players - Microbes and Metabolism

The players

Traditionally, life was placed into two categories – those having a true nucleus (eukaryotes) and those that do not (prokaryotes). This view was dramatically disturbed in 1977 when Carl Woese proposed a third domain, the archaebacteria, now described as archaea, arguing that although apparently prokaryote at first glance they contain sufficient similarities with eukaryotes, in addition to unique features of their own, to merit their own classification (Woese and Fox 1977, Woese, Kandler and Wheelis 1990). The arguments raised by this proposal con-tinue (Cavalier-Smith 2002) but throughout this book the classification adopted is that of Woese, namely, that there are three divisions: bacteria, archaea (which together comprise prokaryotes) and eukaryotes. By this definition, then, what are referred to throughout this work simply as ‘bacteria’ are synonymous with the term eubacteria (meaning ‘true’ bacteria).

 It is primarily to the archaea, which typically inhabit extreme niches with respect to temperature, pressure, salt concentration or osmotic pressure, that a great debt of gratitude is owed for providing this planet with the metabolic capability to carry out processes under some very odd conditions indeed. The importance to environmental biotechnology of life in extreme environments is addressed.

 An appreciation of the existence of these classifications is important, as they differ from each other in the detail of their cell organization and cellular processes making it unlikely that their genes are directly interchangeable. The relevance of this becomes obvious when genetic engineering is discussed later. However, it is interesting to examine the potentially prokaryotic origins of the eukaryotic cell. There are many theories but the one which appears to have the most adherents is the endosymbiotic theory. It suggests that the ‘proto’ eukaryotic cell lost its cell wall, leaving only a membrane, and phagocytosed or subsumed various other bacteria with which it developed a symbiotic relationship. These included an aerobic bacterium, which became a mitochondrion, endowing the cell with the ability to carry out oxidative phosphorylation, a method of pro-ducing chemical energy able to be transferred to the location in the cell where it is required. Similarly, the chloroplast, the site of photosynthesis in higher plants, is thought to have been derived from cyanobacteria, the so-called blue-green algae. Chloroplasts are a type of plastid. These are membrane-bound structures found in vascular plants. Far from being isolated cellular organelles, the plastids com-municate with each other through interconnecting tubules (Kohler¨ et al. 1997). Various other cellular appendages are also thought to have prokaryotic origins such as cilia or the flagellum on a motile eukaryotic cell which may have formed from the fusion of a spirochete bacterium to this ‘proto’ eukaryote. Nuclei may well have similar origins but the evidence is still awaited.

 No form of life should be overlooked as having a potential part to play in environmental biotechnology. However, the organisms most commonly discussed in this context are microbes and certain plants. They are implicated either because they are present by virtue of being in their natural environment or by deliberate introduction.

Microbes

Microbes are referred to as such, simply because they cannot be seen by the naked eye. Many are bacteria or archaea, all of which are prokaryotes, but the term ‘microbe’ also encompasses some eukaryotes, including yeasts, which are unicellular fungi, as well as protozoa and unicellular plants. In addition, there are some microscopic multicellular organisms, such as rotifers, which have an essential role to play in the microsystem ecology of places such as sewage treat-ment plants. An individual cell of a eukaryotic multicellular organism like a higher plant or animal, is approximately 20 microns in diameter, while a yeast cell, also eukaryotic but unicellular, is about five microns in diameter. Although bacterial cells occur in a variety of shapes and sizes, depending on the species, typically a bacterial cell is rod shaped, measuring approximately one micron in width and two microns in length. At its simplest visualisation, a cell, be it a unicellular organism, or one cell in a multicellular organism, is a bag, bounded by a membrane, containing an aqueous solution in which are all the molecules and structures required to enable its continued survival. In fact, this ‘bag’ rep-resents a complicated infrastructure differing distinctly between prokaryotes and eukaryotes (Cavalier-Smith 2002), but a discussion of this is beyond the scope of this book.

 Depending on the microbe, a variety of other structures may be present, for instance, a cell wall providing additional protection or support, or a flagellum,a flexible tail, giving mobility through the surrounding environment. Survival requires cell growth, replication of the DNA and then division, usually sharing the contents into two equal daughter cells. Under ideal conditions of environ-ment and food supply, division of some bacteria may occur every 20 minutes, however, most take rather longer. However, the result of many rounds of the binary division just described, is a colony of identical cells. This may be several millimetres across and can be seen clearly as a contamination on a solid surface, or if in a liquid, it will give the solution a cloudy appearance. Other forms of replication include budding off, as in some forms of yeast, or the formation of spores as in other forms of yeast and some bacteria. This is a type of DNA stor-age particularly resistant to environmental excesses of heat and pH, for example. When the environment becomes more hospitable, the spore can develop into a bacterium or yeast, according to its origins, and the life cycle continues.

 Micro-organisms may live as free individuals or as communities, either as a clone of one organism, or as a mixed group. Biofilms are examples of microbial communities, the components of which may number several hundred species. This is a fairly loose term used to describe any aggregation of microbes which coats a surface, consequently, biofilms are ubiquitous. They are of particular interest in environmental biotechnology since they represent the structure of microbial activity in many relevant technologies such as trickling filters. Models for their organisation have been proposed (Kreft et al. 2001). Their structure, and interaction between their members, is of sufficient interest to warrant at least one major symposium (Allison et al. 2000). Commonly, biofilms occur at a solid/liquid interphase. Here, a mixed population of microbes live in close proximity which may be mutually beneficial. Such consortia can increase the habitat range, the overall tolerance to stress and metabolic diversity of individ-ual members of the group. It is often thanks to such communities, rather than isolated bacterial species, that recalcitrant pollutants are eventually degraded due to combined contributions of several of its members.

 Another consequence of this close proximity is the increased likelihood of bac-terial transformation. This is a procedure whereby a bacterium may absorb free deoxyribonucleic acid (DNA), the macromolecule which stores genetic material, from its surroundings released by other organisms, as a result of cell death, for example. The process is dependent on the ability, or competence, of a cell to take up DNA, and upon the concentration of DNA in the surrounding environ-ment. This is commonly referred to as horizontal transfer as opposed to vertical transfer which refers to inherited genetic material, either by sexual or asexual reproduction. Some bacteria are naturally competent, others exude competence factors and recently, there is laboratory evidence that lightning can impart compe-tence to some bacteria (Demaneche et al. 2001). It is conceivable that conditions allowing transformation prevail in biofilms considering the very high local con-centration of microbes. Indeed there is evidence that such horizontal transfer of DNA occurs between organisms in these communities (Ehlers 2000). In addition to transformation, genes are readily transferred on plasmids as described later. It is now well established that, by one method or another, there is so much exchange of genetic material between bacteria in soil or in aquatic environments, that rather than discrete units, they represent a massive gene pool (Whittam 1992).

 The sliminess often associated with biofilms is usually attributed to excreted molecules often protein and carbohydrate in nature, which may coat and protect the film. Once established, the biofilm may proliferate at a rate to cause areas of anoxia at the furthest point from the source of oxygen, thus encouraging the growth of anaerobes. Consequently, the composition of the biofilm community is likely to change with time.

 To complete the picture of microbial communities, it must be appreciated that they can include the other micro-organisms listed above, namely, yeasts, protozoa, unicellular plants and some microscopic multicellular organisms such as rotifers.

Plants

In contrast with microbes, the role of plants in environmental biotechnology is generally a structural one, exerting their effect by oxygenation of a microbe-rich environment, filtration, solid-to-gas conversion or extraction of the contaminant. Genetic modifi-cation of crop plants to produce improved or novel varieties is discussed. This field of research is vast and so the discussion is confined to rele-vant issues in environmental biotechnology rather than biotechnology in general.