10. Microbial Ecology and Applied Microbiology

10.4 Biotechnology

Learning Objectives

  • Explain the concept of biotechnology and describe some of the earliest examples
  • Describe the biotechnological applications of rhizobia, Baculoviruses, Bacillus thuringiensis and Agrobacterium tumifaciens
  • Describe the role of genetic modification in agriculture
  • Describe some of the diverse uses of microbes in the pharmaceutical industry and medicine
  • Distinguish between probiotic bacteria, Faecal Microbial Transplants (FMT) and Microbial Ecosystem Therapy (MET)

Human life is only possible due to the action of microbes, both those in the environment and those species that call us home. Internally, they help us digest our food, produce vital nutrients for us, protect us from pathogenic microbes, and help train our immune systems to function properly. In fact, prokaryotes provide essential services not just to humans, but to all other organisms. Atmospheric nitrogen (N2), can be “fixed,” or converted into ammonia (NH3) by certain species of bacteria and archaea, but not by any eukaryotes, despite the fact that nitrogen is a major macroelement for all life, and in limiting concentrations in terrestrial environments.

Humans have been consuming fermented foods and beverages like beer, wine, bread, yogurt, cheese, and pickled vegetables for all of recorded history. Discoveries from several archeological sites suggest that even prehistoric people took advantage of fermentation to preserve and enhance the taste of food. Archaeologists studying pottery jars from a Neolithic village in China found that people were making a fermented beverage from rice, honey, and fruit as early as 7000 BC.[1]  While it is likely that people first learned about fermentation by accident—perhaps by drinking old milk that had curdled or old grape juice that had fermented—they later learned to harness the power of fermentation to make products like bread, cheese, and wine.

According to the United Nations Convention on Biological Diversity, biotechnology is “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.”[2] The concept of “specific use” involves some sort of commercial application. Genetic engineering, artificial selection, antibiotic production, and cell culture are current topics of study in biotechnology and will be described in later chapters.

In more recent times, with the advent of recombinant DNA technology, genomics and our increasing understanding of the essential role of the microbes in any ecosystem, including that of the human gut, biotechnology is finding increasing uses in agriculture and medicine. In agriculture it can provide an organic means of fertilization, enhance resistance to disease, pests, and environmental stress to improve both crop yield and quality. Our understanding and use of probiotics has taken on new meaning with medical interventions to restore balance to the gut microbiota. And knowledge of the genetic makeup of our species combined with our ability to manipulate and fix mutant genes now provides methods to correct genetic diseases where the the nature of the mutation has been identified.

Early Biotechnology: Cheese, Bread, Wine, Beer, and Yogurt

Cheese production began around 4,000 to 7,000 years ago when humans began to breed animals and process their milk. Fermentation in this case preserves nutrients: Milk will spoil relatively quickly, but when processed as cheese, it is more stable. As for beer, the oldest records of brewing are about 6,000 years old and were an integral part of the Sumerian culture. Evidence indicates that the Sumerians discovered fermentation by chance. Wine has been produced for about 4,500 years, and evidence suggests that cultured milk products, like yogurt, have existed for at least 4,000 years. Only in recent years has the practice of pasteurization of yogurt been supplanted by the potential value of the lactic acid bacteria, or probiotic bacteria, used to make the yogurt (Figure 10.17).


Photograph of foods and beverages produced by microbes: cheese, wine, beer and yogurt
Figure 10.17. Some foods produced by microorganisms. Some of the products derived from the use of prokaryotes in early biotechnology include (a) cheese, (b) wine, (c) beer and bread, and (d) yogurt. [Credit bread: modification of work by F. Rodrigo/Wikimedia Commons; credit wine: modification of work by Jon Sullivan; credit beer and bread: modification of work by Kris Miller; credit yogurt: modification of work by Jon Sullivan]

Transgenic Plants

Manipulating the DNA of plants (creating genetically modified organisms, or GMOs) has helped to create desirable traits such as disease resistance, herbicide, and pest resistance, as well as increased nutritional value and shelf life (Figure 10.18). Plants are the most important source of food for the human population. Using selective breeding practices, farmers developed ways to select for plant varieties with desirable traits long before modern-day biotechnology practices were established.

Photograph of different cobs of corn, with different coloured kernels
Figure 10.18. Corn, a major agricultural crop used to create products for a variety of industries, is often modified through plant biotechnology. [Credit: Keith Weller, USDA]

Transgenic plants have received DNA from other species. Because they contain unique combinations of genes and are not restricted to the laboratory, transgenic plants and other GMOs are closely monitored by government agencies to ensure that they are fit for human consumption and do not endanger other plant and animal life. Because foreign genes can spread to other species in the environment, particularly in the pollen and seeds of plants, extensive testing is required to ensure ecological stability. Staples like corn, potatoes, and tomatoes were the first crop plants to be genetically engineered.

Transformation of Plants Using Agrobacterium tumefaciens

In plants, galls are tumours caused by the bacterium Agrobacterium tumefaciens. In nature, A. tumefaciens has a tumour-inducing plasmid, the Ti plasmid, containing genes that integrate into the infected plant cell’s genome.By manipulating the plasmid to carry foreign genes for desired traits, researchers have exploited the natural transfer of DNA from Agrobacterium to introduce DNA fragments of their choice into plant.

Organic and Biological Insecticides

The Gram positive bacterium Bacillus thuringiensis (Bt) produces protein crystals that are toxic to many insect species that feed on plants. Within a few hours of consuming the toxin, the insect stops feeding on the plant and death occurs within a couple of days. The toxin can be purified from the bacterium, dried to a powder, and applied to crops. More recently, the gene for the crystal toxin has been cloned from the bacterium and introduced into plants, allowing plants to produce their own Bt toxin. The toxin is safe for the environment and non-toxic to mammals (including humans). As a result, it has been approved for use by organic farmers as a natural insecticide. There is some concern, however, that insects may evolve resistance to the Bt toxin in the same way that bacteria have evolved resistance to antibiotics.

Baculoviruses are insecticidal viruses belonging to the Baculoviridae family. These have found broad use as organic insecticides. The family currently consists of over 600 recognized species, each of which is highly host-specific, infecting at most, a few closely-related species of Lepidoptera (butterflies and moths), Hymenoptera (sawflies), and Diptera (mosquitoes) insect clades.[3] Baculoviruses have two forms: the budded form that allows the virus to spread within the host cells, and the occlusion-derived virus found in large polyhedral occlusion bodies (OBs). Infection of a susceptible host begins with the consumption of OBs (Figure 10.19). The protein matrix of the OB dissolves upon exposure to the alkaline midgut of the insect, releasing the viral particles and degradative enzymes that break down the peritrophic membrane lining the midgut, allowing the virus to penetrate the midgut epithelial cells. The virus replicates and spreads within the host as budded virus, but during the late stages of infection, occlusion bodies form and the degradative enzymes that facilitated the initial penetration of the host then cause host cell lysis and ultimately, the dissolution of the insect. These OBs are highly stable and are the basis for biocontrol of a variety of insect pests including Gypsy Moth larvae (Lymantria dyspar).

Diagram showing the infectious cycle of baculovirus. The panel on the left shows a cross-section of a caterpillar and the initial stages of infection. The panel on the right shows the life cycle of the virus within a host cell.
Figure 10.19. Baculovirus infectious cycle. (A) Cross sectional schematic of an insect larva. A baculovirus occlusion body (OB) ingested with contaminated food starts a new infectious cycle (1). When OBs pass through the foregut and reach the alkaline midgut the proteinaceous matrix is dissolved (2), releasing ODV (3). The peritrophic membrane is degraded by enzymes present in the OB (4), allowing the ODV to enter the cell; (B) Representation of the virus replication cycle. ODV enters the cell by fusion with epithelial cell microvilli (1), releasing nucleocapsids (NC) into the cytoplasm (2). NC enters the nucleus (3), disassemble and release the circular, double-stranded DNA genome (4). Early genes are transcribed (6) and translated (7). Some of the proteins translocate into the nucleus (8), take part in genome transcription/replication, NC and virion assembly (9). In the first stages of viral infection, NC is transported to the cytoplasm (10), approaches the basolateral cell membrane (CM) (11) and emerges as budded virus (BV) (12) in the spots where the viral envelope fusion protein (EFP) (14) accumulates using the secretory pathway (13). In the very late stages of infection, NC are enveloped in the nucleus and occluded in the polyhedral shaped protein matrix (OB) (15)

Rhizobia and Biofertilization

Nitrogen is an integral component of two of the major cellular polymers: nucleic acids and proteins. It is usually the most limiting element in terrestrial ecosystems, with atmospheric nitrogen, N2, providing the largest pool of available nitrogen. However, eukaryotes cannot use atmospheric nitrogen to synthesize macromolecules. Fortunately, nitrogen can be “fixed” or converted into a more accessible form, ammonia (NH3), either biologically or abiotically.

Abiotic nitrogen fixation occurs as a result of physical processes such as lightning or by industrial processes. Biological nitrogen fixation is carried out exclusively by various species of diazotrophic bacteria and archaea (see Biosynthesis). After photosynthesis, diazotrophy is arguably the most important biological process on Earth. Nitrogen fixation is a series of highly endergonic redox reactions requiring the hydrolysis of large amounts of ATP:

N2 + 16ATP + 8e + 8H+ → 2NH3 + 16ADP + 16Pi + H2

The diazotrophs fix approximately 100 to 180 million metric tons per year, which contributes about 65 percent of the nitrogen used in agriculture. Cyanobacteria are the most important nitrogen fixers in aquatic environments. In soil, members of the genera Clostridium and Azotobacter are examples of free-living, nitrogen-fixing bacteria. Other bacteria, the rhizobia, live symbiotically with the roots of  legume plants. Symbionts may fix more nitrogen in soils than free-living organisms by a factor of 10. The rhizobia form nodules on legume plant roots: specialized structures in which nitrogen fixation occurs (Figure 10.20). Nitrogenase, the enzyme that fixes nitrogen, is inactivated by oxygen, however within the nodule is a form of plant haemoglobin called leghaemoglobin, which binds O2, protecting the nitrogenase.

Symbiotic nitrogen fixation provides a natural and inexpensive plant fertilizer: it reduces atmospheric nitrogen to ammonia, which the plants can use as a nitrogen source. The use of rhizobia and legumes is an excellent alternative to chemical fertilization and is of special interest to sustainable agriculture, which seeks to minimize the use of chemicals and conserve natural resources. Through symbiotic nitrogen fixation, the plant benefits from using an endless source of nitrogen: the atmosphere. The bacteria benefit from the carbohydrates produced by the plant during photosynthesis  and having a protected niche. In addition, the soil benefits from being naturally fertilized, preventing the nitrogenous run-off associated with chemical fertilizers and the resulting eutrophication of local aquatic and marine ecosystems.

Why are legumes so important? Some of the most important legumes consumed by humans are soybeans, peanuts, peas, chickpeas, and beans. Other legumes, such as alfalfa, are used to feed cattle.


Photograph of legume roots with nodules
Figure 10.20. Nitrogen-fixation nodules on soybean roots. Soybean (Glycine max) is a legume that interacts symbiotically with the soil bacterium Bradyrhizobium japonicum to form specialized structures on the roots called nodules where nitrogen fixation occurs. (credit: USDA)
  • List some of the most ancient types of biotechnology and some of the more recent ones
  • What role does the bacterium Agrobacterium tumifaciens play in biotechnology?
  • What is the relevance of the rhizobia to plant growth and agriculture?

Production of Vaccines, Antibiotics, and Hormones

Traditional vaccination strategies use weakened or inactive forms of microorganisms, viruses or toxins to stimulate the immune system. Modern techniques use specific genes of microorganisms cloned into vectors and mass-produced in bacteria to make large quantities of specific substances, “antigens”, to stimulate the immune system. The antigen is then used as a vaccine. Vaccines are described in more detail in Section 19.5

Antibiotics are naturally produced by microorganisms such as fungi; penicillin is perhaps the most well-known example, produced by the mould Penicillium notatum. Other antibiotics are naturally produced by different strains of the actinomycete bacteria. Certain antibiotics are synthesized, or chemically modified, in the organic chemistry lab. Antibiotics are described in more detail in various sections of Chapter 15.

Recombinant DNA technology was used to produce large-scale quantities of the human hormone insulin in E. coli as early as 1978. Previously, it was only possible to treat diabetes with pig insulin, which caused allergic reactions in many humans because of differences in the insulin molecule. In addition, human growth hormone (HGH) is used to treat growth disorders in children. The HGH gene was cloned from a cDNA (complementary DNA) library and inserted into E. coli cells by cloning it into a bacterial vector. Some of these are described further in Chapter 13: Modern Applications of Microbial Genetics.

Genetic Diagnosis and Gene Therapy

Mutations in the BRCA genes may increase the likelihood of developing breast and ovarian cancers in women and some other cancers in women and men. A woman with breast cancer can be screened for these mutations. If one of the high-risk mutations is found, female relatives may also be screened for that mutation. Genetic testing is also offered for fetuses (or embryos with in vitro fertilization) to determine the presence or absence of disease-causing genes in families with specific debilitating diseases. Knowledge of the genetic basis for heritable diseases continues to increase, and with that knowledge, the potential for correcting the defect through gene therapy.

Gene therapy is a genetic engineering technique that, in its simplest form, involves the introduction of a non-mutated gene at a random location in the genome to provide a source of a functional protein. The non-mutated gene is usually introduced into diseased cells as part of a vector transmitted by a virus, such as an adenovirus, that can infect the host cell and deliver the foreign DNA into the genome of the targeted cell (Figure 10.21). To date, gene therapies have been primarily experimental procedures in humans. A few of these experimental treatments have been successful, but the methods may be important in the future as the factors limiting its success are resolved. Gene therapy is discussed in more detail in Section 13.4.

MICRO CONNECTIONS: “C. diff” Infections and Microbial Ecosystem Therapy

The dawning understanding of the importance of the gut microbiota to health is starting to drive a new paradigm in medicine. Faecal Microbial Transplant (FMT) has shown clear benefits in the treatment of one of the diseases known to be caused by a lack of diversity in the gut microbiota – Clostridium difficile infection, or “C. diff”.  This particular diarrhoeal pathogen, which can be an intrinsic part of the gut microbiota or which can be spread from patient to patient within a hospital environment, usually does not cause disease in healthy hosts.  However, the organism forms endospores (see Endospores) and thus resists the effects of the antibiotics. It survives and proliferates after antibiotic treatment, taking advantage of a depleted ecosystem.  The toxins it produces damage the epithelium of the large intestine, giving rise to pseudomembranous colitis and severe diarrhoea (Figure 10.22).
Photograph from a colonoscopy of a patient with pseudomembranous colitis. Yellow plaques are characteristic of the disease and are ulcerations of the colon mucosal epithelium
Figure 10.22. Colonoscopy of a patient with pseudomembranous colitis. Yellow plaques in the mucosa are characteristic of this disease and are the basis for early diagnosis. These plaques are small ulcerations of the mucous membranes. [Credit – Kazanowski et al., 2014]
C. difficile infections can be treated with further doses of antibiotics but no antibiotics are able to target only C.difficile, so there is always collateral damage, further reducing the diversity of the gut microbiota.  If FMT is applied to a patient with a C.difficile infection, this immediately replaces most microbial diversity and forces C. difficile numbers back into a minority, for example through nutrient competition – in fact, FMT has been shown to be over 90% effective for the treatment of recurrent C. difficile infection.  Not surprisingly, however, there are many safety and palatability issues when using human waste as medicine.  Recently, biotechnology companies have started to face this challenge head on, and the development of purified ecosystems for use as new biologic drugs has emerged.  Here, microbial strains from stool are isolated, purified and thoroughly characterized, and then selected and combined to form a microbial ecosystem that is similar to FMT in its application and mechanism of action, but which has been standardized for commercial production and screened for the absence of known virulence determinants.  This approach to correct imbalances in the microbiota is called Microbial Ecosystem Therapy, or MET. The barrier to this approach with diseases caused by loss of gut microbial diversity is the fastidious nature of most of the bacterial species that live within the human gut. However novel biotechnological approaches to solve this problem and allow growth of highly fastidious anaerobes in fermentation chambers under defined conditions are now being realized, as are bioinformatics approaches to try to understand which microbes should be applied to which patients as ‘personalized medicine’.
  • Describe some of the pharmaceutical applications of microbes.
  • What is gene therapy and how do microbes play a role?
  • What is FMT and MET?

Key Takeaways

  • The oldest examples of the deliberate harnessing of microbial processes are the fermented foods and beverages that have been part of our diet for millenia
  • Antibiotics may be produced naturally by actinobacteria and various moulds
  • Vaccines use inactivated microbes, microbial structures or toxins as antigens to generate immunity
  • Hormones and vaccines are examples of products obtained by recombinant DNA technology.
  • The mutualistic relationship of the Rhizobia with legumes has been exploited as a natural alternative to fertilization, by providing fixed nitrogen, in the bioavailable form of NH3, to the plant.
  • Transgenic organisms possess DNA from a different species, usually generated by molecular cloning techniques.
  • Transgenic plants can be created using modified Ti plasmids of Agrobacterium tumefaciens and have been created to improve the characteristics of crop plants. Bt corn is one example.
  • Gene therapy involves the correction of a genetic defect through incorporation of a functioning gene into the genome of individuals with a heritable disease

Multiple Choice

Short Answer

  1. What is Bt corn?
  2. How do legumes provide the necessary conditions for N-fixation by rhizobia?

Critical Thinking

  1. Compare and contrast probiotics, FMT and MET.
  2. Describe 3 diverse uses of microorganisms in the pharmaceutical industry.
  3. Why are Baculovirus OBs so useful as insecticides?



Media Attributions

  • Microbial food products
  • Maize
  • Baculovirus life cycle
  • Rhizobia root nodules
  • Pseudomembranous colitis

  1. P.E. McGovern et al. “Fermented Beverages of Pre- and Proto-Historic China.” Proceedings of the National Academy of Sciences of the United States of America 1 no. 51 (2004):17593–17598. doi:10.1073/pnas.0407921102.
  2. http://www.cbd.int/convention/articles/?a=cbd-02, United Nations Convention on Biological Diversity: Article 2: Use of Terms.
  3. Haase, S., Sciocco-Cap, A., and Romanowski, V. "Baculovirus Insecticides in Latin America: Historical Overview, Current Status and Future Perspectives." PLoS One. 2017 Dec 18;12(12):e0189680. doi: 10.1371/journal.pone.0189680.


Icon for the Creative Commons Attribution 4.0 International License

Microbiology: Canadian Edition Copyright © 2019 by Wendy Keenleyside is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

Share This Book