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About this book

This book is devoted to alkaliphiles, their microbiology, biotechnological applications and adaptive mechanisms. Alkaliphiles are extremophilic organisms that are adapted to thrive in alkaline environments. Over the years, a wide variety of alkaliphiles belonging to domain Bacteria, Archaea and Eukarya have been isolated and studied. These organisms use various adaptive mechanisms to thrive in ‘extreme’ alkaline environments, and some of these adaptive mechanisms are of immense importance to a range of biotechnological applications. In this book, readers will learn about the adaptive strategies of alkaliphiles in colonizing alkaline habitats, with a main focus on: (1) the production of enzymes that are active and stable in the high pH environment, and (2) the production of acids that decrease the pH of their immediate surrounding environment.

Enzymes that are operationally stable at high pH (also known as alkaline active enzymes) are desirable in several applications such as detergent formulating and leather tanning processes, and they are among the major selling enzymes and the most important industrial enzymes. The growing demand in many existing and emerging biotechnological applications led to the discovery, characterization, engineering and evaluation of diverse types of alkaline active enzymes. In addition to the use of these fascinating enzymes in biotechnological applications, readers will discover the mechanisms of action and stability of these enzymes at extreme pH.

Studies have shown that some alkaliphiles decrease the severity of the high pH of their media by producing substantial amount of organic acids, which could be of great interest in various applications presented in this book. In addition to enzymes and organic acids, other products of biotechnological importance such as carotenoids, bioactive substances, and chelators have also attracted researchers’ attention. Whole-cells of alkaliphiles have been used as food and feed, and are also useful in environmental applications such as in waste treatment and construction.

Table of Contents


Alkaliphiles: The Versatile Tools in Biotechnology

The extreme environments within the biosphere are inhabited by organisms known as extremophiles. Lately, these organisms are attracting a great deal of interest from researchers and industrialists. The motive behind this attraction is mainly related to the desire for new and efficient products of biotechnological importance and human curiosity of understanding nature. Organisms living in common “human-friendly” environments have served humanity for a very long time, and this has led to exhaustion of the low-hanging “fruits,” a phenomenon witnessed by the diminishing rate of new discoveries. For example, acquiring novel products such as drugs from the traditional sources has become difficult and expensive. Such challenges together with the basic research interest have brought the exploration of previously neglected or unknown groups of organisms. Extremophiles are among these groups which have been brought to focus and garnering a growing importance in biotechnology. In the last few decades, numerous extremophiles and their products have got their ways into industrial, agricultural, environmental, pharmaceutical, and other biotechnological applications.
Alkaliphiles, organisms which thrive optimally at or above pH 9, are one of the most important classes of extremophiles. To flourish in their extreme habitats, alkaliphiles evolved impressive structural and functional adaptations. The high pH adaptation gave unique biocatalysts that are operationally stable at elevated pH and several other novel products with immense biotechnological application potential. Advances in the cultivation techniques, success in gene cloning and expression, metabolic engineering, metagenomics, and other related techniques are significantly contributing to expand the application horizon of these remarkable organisms of the ‘bizarre’ world. Studies have shown the enormous potential of alkaliphiles in numerous biotechnological applications. Although it seems just the beginning, some fantastic strides are already made in tapping this potential. This work tries to review some of the prominent applications of alkaliphiles by focusing such as on their enzymes, metabolites, exopolysaccharides, and biosurfactants. Moreover, the chapter strives to assesses the whole-cell applications of alkaliphiles including in biomining, food and feed supplementation, bioconstruction, microbial fuel cell, biofuel production, and bioremediation.
Graphical Abstract
Gashaw Mamo, Bo Mattiasson

Isolation and Cultivation of Alkaliphiles

Alkaliphilic microorganisms are ubiquitous and inhabit various econiches on Earth. Alkaline environments suitable for alkaliphilic microbial communities may be created by certain geological processes or human activities. Moreover, a significant contribution to the emergence of alkaline conditions may be due to the activity of neutralophilic microorganisms through certain reactions, which explains the widespread distribution of alkaliphiles. Alkaliphilic microorganisms are part of extremophiles and become interesting and useful in environmental and industrial microbiology. With increasing knowledge of alkaliphiles, we greatly increase their biotechnological and industrial application potential. New microorganisms from natural habitats serve as a practically endless source of new enzymes. This chapter summarizes the scattered data on alkaliphiles isolated from habitats other than soda lakes. The conditions for occurrence of alkaline habitats are also considered. Moreover, the chapter reviews some important features on preparation of media for the isolation and cultivation of alkaliphiles. The chapter also includes relevant information on chromatographic analysis of alkaliphiles culture.
Vadim V. Kevbrin

Challenges and Adaptations of Life in Alkaline Habitats

A vast array of organisms is known thriving in high pH environments. The biotechnological, medical, and environmental importance of this remarkable group of organisms has attracted a great deal of interest among researchers and industrialists. One of the most intriguing phenomena of alkaliphiles that engrossed researchers’ attention is their adaptation to high pH and ability to thrive in the “extreme” condition which is often lethal to other organisms. Studies made in this line revealed that alkaliphiles deployed a range of adaptive strategies to overcome the various challenges of life in high pH environments. This chapter highlights some of the challenges and the most important structural and functional adaptations that alkaliphiles evolved to circumvent the hurdles and flourish in alkaline habitats. The fascinating alkaliphiles’ pH homeostasis that effectively maintains a lower cytoplasmic pH than its extracellular environment and the remarkable bioenergetics that produce ATP much faster than non-alkaliphiles systems are reviewed in detail. Moreover, the adaptive mechanisms that alkaliphiles employ to keep the structural and functional integrity of their biomolecules at elevated pH are assessed.
It is undeniable that our understanding of alkaliphiles adaptation mechanisms to high pH is expanding with time. However, considering that little is known so far about the adaptation of life in alkaline milieu, it seems that this is just the beginning. Probably, there is a lot more waiting for discovery, and some of these issues are raised in the chapter, which not only summarizes the relevant literature but also forwards new insights regarding high pH adaptation. Moreover, an effort is made to include the largely neglected eukaryotic organisms’ adaptation to high pH habitats.
Graphical Abstract
Gashaw Mamo

Genomics of Alkaliphiles

Alkalinicity presents a challenge for life due to a “reversed” proton gradient that is unfavourable to many bioenergetic processes across the membranes of microorganisms. Despite this, many bacteria, archaea, and eukaryotes, collectively termed alkaliphiles, are adapted to life in alkaline ecosystems and are of great scientific and biotechnological interest due to their niche specialization and ability to produce highly stable enzymes. Advances in next-generation sequencing technologies have propelled not only the genomic characterization of many alkaliphilic microorganisms that have been isolated from nature alkaline sources but also our understanding of the functional relationships between different taxa in microbial communities living in these ecosystems. In this review, we discuss the genetics and molecular biology of alkaliphiles from an “omics” point of view, focusing on how metagenomics and transcriptomics have contributed to our understanding of these extremophiles.
Pedro H. Lebre, Don A. Cowan

Metabolites Produced by Alkaliphiles with Potential Biotechnological Applications

Alkaliphiles are a diverse group of relatively less known microorganisms living in alkaline environments. To thrive in alkaline environments, alkaliphiles require special adaptations. This adaptation may have evolved metabolites which can be useful for biotechnological processes or other applications. In fact, certain metabolites are found unique to alkaliphiles or are effectively produced by alkaliphiles. This probably aroused the interest in metabolites of alkaliphiles. During recent years, many alkaliphilic microbes have been isolated, especially in countries having alkaline environments, like soda lakes. Even if the number of such isolated alkaliphiles is large, their metabolites have not yet been extensively analyzed and exploited. This is expected to come in the years ahead. So far, the focus of interests in metabolites from alkaliphiles falls into categories such as organic acids, ingredients for foodstuffs and cosmetics, antibiotics, and substances which modify properties of other materials used in industry. This chapter deals with biotechnologically important metabolites of alkaliphiles including compatible solutes, biosurfactants, siderophores, carotenoids, exopolysaccharides, and antimicrobial agents. It also covers the promising potential of alkaliphiles as sources of bioplastic raw materials. Moreover, an overview of the patent literature related to alkaliphiles is highlighted.
Elvira Khalikova, Susanne Somersalo, Timo Korpela

Alkaliphilic Enzymes and Their Application in Novel Leather Processing Technology for Next-Generation Tanneries

Leather manufacturing involves conversion of raw skin and hides into leather (stable material) through series of mechanical and chemical operations. The leather industry has attracted public outcry due to severe environmental degradation, pollution and health and safety risks. Currently the industry faces serious sustainability challenge due to extensive use of toxic chemicals and generation of hazardous waste. This chapter describes the polluting chemicals consumed in different stages of conventional leather processing and the nature of waste generated. In order to overcome the hazards caused by toxic chemicals in tanneries and protect the environment, enzymes have been identified as a realistic alternate for chemicals used in beam house operation and waste management. Alkaline active proteases of alkaliphiles offer advantages over the use of conventional chemical catalysts for numerous reasons, for example, they exhibit high catalytic activity and high degree of substrate specificity, can be produced in large amounts and are economically viable. This is because the enzymes of these alkaliphiles are capable of catalysing reactions at the extremes of pH, temperature and salinity of leather-manufacturing processes.
The chapter describes how alkaliphilic enzyme can effectively be used in soaking, dehairing, bating and degreasing operations to prevent waste generation, help in recovery of valuable by-products, reduce cost and increase leather quality. It is worth noting that protease has the capability to replace sodium sulphide in the dehairing process. In addition, alkaline proteases have shown remarkable ability in bioremediation of waste generated during the industrial processes. Intensive efforts are being directed towards chemical-based industries to use viable clean technology in their operation to reduce their negative impact on the environment. Similarly, leather industry should adopt the use of eco-friendly reagents such as enzymes to achieve long-term sustainability and clean environment and avert health hazards. Application of enzyme technology in clean leather processing strongly depends on legislation, political will and allocation of financial resources in research, development and implementation of this potentially powerful technology.
Wycliffe C. Wanyonyi, Francis J. Mulaa

Starch-Modifying Enzymes

Starch is a carbohydrate polymer found abundantly on earth. It is synthesized in plants as a short-term storage compound for respiration in the leaves and for long-term storage in the tubers, seeds and roots of plants. A wide variety of enzymes modify or convert starch into various products. The classes of enzymes that act on starch include endoamylases, exoamylases, debranching enzymes and transferases. Starch-modifying enzymes of microbial origin are utilized in a wide variety of industrial applications. Alkaline-active amylases are diverse in terms of optimum reaction conditions, substrate and product specificity. Amylases that are active at lower temperatures and alkaline conditions are most suited for detergent formulation. Other notable starch-modifying enzymes from alkaliphiles include maltooligosaccharide-forming amylases and cyclodextrin glycosyltransferases (CGTases), which produce a variety of maltooligosaccharides and cyclodextrins, respectively. Such compounds are used in the food, fine chemical, pharmaceutical and cosmetic industries, among others. Alkaline-active amylases are also applicable in the paper, textile and leather industries and also in bioremediation and alkaline waste water treatment. Their application in these fields is further enhanced through stabilization and improving their specificity and catalytic action by employing nanotechnology and genetic engineering.
Suhaila Omar Hashim

Alkaline Active Hemicellulases

Xylan and mannan are the two most abundant hemicelluloses, and enzymes that modify these polysaccharides are prominent hemicellulases with immense biotechnological importance. Among these enzymes, xylanases and mannanases which play the vital role in the hydrolysis of xylan and mannan, respectively, attracted a great deal of interest. These hemicellulases have got applications in food, feed, bioethanol, pulp and paper, chemical, and beverage producing industries as well as in biorefineries and environmental biotechnology. The great majority of the enzymes used in these applications are optimally active in mildly acidic to neutral range. However, in recent years, alkaline active enzymes have also become increasingly important. This is mainly due to some benefits of utilizing alkaline active hemicellulases over that of neutral or acid active enzymes. One of the advantages is that the alkaline active enzymes are most suitable to applications that require high pH such as Kraft pulp delignification, detergent formulation, and cotton bioscouring. The other benefit is related to the better solubility of hemicelluloses at high pH. Since the efficiency of enzymatic hydrolysis is often positively correlated to substrate solubility, the hydrolysis of hemicelluloses can be more efficient if performed at high pH. High pH hydrolysis requires the use of alkaline active enzymes. Moreover, alkaline extraction is the most common hemicellulose extraction method, and direct hydrolysis of the alkali-extracted hemicellulose could be of great interest in the valorization of hemicellulose. Direct hydrolysis avoids the time-consuming extensive washing, and neutralization processes required if non-alkaline active enzymes are opted to be used. Furthermore, most alkaline active enzymes are relatively active in a wide range of pH, and at least some of them are significantly or even optimally active in slightly acidic to neutral pH range. Such enzymes can be eligible for non-alkaline applications such as in feed, food, and beverage industries.
This chapter largely focuses on the most important alkaline active hemicellulases, endo-β-1,4-xylanases and β-mannanases. It summarizes the relevant catalytic properties, structural features, as well as the real and potential applications of these remarkable hemicellulases in textile, paper and pulp, detergent, feed, food, and prebiotic producing industries. In addition, the chapter depicts the role of these extremozymes in valorization of hemicelluloses to platform chemicals and alike in biorefineries. It also reviews hemicelluloses and discusses their biotechnological importance.
Graphical Abstract
Gashaw Mamo

Alkaliphiles: The Emerging Biological Tools Enhancing Concrete Durability

Concrete is one of the most commonly used building materials ever used. Despite it is a very important and common construction material, concrete is very sensitive to crack formation and requires repair. A variety of chemical-based techniques and materials have been developed to repair concrete cracks. Although the use of these chemical-based repair systems are the best commercially available choices, there have also been concerns related to their use. These repair agents suffer from inefficiency and unsustainability. Most of the products are expensive and susceptible to degradation, exhibit poor bonding to the cracked concrete surfaces, and are characterized by different physical properties such as thermal expansion coefficients which are different to that of concrete. Moreover, many of these repair agents contain chemicals that pose environmental and health hazards. Thus, there has been interest in developing concrete crack repair agents that are efficient, long lasting, safe, and benign to the environment and exhibit physical properties which resemble that of the concrete. The search initiated by these desires brought the use of biomineralization processes as tools in mending concrete cracks. Among biomineralization processes, microbially initiated calcite precipitation has emerged as an interesting alternative to the existing chemical-based concrete crack repairing system. Indeed, results of several studies on the use of microbial-based concrete repair agents revealed the remarkable potential of this approach in the fight against concrete deterioration. In addition to repairing existing concrete cracks, microorganisms have also been considered to make protective surface coating (biodeposition) on concrete structures and in making self-healing concrete.
Even though a wide variety of microorganisms can precipitate calcite, the nature of concrete determines their applicability. One of the important factors that determine the applicability of microbes in concrete is pH. Concrete is highly alkaline in nature, and hence the microbes envisioned for this application are alkaliphilic or alkali-tolerant. This work reviews the available information on applications of microbes in concrete: repairing existing cracks, biodeposition, and self-healing. Moreover, an effort is made to discuss biomineralization processes that are relevant to extend the durability of concrete structures.
Gashaw Mamo, Bo Mattiasson


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