Bacterial Nanocellulose: From Biotechnology to Bio-Economy

Bacterial Nanocellulose: From Biotechnology to Bio-Economy presents an overview on the current and future applications of bacterial nanocellulose, perspectives on the ecology and economics of its production, and a brief historic overview of BNC related companies.

• Discusses recent progresses on the molecular mechanism of BNC biosynthesis, its regulation, and production techniques
• Covers advances in the use of BNC in bio- and nano-polymer composite materials
• Presents a detailed economic analysis of BNC production
• Provides an overview on the regulatory framework on the food and biomedical fields
• Reviews current research in the biomedical and food industries, identifies gaps, and suggests future needs
• Raises awareness about this material and its potential uses in emergent fields, such as the development of aerogels and optoelectronic devices

• Language: English
• Publisher: Elsevier Science
• Release date: Jul 12, 2016
• ISBN: 9780444634665

Book preview

Bacterial NanoCellulose

From Biotechnology to Bio-Economy

Edited by

Miguel Gama

Minho University, Biological Engineering Department

Campus de Gualtar, Braga, Portugal

Fernando Dourado

Minho University, Biological Engineering Department

Campus de Gualtar, Braga, Portugal

Stanislaw Bielecki

Lodz University of Technology, Institute of Technical Biochemistry

Lodz, Poland

Table of Contents

Cover

Title page

Copyright

List of Contributors

Preface

Chapter 1: Taxonomic Review and Microbial Ecology in Bacterial NanoCellulose Fermentation

Abstract

Acetic Acid Bacteria

The nata Organism

Conclusions

Chapter 2: Bacterial NanoCellulose Synthesis, Recent Findings

Abstract

Introduction

The Role of the Proteins Encoded in the Cellulose Synthase Operon and its Flanking Regions in the Biosynthesis of Cellulose

Metabolic Point of View on BNC Production

Genetic Modification of Bacteria of the Genus Gluconacetobacter

Chapter 3: Molecular Control Over BNC Biosynthesis

Abstract

Introduction: BNC Can Be Regarded as a Sort of a Biofilm

The Cyclic di-GMP Signaling Network Ruling Over Cellulose Production and Bacterial Life-Style Switch

Beyond c-di-GMP Signaling—Potential Importance of General Regulatory Pathways in Cellulose Biosynthesis Control

Regulatory Mechanisms Involved in Cellulose Synthesis Identified Recently

Summary: Can We Expect Any Practical Conclusions?

Chapter 4: Bacterial NanoCellulose Characterization

Abstract

Surface Characterization of Bacterial NanoCellulose

FTIR Analysis of BNC

Bio Nanocellulose as a Single Nanofiber Prepared from BNC Pellicle

Chapter 5: Bacterial NanoCellulose Aerogels

Abstract

A Brief Overview on 85 Years of Research on Man-Made Aerogels

Bacterial NanoCellulose: A Green, Cheap, and Mass Producible Porous Material

Conversion of Bacterial NanoCellulose Aquogels to Aerogels

Properties of Bacterial NanoCellulose Aerogels

Reinforcement of Bacterial NanoCellulose Aerogels

Potential Applications of Bacterial NanoCellulose Aerogels

Outlook

Chapter 6: Bacterial NanoCellulose as Reinforcement for Polymer Matrices

Abstract

Introduction

Tensile Properties of (Nano)cellulose

NFC and BNC: Similarities and Differences

Bacterial NanoCellulose as Reinforcement for Polymers

Outlook

Acknowledgment

Chapter 7: Celluloses as Food Ingredients/Additives: Is There a Room for BNC?

Abstract

Hydrocolloids in Food Technology

Cellulose Derived Hydrocolloids and Microcrystalline Cellulose

Nata de coco

BNC as a Food Additive

BNC Dietetic Information

Conclusions

Chapter 8: European Regulatory Framework on Novel Foods and Novel Food Additives

Abstract

Food Safety and Consumers Health Protection in the European Community

Novel Food Regulation

General Application Procedure

Simplified Notification Procedure

Food Additives

Concluding Remarks

Chapter 9: Medical and Cosmetic Applications of Bacterial NanoCellulose

Abstract

Overview of Biomedical Applications

Cosmetic Applications of Bacterial NanoCellulose

Chapter 10: Medical Devices Regulation

Abstract

Introduction

Obligations of Manufacturers, Authorized Representatives, Importers, Distributors

Definition of Medical Device and its Classification

Conformity Assessment Procedures Prior to Putting a Medical Device into Service and Use

Glossary (given in accordance with directive: MDD, AIMD, 93/42/EEC and directive: 2007/47/EC)

Chapter 11: Optoelectronic Devices from Bacterial NanoCellulose

Abstract

Introduction

Structural, Morphological and Thermal Properties of Bacterial NanoCellulose

Application to Electronic and Optoelectronic Devices

Conclusions

Acknowledgments

Chapter 12: Process Modeling and Techno-Economic Evaluation of an Industrial Bacterial NanoCellulose Fermentation Process

Abstract

Introduction

Process Simulation Software

Cost Estimations

Process Simulation: Bacterial NanoCellulose Production

Concluding Remarks

Chapter 13: Nata de Coco Industry in the Philippines

Abstract

Introduction

Methodology

Nata de Coco Trade Business

Nata de Coco Production

Raw Nata de Coco Producers

Production and Market Outlets

Selling Prices

Profitability of Raw Nata de Coco Production

Problems in Nata de Coco Production

Conclusions

Chapter 14: Nata de coco Industry in Vietnam, Thailand, and Indonesia

Abstract

Nata de coco Value Chain Linkages in Vietnam

Nata de Coco Market in Thailand

Nata de Coco Market in Indonesia

Conclusions

Acknowledgments

Subject Index

Copyright

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List of Contributors

Hugo Águas,     i3N/CENIMAT, Department of Materials Science, Faculty of Science and Technology, Universidade NOVA de Lisboa and CEMOP/UNINOVA, Lisbon, Portugal

Stanislaw Bielecki,     Lodz University of Technology, Institute of Technical Biochemistry, Lodz, Poland

Alexander Bismarck

Polymer and Composite Engineering (PaCE) Group, Institute of Materials Chemistry and Research, Faculty of Chemistry, University of Vienna, Vienna, Austria

Polymer and Composite Engineering (PaCE) Group, Department of Chemical Engineering, Imperial College London, London, United Kingdom

Rusdianto Budiraharjo,     Department of Biotechnology, Indonesia International Institute for Life Sciences (i3L), Jakarta, Indonesia

Son Chu-Ky,     School of Biotechnology and Food Technology, Hanoi University of Science and Technology, Hanoi, Vietnam

Ana Cristina Rodrigues,     Minho University, Biological Engineering Department, Campus de Gualtar, Braga, Portugal

Fernando Dourado,     Minho University, Biological Engineering Department, Campus de Gualtar, Braga, Portugal

Paulo Duarte,     i3N/CENIMAT, Department of Materials Science, Faculty of Science and Technology, Universidade NOVA de Lisboa and CEMOP/UNINOVA, Lisbon, Portugal

Gabriella Gita Febriana,     Department of Biomedicine, Indonesia International Institute for Life Sciences (i3L), Jakarta, Indonesia

Ana Fontão,     Minho University, Biological Engineering Department, Campus de Gualtar, Braga, Portugal

Elvira Fortunato,     i3N/CENIMAT, Department of Materials Science, Faculty of Science and Technology, Universidade NOVA de Lisboa and CEMOP/UNINOVA, Lisbon, Portugal

Miguel Gama,     Minho University, Biological Engineering Department, Campus de Gualtar, Braga, Portugal

Diana Gaspar,     i3N/CENIMAT, Department of Materials Science, Faculty of Science and Technology, Universidade NOVA de Lisboa and CEMOP/UNINOVA, Lisbon, Portugal

Marzena Jedrzejczak-Krzepkowska,     Lodz University of Technology, Institute of Technical Biochemistry, Lodz, Poland

Marek Kolodziejczyk,     Lodz University of Technology, Institute of Technical Biochemistry, Lodz, Poland

Tetsuo Kondo,     Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Japan

Katarzyna Kubiak,     Lodz University of Technology, Institute of Technical Biochemistry, Lodz, Poland

Marta Leal,     Minho University, Biological Engineering Department, Campus de Gualtar, Braga, Portugal

Koon-Yang Lee,     The Composites Centre, Department of Aeronautics, Imperial College London, London, United Kingdom

Falk Liebner,     Division of Chemistry of Renewable Resources, University of Natural Resources and Life Sciences Vienna, Tulln, Austria

Karolina Ludwicka,     Lodz University of Technology, Institute of Technical Biochemistry, Lodz, Poland

Daniela Martins,     Minho University, Biological Engineering Department, Campus de Gualtar, Braga, Portugal

Rodrigo Martins,     i3N/CENIMAT, Department of Materials Science, Faculty of Science and Technology, Universidade NOVA de Lisboa and CEMOP/UNINOVA, Lisbon, Portugal

Danh-Nguyen Nguyen,     School of Economics and Management, Hanoi University of Science and Technology, Hanoi, Vietnam

Teresa Pankiewicz,     Lodz University of Technology, Institute of Technical Biochemistry, Lodz, Poland

Luís Pereira,     i3N/CENIMAT, Department of Materials Science, Faculty of Science and Technology, Universidade NOVA de Lisboa and CEMOP/UNINOVA, Lisbon, Portugal

Muenduen Phisalaphong,     Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand

Ma. Eden S. Piadozo,     Department of Agricultural and Applied Economics, College of Economics and Management, University of the Philippines Los Baños, Los Baños, Philippines

Nicole Pircher,     Division of Chemistry of Renewable Resources, University of Natural Resources and Life Sciences Vienna, Tulln, Austria

Thomas Rosenau,     Division of Chemistry of Renewable Resources, University of Natural Resources and Life Sciences Vienna, Tulln, Austria

Malgorzata Ryngajllo,     Lodz University of Technology, Institute of Technical Biochemistry, Lodz, Poland

Przemysław Rytczak,     Lodz University of Technology, Institute of Technical Biochemistry, Lodz, Poland

Siriporn Taokaew,     Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand

Tien-Khai Tran,     School of Economics, University of Economics Ho Chi Minh City, Vietnam

Claudia van den Berg,     TNO, The Netherlands

António Vicente,     i3N/CENIMAT, Department of Materials Science, Faculty of Science and Technology, Universidade NOVA de Lisboa and CEMOP/UNINOVA, Lisbon, Portugal

Preface

Bioeconomy is based on the efficient use of diverse natural substrates and processes, for the production of food, feed, bio-based products, and bioenergy. An example of the rational usage of natural resources is the fabrication of Bacterial NanoCellulose (BNC), which may be produced from various wastes or biomass.

BNC is a natural polymer, synthesized by a number of species, among which acetic acid bacteria (AAB) of the species Gluconoacetobacter xylinus and Ga. hansenii are its best recognized and most efficient producers. These bacteria produce an extracellular, chemically pure β-glucan, supporting their survival in the natural environment since the cells are kept at the surface of culture media, being entrapped inside gelatinous, skin-like membranes, consisting of entangled cellulose fibers. Such self-immobilization of the cells promotes efficient transport of nutrients and oxygen, which is essential for these aerobic bacteria. Owing to the high water holding capacity (water accounts for around 98% wet membrane weight) this polysaccharide protects its producers from desiccation. Cellulose matrix shields these bacteria also from other adverse environmental factors, like UV radiation. Bacteria synthesizing cellulose adhere to its surface and are relatively motile, leading to successful colonization of ecological niches. This in turn reduces available space and supply of nutrients for other microorganisms populating the same habitats.

BNC synthesized by Gluconoacetobacter species is characterized by unique features, resulting from its natural biological role, such as high hydrophilicity, crystallinity, purity and water holding capacity, mechanical durability and resistance to degradation, excellent biocompatibility and lack of cytotoxicity and allergenicity. Because of these properties and susceptibility to biological, chemical, and physical modifications, this natural biomaterial found numerous applications in fabrication of bioproducts and is considered a bio-base for the development of novel materials in various fields, like food processing, electronics, paper making, chemical and textile industries as well as in medicine. Increasing applications of bacterial cellulose and its derivatives in various branches of industry and in medicine gave rise to intensive studies on the improvement of its production efficiency, while attempting to lower the costs of BC biosynthesis and modifications.

AAB have a long history of use in several fermentation processes. Their exploitation gradually emerged in biotechnological applications, especially in the biosynthesis of useful chemicals and processes for the manufacture of several fermented food products. Taxonomic studies, from traditional to polyphasic approaches, have gradually allowed the proper classification of several ABB into distinct genera and species, among them, the BNC producers, notably G. xylinus. Chapter 1 first reviews the main historical steps involved in the taxonomic classification of AAB. It then addresses the lying potential behind mixed microbial fermentations, from kombucha to nata de coco, both sharing in common, the contribution of cellulose-producing bacteria for the fermentation process.

Recent advances in molecular biology studies on Gluconacetobacter species metabolism are presented in Chapter 2. Its readers will find the map of metabolic pathways of these bacterial species, information about utilization of various wastes for BNC biosynthesis, novel findings related to the structure of cellulose synthase operon and flanking sequences in Gluconacetobacter species and other microbial cellulose producers as well as the explanation of roles played by proteins that are encoded by these sequences in cellulose biosynthesis. Also, crystallographic structures of A and B subunits of cellulose synthase and its complex with c-di-GMP, which were resolved in 2013 and 2014, are presented in this chapter. Examples of genetic modifications of Gluconacetobacter species, with particular emphasis on genetic tools applied, and their effect on BNC biosynthesis are also included.

Chapter 3 describes mechanisms of bacterial cellulose biosynthesis regulation, paving way to further genetic studies, leading to better comprehension of molecular control of BNC secretion.

Chapter 4 summarizes analytical techniques that are used to characterize BNC and presents common physical, chemical methods enabling for a detailed description of the properties of native and modified BNC.

Chapter 5 describes the intriguing properties of BNC aerogels and the way they can be obtained. These aerogels are expected to find use in high-performance thermal insulation, as matrix material for gas separation, carrier for magnetic particles (electro actuators), catalysts, quantum dots (bio-sensing, volumetric displays), or bioactive compounds (controlled drug release). BNC aerogels are furthermore promising cell scaffolds (tissue engineering) and precursor materials for the manufacture of carbon aerogels (electrochemical applications).

BNC is a promising material for the production of high performance renewable composites because of its high tensile properties, low density, and low toxicity. Chapter 6 starts with the discussion of both theoretical and experimental tensile properties of nanometre-scale cellulose fibrils, more commonly known as nanocellulose. This is then followed by what neat BNC offers as nanoreinforcement for polymers. The tensile properties of various neat BNC-reinforced polymer nanocomposites published in the literature to date are reviewed and are tabulated. In addition, the micromechanical models that are suitable to describe the tensile properties of BNC-reinforced polymer nanocomposites are critically discussed.

The use of BNC as a food product, and particularly its potential as a novel food additive, is reviewed in Chapter 7. Its technological potential as a novel hydrocolloid for the modification of textural properties of food products is addressed. This work briefly reports on the already commercially available cellulose based hydrocolloids, namely colloidal microcrystalline cellulose, then reviewing the studies which demonstrate the potential of BNC in this field. Chapter 8 overviews the European Union (EU) legislative framework of Novel Foods/Novel Food Ingredients and Food additives, to better familiarize the reader of the general steps involved in a premarket approval within these regulatory frameworks.

Chapter 9 describes the medical and cosmetic applications of BNC, starting from the most known never-dried wound dressings and facial masks, going through BNC internal uses as implantable material, like artificial heart valves, blood vessels or dura mater, and finally covering the topic of cellulose numerous modifications for the use as, inter alia, substitute of cartilage, tubes for nerves regeneration, or composites with porous materials for meshes preparation. Potential usage of this natural biomaterial in other fields of medical sciences, like tissue engineering exploiting the forms of porous scaffolds, as well as drug delivery sector applying BNC-based release systems, is also presented.

Chapter 10 provides the readers with the necessary basic knowledge for the implementation of original, innovative technological solutions, based on results of scientific research. This knowledge may be used for faster and more precise presentation of data and collection of suitable documentation for the certification processes that are obligatory for commercial products, based on BNC. This chapter includes the description of relevant definitions and classification of medical products, principal requirements, conformity assessment procedures, obligations of manufacturers and other information related to medical applications of the biomaterial in order to present readers with a clearer picture of the issues related to obtaining the necessary certificates for medical devices before placing them onto the market.

Chapter 11 reviews the main applications of BNC in electronics, either as a substrate (passive) or as a real electronic material (active), and discusses the advantages of BNC in the field of Paper Electronics.

Chapter 12 explores the process and economics of a computer simulated large scale production of BNC by static culture conditions. A comparative economic analysis between modern and traditional plants is not straightforward due to differences in local feedstock costs, energy, equipments, taxes, labor, currency, and so forth. However, data here gathered showed that even if considering the use of low cost substrates, the biotechnological fermentation of BNC is markedly expensive and inefficient, as compared to traditional fermentation. The high capital investment and high production costs increased by almost two orders of magnitude the selling price of BNC produced in a modern technological set, which would limit the scope of market penetration.

Finally, Chapters 13 and 14 overview, perhaps for the first time, the nata de coco business in the Philippines, Thailand, Vietnam, and Indonesia, providing an insight into the current trade situation, including exports and import analysis, identify the major raw nata producers, their production practices, marketing outlets, and their selling price. The profitability of growing raw nata de coco business is also analyzed.

Chapter 1

Taxonomic Review and Microbial Ecology in Bacterial NanoCellulose Fermentation

Fernando Dourado*

Malgorzata Ryngajllo**

Marzena Jedrzejczak-Krzepkowska**

Stanislaw Bielecki**

Miguel Gama*

*    Minho University, Biological Engineering Department, Campus de Gualtar, Braga, Portugal

**    Lodz University of Technology, Institute of Technical Biochemistry, Lodz, Poland

Abstract

Acetic acid bacteria (AAB) have a long history of use in several fermentation processes. Their exploitation gradually emerged in biotechnologic applications, especially in the biosynthesis of useful chemicals and processes for the manufacture of several fermented food products. Taxonomic studies, from traditional to polyphasic approaches, have gradually allowed the proper classification of several ABB into distinct genera and species, among them, the bacterial nanocellulose (BNC) producers, notably Komagataeibacter xylinus. Despite the advantages in using specific (isolated) strains for biotechnologic processes toward controlling the kinetics and process yield, mixed culture fermentations may provide an interesting approach to tailoring the properties of BNC and to increase the product yield when aiming at industrial scale. Microbial population dynamics may play a synergistic role in the coordinative substrate consumption and metabolites’ production, especially if using complex media (as is the case with low cost substrates, eg, residues from other processes). This chapter will first review the main historic steps involved in the taxonomic classification of AAB. It will then address the lying potential behind mixed microbial fermentations, from kombucha to nata de coco, both sharing in common, the contribution of cellulose-producing bacteria for the fermentation process.

Keywords

acetic acid bacteria

taxonomy

Acetic Acid Bacteria

Acetic acid bacteria (AAB) are well-known producers of certain foods and drinks, such as vinegar, kombucha tea, and cocoa. They are also known for being spoilers of other food products such as wine, beer, soft drinks, and fruits. Cellulose is also a specific product from AAB metabolism. The AAB name derives from the bacteria’s ability to oxidize ethanol into acetic acid.

The Acetobacteraceae family consist of a wide group of strictly aerobic, Gram negative, AAB, endowed with the ability to oxidize a wide variety of carbohydrates, alcohols, and sugar alcohol into acetic acid and other organic acids (such as gluconic, fumaric, citric, oxoacids, and ketones) and even amino acids. Among the several genera of this family, the Acetobacter and Komagataeibacter genus are the most notable acetic acid producers; also they show rather high tolerance to acidic and alcoholic environments, both scenarios highly prohibitive of the growth of other microorganisms. Acetobacter strains have a higher capacity for acetic acid production from ethanol, whereas Komagataeibacter oxidize sugars better [1–5]. Both genera typically display a diauxic growth curve when cultured in a medium containing ethanol, the first phase being characterized by ethanol oxidation to acetic acid, while in a second stage (overoxidation phase) acetic acid is oxidized to water and carbon dioxide, for further growth [6].

The Taxonomic Classification of Acetic Acid Bacteria

The taxonomic classification of AAB at the species level has been an evolving subject not only because of the methodologies used but also due to these bacteria’s tendency to undergo spontaneous mutations. Initially, taxonomic classification was based on morphologic, physiologic and biochemical characteristics (classic taxonomy) [7]. Nowadays, it also considers information derived from microbial metabolism, ecology, genome characterization, and phylogeny (polyphasic approach, Fig. 1.1).

Figure 1.1   Diagrammatic representation of techniques and markers used in modern polyphasic approach for resolving the bacterial hierarchy. Reproduced from Ref. [8] with permissions.

Several methods used for the taxonomic classification of AAB include 16S rRNA gene sequencing analysis (a highly preserved region of the gene in which small changes can be characteristic of different species), % base ratio determination (one of the first molecular tools used in bacterial taxonomy that calculates the percent of G + C (guanine + cytosine) in the bacterial genome), DNA–DNA hybridization (a widely used technique for describing new species within bacterial groups; it measures the degree of similarity of the genomes of different species), analysis of molecular markers and signature patterns. These culture-independent methods allow to overcoming the limited to inexistent growth of certain strains once isolated from their natural habitats; they allow for a more reliable classification and reclassification of several species and to better determining the relationships between different organisms within a microbial community [8,9]. Recent reviews on the molecular techniques for the identification of ABB can be found in Refs. [8–12].

Fig. 1.2 displays a flow diagram depicting the sequential approaches in the taxonomic characterization of newly isolated strains followed by its deposition in culture collection centers and publication. The International Code of Nomenclature of Bacteria (ICNB) regulates the scientific naming of bacteria, their changes and proposals of new names [13]. The "International Journal of Systematics and Evolutionary Microbiology (IJSEM) compiles and publishes new taxa of bacteria and yeasts. It is the official journal of record for bacterial names of the International Committee on Systematics of Prokaryotes (ICSP) of the International Union of Microbiological Societies (IUMS). The National Center for Biotechnology Information (NCBI) taxonomy database (http://www.ncbi.nlm.nih.gov/taxonomy) is the standard nomenclature and classification repository for the International Nucleotide Sequence Database Collaboration (INSDC), which comprises the databases from the GenBank, the European Nucleotide Archive (ENA), and the DNA Data Bank of Japan (DDBJ). NCBI’s database includes organism names and taxonomic lineages for each of the sequences represented in the INSDC’s nucleotide and protein sequence databases [14]. The Bergey’s Manual of Systematic Bacteriology is the main monographic work in the field of prokaryotic biology. Under periodic revisions, the manual includes every characterization method/technique for determining bacterial identity.

Figure 1.2   Flow diagram depicting the step-by-step procedure for taxonomic characterization of newly isolated strains followed by its deposition in culture collection centers and publication. Reproduced from Ref. [8] with permissions.

Fig. 1.3 shows a phylogenetic tree reflecting the relationships between the members of the Acetobacteraceae family. Further phylogenetic information can be found in Ludwig [10] and more recently by Matsutani et al. [15] and Yamada et al. [16].

Figure 1.3   Maximum likelihood phylogenetic tree calculated with MUSCLE from predicted (Barrnap—http://www.vicbioinformatics.com/software.barrnap.shtml) 16S rRNA gene sequences of Acetobacteraceae genomes.

The numbers at branching points represent percentage bootstrap values from 500 replications. The bar represents 1% sequence divergence. Phylogenetic tree was constructed using MEGA6.06.

An overview of some of the most important milestones in AAB taxonomy can be found in Table 1.1 [9,11,17]. The main genera of AAB have long been the Acetobacter and Gluconobacter. By 1898, the genus Acetobacter, with a single species (Acetobacter aceti) was established. By 1925, Visser’t Hooft [18] was the first to propose a taxonomic classification of AAB based on biochemical and physiologic criteria. Later on in 1935, Asai [19] further proposed that the genus Gluconobacter should include those bacteria capable of oxidizing glucose to gluconic acid; Asai also proposed that the genus Gluconobacter should include those species that could not oxidize acetic acid. By 1950, physiology studies further led to the classification of bacteria based on morphologic and physiologic features. Frateur [20] divided the Acetobacter genera into four groups: peroxydans, oxydans, mesoxydans, and suboxydans. Leifson (by 1954) also further separated the flagellated AAB and capable of oxidizing ethanol into the genus Acetobacter from those with polar flagella but unable to completely oxidize ethanol into the genera Gluconobacter [9,21]. De Ley and Schell [22] studied the base composition of DNA from 28 strains of AAB, and proposed a close relationship and a possible common phylogenetic origin for Acetobacter and Gluconobacter.

Table 1.1

Main chronological phases of the study of AAB systematics

Adapted from Ref. [9] with permissions.

By 1983 and 1984, Yamada et al. [1,16,24,25] proposed a division of the genus Acetobacter into two subgenera, based on differences in the bacteria’s ubiquinone system: Acetobacter was thus characterized by a Q-9 ubiquinone as the major respiratory quinone, while the proposed type subgenus Gluconacetobacter mainly uses a Q-10 ubiquinone. As a follow up, several ubiquinone Q-10 containing Acetobacter species were transferred to the subgenus Gluconacetobacter, namely A. diazotrophicus, A. europaeus, A. hansenii, A. liquefaciens, and A. xylinus [3,4,26–28]. By 1998, and based on partial 16S rDNA sequences, Yamada et al. [1] proposed the elevation of the subgenus Gluconacetobacter to the genus category. However, the growing number of identified Gluconacetobacter species lead to further division into Ga. liquefaciens and Ga. xylinus groups [17]. The main phenotypic difference between these two groups was that the members of the Ga. liquefaciens group were motile, with peritrichous flagella, whereas Ga. xylinus group lacked any flagellation and was thus nonmotile. Phylogenetic division in Gluconacetobacter genus was supported from sequence analyses of three housekeeping genes (dnaK, groEL, and rpoB) by Cleenwerck et al. [29]. With this collective evidence, Yamada et al. [30] proposed the classification of the Ga. xylinus group into a separate new genus— Komagataeibacter.

Perhaps, other sequences maybe explored to achieve better separation of these two groups. One such good example could be sequence of PEPCase, since it is a central metabolism enzyme and the degree of separation of species is even stronger than when using dnaK sequence (Fig. 1.4A and B). Furthermore, this sequence enables some degree of separation even between the strains within the group.

Figure 1.4   ML phylogenetic tree calculated from predicted gene sequences of (A) dnaK (together with partial sequences of genes from liquefaciens group) and (B) PEPCase for Gluconacetobacter species.

The numbers at branching points represent percentage bootstrap values from 500 replications, for A and B. The bar represents 10% and 5% sequence divergence, respectively.

The Acetobacteraceae family (Fig. 1.3) now accounts for 25 genera, 6 of which are monotypic (Acidomonas, Kozakia, Swaminathania, Saccharibacter, Neoasaia, and Granulibacter) [17,31]. Acetobacter (with a record of 24 identified species in http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=434) and Gluconacetobacter (with 23 species; http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=89583) are among the most widely known and used in industry [32]. GC content of AAB is not homogenous and ranges between