found in polluted soils and in effluents," says Anil Gupta. "Bacteria for instance, are capable of growing in extreme environments like hypersaline lakes, hot springs, leaching environments like acid mine waters, swamps, estuaries, in the deep sea and other hostile areas. It is from environments like this that potential commercial products can be found," he adds (See box: Hot wealth). While even a conservative estimate may be way off target, due attention should be paid to these areas when drafting any national, regional or international plans for their protection.

Currently more than 69,000 species of fungi are known worldwide, 24,000 of which occur in India. Some species with common structural characteristics or genera like Cercospora, Fusarium and rust fungi are more predominant in India, than in any other part of the world. "Indian efforts in conserving fungal biodiversity are still inadequate, given the vast country and its diverse nature," feels Anupam Varma, dean and joint director, Indian Agricultural Research Institute, New Delhi. "Taking all the culture collections together, accessions of fungi number about 9,000 - a small fraction of what is estimated to exist in India," he says (See box: Saprophyte abundance).Studies on the biodiversity and roles of almost all microorganism groups are frustrated by an inadequate taxonomic base - the assigning of organisms to identified classified units. "India is seriously hampered by a lack of qualified taxonomists capable of assigning the microbes to these taxa, which is also a global problem," says B B Chattoo, head of the biotech centre at Maharaja Sayaji University, Baroda. "Out of the approximately 30,000 members of the American Society of Microbiologists in the US, taxonomists number about a 100," he adds. "Attention needs to be focused on strengthening existing centres of expertise, developing international linkages and establishing networks of centres and specialists to make research complimentary and collaborative." It is precisely this approach that Anil Gupta is adopting (See box: Treasure down under).

Bioprospecting and microorganisms
Microbes are used in various commercial products with a total value amounting to billions of dollars. The search for microbes of potential commercial value is part of bioprospecting. Though bio-prospecting is not new, what is current is the increasingly widespread use of biotechnology to discover new uses of biodiversity along with the technical and ethical questions they are raising. Direct isolation of DNA from the environment offers a rapid means for microbial biodiversity fingerprinting, comparing several environments and detecting major shifts in species of microbial population. This technology has been successfully used to assess the biodiversity from sites ranging from mine wastes to geothermal takes to waste waters from fertifiser factories. Microbes with their ability to adapt rapidly to their environment can perform various unique functions. Recombinant Biocatalysts Incorporated (RBI), a biotechnology company in the US, is currently looking into these unique properties. It has developed a strategy wherein DNA is directly isolated from environment samples of microbes. These are then screened for desirable products. RBI has acquired exclusive rights to organisms like Pyrolus fumarius, a bacteria, which grows optimally at 108oC and an Icelandic collection of extremophiles (microbes which grow in extreme environments).

Says Manju Sharma, secretary, DBT, "We have identified biodiversity conservation as a high priority area and, under the national biodiversity conservation task force, have chalked out plans for the next five years." DBT has set aside funds for bioprospecting of new molecules, genes, biopesticides and medicinally important organisms. This project will involve DBT, Department of Space, National Chemical Laboratory, Pune, North Eastern Hill University, Shillong, Tropical Botanical Garden Research Institute, Thiruvananthapuram, and G B Pant University, Pantnagar.

According to the Convention on Biological Diversity (CBD), a nation has sovereign rights over its genetic resources and must protect them. India, therefore, urgently needs to characterise, conserve and use its resources. Officials in the mhr agree, but are unable to give any time- frame, "We are aware of the problem, but you must appreciate the magnitude of efforts and finances required, given the vast and diverse nature of our country," says Vinod Vaishya. "The situation is even more complicated for microbial diversity. Rather than first identifying the diversity, maybe under the CBD we can have some-arrangement where bioprospecting can be in the form of a joint venture with the foreign collaborator having limited access to our resources. This way, both the parties would benefit," he adds.

MEF's approach only highlights the weaknesses in our ability to carry out front-line research and DBT's approach seems to be biased towards identifying predetermined requirements, rather than mapping and assessing the biodiversity. "We need to realise that taxonomy can be linked with microbial research in such a way that basic questions can be answered along the way. Awareness of this approach would greatly motivate people to get into this area," says Chattoo.

Losing out
Commercially useful biomaterials from India deposited in theAmerican Type Culture Collection (ATCC), US and now excludedfrom the Convention on Biological Diversity

 

As this material was collected from the donor country prior to the Convention coming into force, the material is thus the property of the depositor and not the donor. Any royalty claims against patents will be paid to the depositor and not the donor.
   Till 1992, 35 microbial samples from India were recorded in the database of ATCC. Of this, 29 were awarded US patents and patent claims for 6 are pending. The main depositors include:
Bristol Myers 24
Lepetit labs 4
Pfizer 4
Merck 2
Lederle labs 1
Patenting microbes
Patent applications in biotechnology usually involve depositing the biological material in 'culture collections' - institutes designed to preserve biological materials in perpetuity. Patent laws in the US and most countries require that the inventor gives a full disclosure of his or her invention to the patent office. For the patenting of microorganisms, the patent law requires that a sample containing the microorganism be deposited with a recognised patent culture depository, regulated internationally by the Budapest Treaty on the International Recognition of the deposit of Microorganisms for the purpose of Patent Procedure, administered by the World Intellectual Property Organization in Geneva. Under this treaty, 26 institutes in 15 countries have been recognised as 'International Depository Authorities'. Out of the 15 countries, 14 are from the North, housing 23 of the 26 institutes.

Samples of genetic materials deposited in culture collections are not always freely available to individuals or institutes requesting them. At the American Type Culture Collection (ATCC), Maryland, US, the world's largest depository, written permission from the depositor must be obtained. The language for meeting patenting criteria under the Budapest Treaty is rather vague. In its strict sense, industrial patent law does differentiate between a 'discovery' and an 'invention'. A previously and naturally occurring microorganism is recognised as a discovery and is thus non-patentable. Due to existing loopholes in various countries, however, depending upon the degree of human intervention, discoveries can be turned into patents. In Britain and the European Patent Office, for example, previously undiscovered microorganisms can be patented. In Germany, isolating a strain of a microorganism allows its patenting. In the US, a 'biologically pure culture' could be patented, provided certain patentable criteria are met. If these are not met, their products such as enzymes and antibiotics are patentable, so that, if directly the microbe is not patentable, indirectly it can end up being patented.

The Indian perspective
Considerable confusion exists amongst policymakers and scientists about the possibility of patenting microorganisms isolated in India. The Indian Patent Law of 1970 does not allow patenting of live forms, and consequently of microbes. When asked about India's stand on patenting microbes in India, Manju Sharma said: "We can always publish our findings in a journal of repute. Once published, the scientist has automatically established his claim internationally over its future commercial use." Keith Percy, a chartered British and European patent attorney of the British Technology Group, however, differs, "When is it safe to publish? Only after filing an application for a patent. If you publish, you are going to destroy the novelty of any subsequent application you file."

Biopiracy: threat to nations' resources
Though the CBD recognises that a country has sovereign rights over the biodiversity existing in its geographical limits, it conveniently excludes from its purview all germplasm collections before it (the treaty) came into being. Thus, all germplasm collected before 1993, the year when CBD took shape, are now the property of the depositor rather than the country from where the germplasm originated. For microbes, this means that all the microbes collected by northern countries from the southern region and being preserved there belong to the former.

The irony of the CBD lies in the fact that the depositor or the person who deposits the microbe in an inter nationally recognised depository as a requirement for filing a patent, may not recognise or disclose the source of the microbe he or she has deposited. The donor country or the source from where the microbe has been taken, however, has to acknowledge the rights of the depositor. Patent holders are not obliged to divulge the original source of either a naturally occurring organism or one that has been altered after it was isolated from nature. As a result, it is almost impossible to estimate the number of microbes originating from the South and currently held in the North. According to the RAFI Communique, a newsletter of the Rural Advancement Foundation International, an NGO based in Canada, a search of the ATCC databases reveals that a considerable number of microbes from the South are held in the North and are subject to exclusive monopoly under the industrial patent claims (See box. Losing out).

According to P K Ghosh, it is wishful thinking on our part that we have the resources to guard out assets. "The only alternative appears to be to channelise and utilise our limited resources in selected fields so that we become experts in them, rather than making our prescnce in every arena." Adds Vaishya, "Rather than looking into the past, it is better to look at the future." It seems a moot point, but MEF appears to be doing neither. No plans, not even of a tentative nature, for conserving and protecting our microbial diversity seem to be on the anvil. "We have the best brain-power in our country advising us," says Vaishya, "I am sure we will do something."

Given the potential importance of microbes and their diversity in ecosystems, "We need to carry out taxonomy at the local level, and then maintain the cultures according to internationally acceptable standards, since exchange of microbes is increasingly going to depend on what we are able to give in return," feels Chattoo. "We also need to develop a holistic approach, so that we conserve, bioprospect and look for answers to understanding the ecosystem. We also need to make taxonomy more interesting." Chattoo has attempted to develop interest amongst students by asking them to go out and collect samples from the environment and then do some basic analysis on what they have got. "Initially reluctant, the students are now excited about what they see in the lab with 'their samples'."

Though strapped by lack of qualified experts, infrastructure and funds, now that India has ratified the CBD, urgent steps are needed to be taken to identify, conserve, utilise and claim rights over the country's resources. "We have such a large genetic resource, how can we immediately take care of all that we have? Give us time. What is gone is gone," says a matter-of-fact Sharma. The projects initiated by the DBT are directed at bioprospecting for specific properties or products rather than for assessing the total biodiversity. Though a start has been made, lessons of a more holistic approach could be learnt from Anil Gupta's interactive and voluntary approach.

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