**Cultural, Physiological, and Biochemical Identification of Actinobacteria**

Qinyuan Li, Xiu Chen, Yi Jiang and Chenglin Jiang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61462

#### **Abstract**

[58] Chater KF, Chandra G. The evolution of development in *Streptomyces* analysed by ge‐ nome comparisons. FEMS Microbiol Rev 2006;30:651-72. DOI: 10.1111/j.

[59] Flärdh K, Richard DM, Hempel AM, Howard M, Buttner MJ. Regulation of apical growth and hyphal branching in *Streptomyces*. Curr Opin Microbiol 2012;15:737-43.

[60] Hengst CD, Tran NT, Bibb MJ, Chandra G, Leskiw BK, Buttner MJ. Genes essential for morphological development and antibiotic production in *Streptomyces coelicolor* are targets of BldD during vegetative growth. Molecul Microbiol 2010;78:361-79.

[61] Hillerich B, Westpheling J. A new TetR family transcriptional regulator required for morphogenesis in *Streptomyces coelicolor.* J Bacteriol 2008;190:61-7. DOI: 10.1128/JB.

[62] Petrus MLC, Claessen D. Pivotal roles for *Streptomyces* cell surface polymers in mor‐ phological differentiation, attachment and mycelial architecture. Antonie van Leeu‐

wenhoek 2014;106:127-39. DOI 10.1007/s10482-014-0157-9

1574-6976.2006.00033.x

86 Actinobacteria - Basics and Biotechnological Applications

01316-07

DOI: 10.1016/j.mib.2012.10.012

DOI: 10.1111/j.1365-2958.2010.07338.x

The traditional phenotypic tests are commonly used in actinobacterial identifica‐ tion. They constitute the basis for the formal description of taxa, from species and subspecies up to genus and family. The classical phenotypic characteristics of acti‐ nobacteria comprise morphological, physiological, and biochemical features. The morphology of actinobacteria includes both cellular and colonial characters. The physiological and biochemical features include data on growth at different tempera‐ tures, pH values, salt concentrations, or atmospheric conditions, and data on growth in the presence of various substances such as antimicrobial agents, the pres‐ ence or activity of various enzymes, and with respect to metabolization of com‐ pounds. The phenotype is the observable expression of the genotype. Gene expression is directly related to the environmental conditions. Actinobacterial phe‐ notype cannot be based on the simple observation of the organism. Strains of the most closely related taxa should be compared in their phenotypic analysis using identical methods. The comparisons must include the type strain of the type species of the appropriate genera. Furthermore, with the development of technology, mi‐ crobial physiological and biochemical identification technology is becoming fast, simple, and automated.

**Keywords:** Phenotype, Physiological and biochemical characteristics, Automatic identifi‐ cation system

### **1. Introduction**

The polyphasic approach [1], the comprehensive results of various methods, such as morpho‐ logical, physiological, rRNA gene sequencing, chemotaxonomic markers and pathogenicity,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

are used to compile a description of a new species. Classification of actinobacteria is now based largely on analysis of the nucleotide sequences, especially 16S rRNA genes [2]. However, a comprehensive characterization of a new species should still be identified, because from even a complete genomic sequence, it would be difficult to predict many of the phenotypic features of a new species [3]. Physiological and biochemical characteristics are directly related to the activity of microbial enzymes and regulatory proteins. Enzymes and proteins are gene products; so the comparison on physiological and biochemical characteristics of actinobacteria is the indirect comparison of genome, and determination of physiological and biochemical characteristics is much easier than direct analysis of the genome. Therefore, the physiological and biochemical characteristics in actinobacterial systematics and identification are still meaningful [4]. In addition to a thorough phenotypic characterization of a new species, it is important to determine which phenotypic features are the ones most useful for identifying new species. Moreover, the particular methods used for characterizing an organism should always be stated, because the results of phenotypic tests can vary with methodology.

Phenotypic analysis is a very tedious task in the classification of actinobacteria. The classical phenotypic characteristics of actinobacteria comprise morphological, physiological, and biochemical features. Individually sufficient as parameters for genetic relatedness, yet as a whole, they provide descriptive information enabling us to recognize taxa [5]. The morpho‐ logical traits include both cellular (cell shape and size, spore, sporangia, sporangiospore, the location of the spores or sporangia and their size, flagella, motility, intracellular structures, etc.) and colonial characters (shape and size, color, dimensions, form, etc.). The physiological and biochemical traits include data on growth at different temperatures, pH values, salt concentrations, atmospheric conditions (aerobic/anaerobic), growth in the presence of various substances such as antimicrobial agents, and data on the presence or activity of various enzymes, metabolization of compounds, and so on (Table 1). Physiological and biochemical tests should be carried out in test media and under conditions that are identically standard or at least comparable. There are three major inter-related areas in actinobacterial identification: taxonomic relevance; methodological reliability and cost effectiveness; and data portability [6]. It must be noted that novel taxa should be described based on the characteristics of more than one related strain and the type strain of the type species of the appropriate genera [7]. It should be emphasized that some phenotypes are encoded by extrachromosomal inheritance factors and the influencing factors that affect the expression of physiological and biochemical traits are complicated. To determine the genetic relationships based on physiological and biochem‐ ical characteristics,systematic classification, must be integrated with other characteristics, particularly genotype characteristics analysis.

Identification of a species is a constant basic work in any microbiology laboratory. Regardless of the type of microorganisms, the working steps are inseparable from the following three items: (i) to obtain the pure cultures of microorganism, (ii) to determinate the necessary appraisal indicators, (iii) to find authoritative identification manuals and publications, and related site information. Different organisms often have their own different identification priorities. For example, the identification of microorganisms with rich morphological charac‐ ters, such as fungi, often bases on their morphological features as the main indicators; the identification of actinobacteria and yeasts, synthesizes the morphological, physiological and biochemical characteristics; the identification of bacteria lacking morphological difference, often uses more physiological, biochemical, and genetic parameters.


**Table 1.** Common physiological and biochemical characteristics used for classification and identification of actinobacteria

### **2. Cultural characteristics of actinobacteria**

are used to compile a description of a new species. Classification of actinobacteria is now based largely on analysis of the nucleotide sequences, especially 16S rRNA genes [2]. However, a comprehensive characterization of a new species should still be identified, because from even a complete genomic sequence, it would be difficult to predict many of the phenotypic features of a new species [3]. Physiological and biochemical characteristics are directly related to the activity of microbial enzymes and regulatory proteins. Enzymes and proteins are gene products; so the comparison on physiological and biochemical characteristics of actinobacteria is the indirect comparison of genome, and determination of physiological and biochemical characteristics is much easier than direct analysis of the genome. Therefore, the physiological and biochemical characteristics in actinobacterial systematics and identification are still meaningful [4]. In addition to a thorough phenotypic characterization of a new species, it is important to determine which phenotypic features are the ones most useful for identifying new species. Moreover, the particular methods used for characterizing an organism should

always be stated, because the results of phenotypic tests can vary with methodology.

particularly genotype characteristics analysis.

88 Actinobacteria - Basics and Biotechnological Applications

Phenotypic analysis is a very tedious task in the classification of actinobacteria. The classical phenotypic characteristics of actinobacteria comprise morphological, physiological, and biochemical features. Individually sufficient as parameters for genetic relatedness, yet as a whole, they provide descriptive information enabling us to recognize taxa [5]. The morpho‐ logical traits include both cellular (cell shape and size, spore, sporangia, sporangiospore, the location of the spores or sporangia and their size, flagella, motility, intracellular structures, etc.) and colonial characters (shape and size, color, dimensions, form, etc.). The physiological and biochemical traits include data on growth at different temperatures, pH values, salt concentrations, atmospheric conditions (aerobic/anaerobic), growth in the presence of various substances such as antimicrobial agents, and data on the presence or activity of various enzymes, metabolization of compounds, and so on (Table 1). Physiological and biochemical tests should be carried out in test media and under conditions that are identically standard or at least comparable. There are three major inter-related areas in actinobacterial identification: taxonomic relevance; methodological reliability and cost effectiveness; and data portability [6]. It must be noted that novel taxa should be described based on the characteristics of more than one related strain and the type strain of the type species of the appropriate genera [7]. It should be emphasized that some phenotypes are encoded by extrachromosomal inheritance factors and the influencing factors that affect the expression of physiological and biochemical traits are complicated. To determine the genetic relationships based on physiological and biochem‐ ical characteristics,systematic classification, must be integrated with other characteristics,

Identification of a species is a constant basic work in any microbiology laboratory. Regardless of the type of microorganisms, the working steps are inseparable from the following three items: (i) to obtain the pure cultures of microorganism, (ii) to determinate the necessary appraisal indicators, (iii) to find authoritative identification manuals and publications, and related site information. Different organisms often have their own different identification priorities. For example, the identification of microorganisms with rich morphological charac‐ ters, such as fungi, often bases on their morphological features as the main indicators; the Cultural characteristics of actinobacteria refer to the growth characteristics and morphology in various kinds of culture media. Pure culture should be taken before morphological obser‐ vation. The pure culture of actinobacteria can be obtained through the use of spread plates, streak plates, or pour plates and are required for the careful study of an individual microbial species [Figure 1]. Cultural characteristics on 4 to 6 media are usually determined after incubation 14 to 28 days at 28°C strictly according to the methods used in the *International Streptomyces Project* (ISP) [8]. Sometimes, other media can be chosen, such as nutrient agar and czapek's agar. The colors of substrate and aerial mycelia and any soluble pigments produced were determined by comparison with chips from the ISCC-NBS color charts [9].


**Figure 1.** Acquisition of pure culture

### **3. Physiological and biochemical characteristics for identification of actinobacteria**

Some phenotypic characteristics of actinobacteria are of such primary importance to a genus or species description. Several problems must be considered when planning the physiological and biochemical tests of actinobacteria. One of them, according to the phylogenic information based on 16S rRNA analyses, strains of the most closely related taxa and the type strain of the type species of the appropriate genera should be chosen for comparison in their phenotypic traits. Other problems are concerned with methodology. In classifying actinobacteria, it is desirable to use an established approach based on common sense, and to use tests that are pertinent. If novel methods are used, the researcher must provide evidence that the new methods produce comparable results to established methods. Furthermore, phenotypic characteristics of actinobacteria are influenced by cultural conditions and other factors, so tests should be performed in duplicate or triplicate. More importantly, design reasonable positive and negative controls in the experiments.

### **3.1. Temperature range and optima for growth**

**Nutrient agar medium (G/Liter)** Peptone 10.0 g Beef extract/yeast extract 3.0 g NaCl 5.0 g Agar 15.0 g Final pH (at 25°C) 7.0±0.2 **Czapek's agar medium (G/Liter)** Sucrose 30.0 g NaNO3 3.0 g MgSO4·7H2O 0.5 g KCl 0.5 g FeSO4·4H2O 0.01 g K2HPO4 1.0 g Agar 15.0 g Final pH (at 25°C) 7.2±0.2

90 Actinobacteria - Basics and Biotechnological Applications

**Figure 1.** Acquisition of pure culture

**actinobacteria**

**3. Physiological and biochemical characteristics for identification of**

Some phenotypic characteristics of actinobacteria are of such primary importance to a genus or species description. Several problems must be considered when planning the physiological and biochemical tests of actinobacteria. One of them, according to the phylogenic information based on 16S rRNA analyses, strains of the most closely related taxa and the type strain of the type species of the appropriate genera should be chosen for comparison in their phenotypic traits. Other problems are concerned with methodology. In classifying actinobacteria, it is desirable to use an established approach based on common sense, and to use tests that are pertinent. If novel methods are used, the researcher must provide evidence that the new methods produce comparable results to established methods. Furthermore, phenotypic

Incubate cultures at a range of temperatures; using constant temperature incubators or water baths, measure the growth response of the actinobacteria. The tested temperature range is usually from 0°C to 75°C. In general, temperature experiments employ solid medium instead of broth in order to better observe. The basal medium is Bennett's medium or YIM38 medium or nutrient medium.


Vitamin mixture (0.5 mg each of thiamine-HCl, riboflavin, niacin, pyridoxine-HCl, inositol, calcium pantothenate, and p-aminobenzoic acid and 0.25 mg biotin)


Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 min. Mix well and pour into sterile Petri plates.

Note: If the temperature is above 100°C, use screw-cap culture tubes or screw-cap glass bottles and seal the screw caps to prevent the evaporation of the medium. Incubate the cultures in commercial water baths filled with dimethyl silicone oil. If the temperature is below 0°C, use an ethylene glycol water bath. For marine actinobacteria that require seawater, open ocean seawater has NaCl concentration of about 35 g/L or 3.5% (wt/vol).

### **3.2. Optimum pH and pH range for growth**

An essential part of the description of any actinobacteria is the range of pH values at which it can grow, as well as the optimal pH for growth. Measure growth responses from a standar‐ dized inoculum using basic medium (Bennett's medium or YIM38 medium or nutrient medium) at various pH values. Liquid medium is to be used for pH tests, to measure the growth responses turbidimetrically. The selection of buffer is critical. A buffer should be used in most media to maintain a stable pH for growth of the test strain. Buffers are most effective at their p*Ka* values and should be chosen with this in mind. Some useful biological buffers are listed in Table 2. Some buffers such as citrate, succinate, or glycine may be metabolized by the test organism. Others may be toxic. Sometimes, a combination of buffers may be helpful. Certain buffers (Good buffer) are non-metabolizable, non-toxic, have low reactivity with metal ions, and have other desirable features [10]. Phosphate salts are most commonly used because they are effective in the growth range of most bacteria, are usually non-toxic, and provide a source of phosphorus for the organism.


**Table 2.** Common biological buffers, their effective range, and their pK*a* values at 25°C

Usually, it is necessary to test the growth of actinobacteria from pH 4.0 to 13.0 and determine the strain growth pH range and the optimum pH value by using the following buffer system: pH 4.0–5.0: 0.1 M citric acid/0.1 M sodium citrate; pH 6.0–8.0: 0.1 M KH2PO4/0.1 M NaOH; pH 9.0–10.0: 0.1 M NaHCO3/0.1 M Na2CO3; pH 11.0: 0.05 M Na2HPO4/0.1 M NaOH; pH 12.0–13.0: 0.2 M KCl/0.2 M NaOH [11]. Negative controls for each buffer were used and the final pH was determined by using an indicator of acidity.

### **3.3. NaCl ranges and optima for growth**

dized inoculum using basic medium (Bennett's medium or YIM38 medium or nutrient medium) at various pH values. Liquid medium is to be used for pH tests, to measure the growth responses turbidimetrically. The selection of buffer is critical. A buffer should be used in most media to maintain a stable pH for growth of the test strain. Buffers are most effective at their p*Ka* values and should be chosen with this in mind. Some useful biological buffers are listed in Table 2. Some buffers such as citrate, succinate, or glycine may be metabolized by the test organism. Others may be toxic. Sometimes, a combination of buffers may be helpful. Certain buffers (Good buffer) are non-metabolizable, non-toxic, have low reactivity with metal ions, and have other desirable features [10]. Phosphate salts are most commonly used because they are effective in the growth range of most bacteria, are usually non-toxic, and provide a

**range**

**pKa (25°C)**

**Buffer Effective pH**

**Table 2.** Common biological buffers, their effective range, and their pK*a* values at 25°C

Maleate (Salt of maleic acid) 1.2–2.6 1.97 (p*Ka*1) Phosphate (Salt of phosphoric acid) 1.7–2.9 2.15 (p*Ka*1) Glycine 2.2–3.6 2.35 (p*Ka*1) Citrate (Salt of citric acid) 2.2–3.5 3.13 (p*Ka*1) Malate (Salt of malic acid) 2.7–4.2 3.40 (p*Ka*1) Citrate (Salt of citric acid) 3.0–6.2 4.76 (p*Ka*2) Succinate (Salt of succinic acid) 3.2–5.2 4.21 (p*Ka*1) Acetate (Salt of acetic acid) 2.6–5.6 4.76 Malate (Salt of malic acid) 4.0–6.0 5.13 (p*Ka*2) Succinate (Salt of succinic acid) 5.5–6.5 5.64 (p*Ka*2) MES 2-(*N*-Morpholino)-ethanesulfonic acid 5.5–6.7 6.10 Maleate (Salt of maleic acid) 5.5–7.2 6.24 (p*Ka*2) Citrate (Salt of citric acid) 5.5–7.2 6.40 (p*Ka*3) ACES (*N*-(2-Acetamido)-aminoethanesulfonic acid) 6.1–7.5 6.78 BES (*N,N*-Bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid) 6.4–7.8 7.09 MOPS (3-(*N*-Morpholino)-propanesulfonic acid) 6.5–7.9 7.14 HEPES (*N*-(2-Hydroxyethyl)-piperazine-*N'*-ethanesulfonic acid) 6.8–8.2 7.48 Phosphate (Salt of phosphoric acid) 5.8–8.0 7.20 (p*Ka*2) Imidazole 6.2–7.8 6.95 TES (2-[Tris(hydroxymethyl)-methylamino]-ethanesulfonic acid) 6.8–8.2 7.40 Tricine (N-[Tris(hydroxymethyl)-methyl]-glycine) 7.4–8.8 8.05 Tris (Tris(hydroxymethyl)-aminomethane) 7.5–9.0 8.06 TABS (*N*-tris[hydroxymethyl]-4-amino-butanesulfonic acid) 8.2–9.6 8.90 CHES (Cyclohexylaminoethanesulfonic acid) 8.6–10.0 9.50 Glycine 8.8–10.6 9.78 (p*Ka*2) CAPSO (3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid) 8.9–10.3 9.60 CAPS (3-(Cyclohexylamino)-propanesulfonic acid) 9.7–11.1 10.40

source of phosphorus for the organism.

92 Actinobacteria - Basics and Biotechnological Applications

Salt tolerance experiments mainly test the tolerance ability of the organism to NaCl and other salts, and determine the optimum concentration for growth. Inoculate liquid media containing a range of NaCl (usually 0–30%, W/V, or relative molar concentrations) concentrations and measure the growth response turbidimetrically. Bennett's medium or YIM38 medium or nutrient medium can be used as basal medium.

For some marine actinobacteria, NaCl alone may not substitute for filtered seawater, which should be sterilized by filtration and added aseptically to the sterilized medium. Even if seawater is used, it may need to be aged for a few weeks in a glass vessel in the dark to be effective. Seawater contains 3% NaCl, and testing marine organisms for growth at levels below this can be done by using various proportions of distilled water to seawater in the medium, or by using artificial seawater in which the level of NaCl can be varied.

#### **3.4. Utilization of carbon source**

Utilization of carbon source tests usually uses turbidimetric method. Use a chemically defined basal medium that lacks a carbon source, but otherwise is suitable for growth of the actinobacteria being tested. The basal medium is Pridham and Gottlieb carbon utiliza‐ tion medium [8]. Add carbon sources to a concentration (sugar alcohols 0.5–1%, others 0.1– 0.2%). After growth has occurred, measure the growth response turbidimetrically with a spectrophotometer.


#### Note:


#### **3.5. Utilization of nitrogen source**

Use the turbidimetric method to test the utilization of nitrogen source, especially sole nitrogen sources. Basal medium that omit nitrogen source but include a suitable carbon source are used. Add nitrogen source to a concentration (usually 0.5%).


Note:


#### **3.6. Enzymological characteristics**

Some enzymological characteristics are of such primary importance to a genus or species description that they must appear in every published description.

### **Oxidase test**

Note:

**1.** Use liquid medium to avoid the influence of agar.

94 Actinobacteria - Basics and Biotechnological Applications

Add nitrogen source to a concentration (usually 0.5%).

**Basic medium for nitrogen source utilization (G/Liter)** D-Glucose 1.0 g MgSO4·7H2O 0.05 g NaCl 0.05 g FeSO4·7H2O 0.001 g K2HPO4 0.01 g Final pH (at 25°C) 7.2–7.4

**1.** Use liquid medium to avoid the influence of agar.

**3.6. Enzymological characteristics**

**3.** Controls required for the test: negative control (no carbon source).

description that they must appear in every published description.

of the carbon source).

the media by filtration.

**3.5. Utilization of nitrogen source**

control).

Note:

**2.** Thermolabile carbon sources should be sterilized by filtration (filter sterilize 10% solution through bacteriological filter) or ether sterilization (weigh an appropriate amount of the dry carbon source and spread in a pre-sterilized Erlenmeyer flask fitted with a loose cotton plug. Add sufficient acetone-free ethyl ether to cover the carbohydrate. Allow ether to evaporate at room temperature under a ventilated fume hood overnight or longer. When all ether has evaporated, add sterile distilled water aseptically to make a 10% w/v solution

**3.** Controls required for the test: No carbon source (negative control); D-glucose (positive

**4.** For marine actinobacteria, instead of distilled water, use a synthetic seawater and sterilize

Use the turbidimetric method to test the utilization of nitrogen source, especially sole nitrogen sources. Basal medium that omit nitrogen source but include a suitable carbon source are used.

**2.** Thermolabile nitrogen sources should be sterilized by filtration or ether sterilization.

Some enzymological characteristics are of such primary importance to a genus or species

This method tests for an enzyme that transfers electrons from a donor molecule to O2, thereby forming H2O. Oxidase-positive organisms are usually aerobes or microaerophiles that can use O2 as their final electron acceptor. The test reagent, N,N,N′,N′-tetramethyl-*p*-phenylenedia‐ mine (TMPD), acts as an artificial electron acceptor for the oxidase and the reduced form is the colored compound indophenol blue.

Prepare a 1% (wt/vol) solution of TMPD in certified-grade dimethylsulfoxide (DMSO). The solution is stable for at least a month under refrigeration. Test methods:


#### Note:


#### **Catalase test**

Catalase catalyzes the disproportionation reaction 2H2O2 → 2H2O + O2, thereby helping to prevent oxidative damage to cells caused by H2O2. Add 0.2 ml of a 3–10% H2O2 solution to a screw-cap test tube. Using a platinum loop, disposable plastic loop, or glass rod, remove some growth from a colony or agar slant and rub the growth on the inner wall of the tube. Cap the tube (to prevent escape of aerosols) and slant it, so that the H2O2 solution covers the growth. Effervescence within 30 sec indicates a positive reaction.

Note: For this test, do not use cultures grown on blood-containing media, as blood contains catalase; however, cultures grown on a medium containing heated blood, such as chocolate agar, can be used. Some bacteria can make catalase only when provided with heme; these organisms are negative when cultured on media lacking blood, but are positive when cultured on chocolate agar. Some bacteria make a pseudocatalase (a non-heme catalase) when grown on media lacking blood, but containing little or no glucose; they are negative for catalase when cultured on media containing 1% glucose. With anaerobic organisms, expose the culture to air for 30 min before performing the test, as some anaerobes have an inducible catalase.

### **Urease test**

Urease test check he ability of an organism to produce an exoenzyme, called urease. Urease catalyzes the reaction (NH2)2CO + H2O → 2NH3 + CO2. The ammonia that is formed causes the medium to become alkaline: NH3 + H2O → NH4 + + OH– . The alkalinity can be detected with a pH indicator.

Prepare the following medium (per liter of distilled water): peptone 1 g; NaCl 5 g; glucose 1 g; KH2PO4 2 g; phenol red 0.012 g; agar 15 g; pH 6.8–6.9. Autoclave and cool to 55°C. Add 30% (W/V) filter-sterilized (or ether-sterilized) solution of urea to make final concentration of 2%. Mix and dispense appropriate portions into tubes or plates. Prepare a control medium lacking urea. Inoculate the surface of the slant or plate for 4 days. Look for development of a red-violet color compared to an uninoculated control. The medium can also be prepared as a liquid medium by omitting the agar [14].

For other organisms that grow poorly or not at all on Christensen's medium, use a test medium of the composition (per liter of distilled water): BES buffer 1.065 g; urea 20.0 g; phenol red 0.01 g; pH 7.0. Also prepare a control medium lacking urea. Sterilize both media by filtration and dispense 2.0-ml portions into sterile tubes. Culture the test organism in a suitable liquid medium. Centrifuge the cells and suspend them in sterile distilled water to a dense concen‐ tration. Add 0.5 ml of the suspension to the test medium and the control medium. Incubate the tubes for 24 h at the optimal temperature for the organism. Look for the development of a red-violet color in the test medium, but not in the control medium [15].

### **Lipase test**

Lipase activity can be shown by using Tweens, for example, Tween 80 (polyethylene sorbitan monooleate, an oleic acid ester), Tween 40 (a palmitic acid ester), and Tween 20 (a stearic acid ester). Lipolytic organisms split off the fatty acid, and the calcium salts of the fatty acids produce opaque zones around the colonies.

Prepare a basal medium containing the following (per liter of distilled water): peptone 10.0 g; NaCl 5.0 g; CaCl2 2H2O 0.1 g; agar 9.0 g; pH 7.4. Sterilize by autoclaving (121°C, 20 min). Autoclave the desired Tween separately (121°C, 20 min). Cool the basal medium to 45– 50°C, add the Tween to give a final concentration of 1.0%, shake until the Tween is completely dissolved and pour into plates. Inoculate the cultures as lines on the surface of the agar. Incubate for up to 7 to 14 days, inspecting daily. Look for an opaque halo around the growth [16].

### **Gelatin liquefaction**

Note: For this test, do not use cultures grown on blood-containing media, as blood contains catalase; however, cultures grown on a medium containing heated blood, such as chocolate agar, can be used. Some bacteria can make catalase only when provided with heme; these organisms are negative when cultured on media lacking blood, but are positive when cultured on chocolate agar. Some bacteria make a pseudocatalase (a non-heme catalase) when grown on media lacking blood, but containing little or no glucose; they are negative for catalase when cultured on media containing 1% glucose. With anaerobic organisms, expose the culture to air

Urease test check he ability of an organism to produce an exoenzyme, called urease. Urease catalyzes the reaction (NH2)2CO + H2O → 2NH3 + CO2. The ammonia that is formed causes the

Prepare the following medium (per liter of distilled water): peptone 1 g; NaCl 5 g; glucose 1 g; KH2PO4 2 g; phenol red 0.012 g; agar 15 g; pH 6.8–6.9. Autoclave and cool to 55°C. Add 30% (W/V) filter-sterilized (or ether-sterilized) solution of urea to make final concentration of 2%. Mix and dispense appropriate portions into tubes or plates. Prepare a control medium lacking urea. Inoculate the surface of the slant or plate for 4 days. Look for development of a red-violet color compared to an uninoculated control. The medium can also be prepared as a liquid

For other organisms that grow poorly or not at all on Christensen's medium, use a test medium of the composition (per liter of distilled water): BES buffer 1.065 g; urea 20.0 g; phenol red 0.01 g; pH 7.0. Also prepare a control medium lacking urea. Sterilize both media by filtration and dispense 2.0-ml portions into sterile tubes. Culture the test organism in a suitable liquid medium. Centrifuge the cells and suspend them in sterile distilled water to a dense concen‐ tration. Add 0.5 ml of the suspension to the test medium and the control medium. Incubate the tubes for 24 h at the optimal temperature for the organism. Look for the development of a

Lipase activity can be shown by using Tweens, for example, Tween 80 (polyethylene sorbitan monooleate, an oleic acid ester), Tween 40 (a palmitic acid ester), and Tween 20 (a stearic acid ester). Lipolytic organisms split off the fatty acid, and the calcium salts of the fatty acids

Prepare a basal medium containing the following (per liter of distilled water): peptone 10.0 g; NaCl 5.0 g; CaCl2 2H2O 0.1 g; agar 9.0 g; pH 7.4. Sterilize by autoclaving (121°C, 20 min). Autoclave the desired Tween separately (121°C, 20 min). Cool the basal medium to 45– 50°C, add the Tween to give a final concentration of 1.0%, shake until the Tween is completely dissolved and pour into plates. Inoculate the cultures as lines on the surface of the agar. Incubate for up to 7 to 14 days, inspecting daily. Look for an opaque halo around

red-violet color in the test medium, but not in the control medium [15].

+ + OH–

. The alkalinity can be detected with a

for 30 min before performing the test, as some anaerobes have an inducible catalase.

**Urease test**

pH indicator.

**Lipase test**

the growth [16].

medium to become alkaline: NH3 + H2O → NH4

96 Actinobacteria - Basics and Biotechnological Applications

medium by omitting the agar [14].

produce opaque zones around the colonies.

The gelatin hydrolysis tests for an organism's ability to break down the protein gelatin, which is derived from collagen. Gelatin causes the media to thicken, especially at cooler (below 28°C) temperatures. If the organism can release gelatinase enzymes, the gelatin is broken down or liquefied. The media is checked over a period of about a week after inoculation and incubation at room temperature, for gelatinase activity. The tube is placed on ice for a few minutes; and if the media fails to solidify, it is considered a positive test. The gelatinase reaction may be slow or incomplete.

The conventional methods require long periods of growth, long periods for development, or are difficult to interpret. Now, commonly use the trichloroacetic acid (TCA) enhancement to be more rapid and sensitive [17]. Prepare gelatin agar plates (per liter of deionized water): tryptic soy agar powder 40.0 g, gelatin 16.0 g. Make a single streak or spot of the microorganism from a stock culture onto a gelatin agar plate and/or casein agar plate and incubate at 30–35°C. Prepare a stock solution of 35% (W/V) TCA in deionized water. After incubation for 3 h (or 24 h for the casein hydrolysis test), flood the plate with the TCA solution. Look for occurrence of a clear zone around the growth within at least 4 min. With casein hydrolysis, clear zones may be visible without adding TCA, but the TCA enhances the visibility.

### **Coagulation and peptonization of milk**

Milk coagulation and peptonization test the ability of actinobacteria to produce protease. Coagulation is that mild protein is preliminarily degraded into big pieces by organism. Further degradation is peptonization.

Prepare milk coagulation and peptonization medium (per liter of distilled water): skim milk powder 200 g; CaCO3 0.2 g. Dispense 3–5 ml portions into narrow tubes and sterilize by autoclaving (115°C, 15 min) or fractional sterilization for 2–3 times. Inoculate the tubes and observe in 5, 10, 20, 30 days, respectively. Milk solidification occurrence is the phenomenon of coagulation. Clots further hydrolyzed into liquid, is the phenomenon of peptonization. Peptonized exudates is translucent, typically begins after coagulation.

### **Starch hydrolysis**

Starch hydrolysis tests the ability of an organism to produce certain exoenzymes, including aamylase and oligo-1, 6-glucosidase, that hydrolyze starch. Starch molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade starch into subunits that can then be utilized by the organism. Make a single spot of the test organisms on a plate of the agar (within 5 mm in diameter) and incubate.

Prepare a basal medium containing the following (per liter of distilled water): soluble starch 10 g; K2PO4 0.3 g; MgCO3 1 g; NaCl 0.5 g; KNO3 1 g; agar 15 g; pH 7.2–7.4. Prepare Gram's iodine solution by grinding 1.0 g of iodine crystals together with 2.0 g of KI in a mortar (slowly add 300 ml of distilled water while grinding until the iodine is dissolved). After growth occurs, flood the plate with the iodine solution. Starch stains blue with iodine, so look for colorless areas around the microbial growth.

### **Cellulose hydrolysis**

Cellulose hydrolysis tests the ability of an organism to produce cellulase.

Conventional method: Prepare a cellulose hydrolysis medium (per liter of distilled water): MgSO4 0.5 g; NaCl 0.5 g; K2HPO4 0.5 g; KNO3 1 g; pH 7.2. A filter paper (5 × 0.8 cm) submerges in liquid medium. Sterilize by autoclaving (121°C, 20 min). Inoculate the cultures on the filter paper. Incubate for 1 month to observe whether filter paper is decomposed.

Congo red-polysaccharide method [18]:The interaction of the direct dye congo red with intact β-D-glucans provides the basis for a rapid and sensitive assay system for bacterial strains possessing β-(1 → 4),(1 → 3)-D-glucanohydrolase, β-(1 → 4)-D-glucanohydrolase, and β-(1 → 3)-D-glucanohydrolase activities. Prepare basal medium contain cellulose. Inoculate for 7 to 14 days. After growth occurs, flood the plate with the congo red (1 mg/ml) for 10–15 min. Wash with NaCl (1 mol/L) 2–3 times (15 min/time). Cellulose hydrolysis can produce trans‐ parent circle. Congo red can also be added directly to the medium.

Other method [19]: Prepare mineral agar culture media in which cellulose is to be provided as a sole carbon source (per liter of distilled water): KNO3 0.5 g; K2HPO4 1.0 g; KCl 0.5 g; MgSO4 7H2O 0.5 g; and agar, 15.0 g. Add 0.5 ml of a suitable trace metals solution. Autoclave, cool, and dispense into plates. Prepare a series of dilutions of the organism to be tested and spread 25–50 µl portions over the surface of the plates. Place a sterile disc of lens paper on the seeded surface of the plates and incubate for 3–7 days. Look for colonies that form visible holes in the paper. To increase visibility of the holes, stain the paper on the plates or after the paper is removed with 0.2% irgalan black in 2% acetic acid.

#### **Nitrate reduction**

Many organisms can respire anaerobically by using NO3 – as a terminal electron acceptor for an electron transport system (nitrate respiration or dissimulator nitrate reduction). Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase. It also tests the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen. The Griess reaction has more recently been employed to detect nitrite and nitrate as products of nitric oxide synthase in bacterial identification [20].

#### Preparation of recipes:

Nitrate reduction medium (per liter of distilled water): beef (meat) extract 3.0 g; KNO3 1.0 g; peptone 5.0 g; pH 7.2–7.4.

Nitrite reduction medium (per liter of distilled water): beef (meat) extract 3.0 g; KNO2 1.0 g; peptone 5.0 g; pH 7.2–7.4.

For either broth substrate, carefully weigh the ingredients and heat gently into solution. Dispense into test tubes and add inverted Durham tubes. Autoclave for 15 min at 121°C.

Reagent A (Griess A): Sulfanilic acid 0.8 g; acetic acid (5N) 100 ml.

Reagent B (Griess B): N, N-Dimethyl-α-naphthylamine 0.6 ml (replace alpha-naphthylamine); acetic acid (5N) 100 ml. (Fresh reagent has a very slight yellowish color.)

5N acetic acid is prepared by adding 287 ml of glacial acetic acid (17.4 N) to 713 ml of deionized water.

Zinc dust must be nitrate- and nitrite-free.

Protocol:

**Cellulose hydrolysis**

98 Actinobacteria - Basics and Biotechnological Applications

**Nitrate reduction**

identification [20].

Preparation of recipes:

peptone 5.0 g; pH 7.2–7.4.

peptone 5.0 g; pH 7.2–7.4.

Cellulose hydrolysis tests the ability of an organism to produce cellulase.

paper. Incubate for 1 month to observe whether filter paper is decomposed.

parent circle. Congo red can also be added directly to the medium.

is removed with 0.2% irgalan black in 2% acetic acid.

Many organisms can respire anaerobically by using NO3

Reagent A (Griess A): Sulfanilic acid 0.8 g; acetic acid (5N) 100 ml.

Conventional method: Prepare a cellulose hydrolysis medium (per liter of distilled water): MgSO4 0.5 g; NaCl 0.5 g; K2HPO4 0.5 g; KNO3 1 g; pH 7.2. A filter paper (5 × 0.8 cm) submerges in liquid medium. Sterilize by autoclaving (121°C, 20 min). Inoculate the cultures on the filter

Congo red-polysaccharide method [18]:The interaction of the direct dye congo red with intact β-D-glucans provides the basis for a rapid and sensitive assay system for bacterial strains possessing β-(1 → 4),(1 → 3)-D-glucanohydrolase, β-(1 → 4)-D-glucanohydrolase, and β-(1 → 3)-D-glucanohydrolase activities. Prepare basal medium contain cellulose. Inoculate for 7 to 14 days. After growth occurs, flood the plate with the congo red (1 mg/ml) for 10–15 min. Wash with NaCl (1 mol/L) 2–3 times (15 min/time). Cellulose hydrolysis can produce trans‐

Other method [19]: Prepare mineral agar culture media in which cellulose is to be provided as a sole carbon source (per liter of distilled water): KNO3 0.5 g; K2HPO4 1.0 g; KCl 0.5 g; MgSO4 7H2O 0.5 g; and agar, 15.0 g. Add 0.5 ml of a suitable trace metals solution. Autoclave, cool, and dispense into plates. Prepare a series of dilutions of the organism to be tested and spread 25–50 µl portions over the surface of the plates. Place a sterile disc of lens paper on the seeded surface of the plates and incubate for 3–7 days. Look for colonies that form visible holes in the paper. To increase visibility of the holes, stain the paper on the plates or after the paper

an electron transport system (nitrate respiration or dissimulator nitrate reduction). Nitrate broth is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase. It also tests the ability of organisms to perform nitrification on nitrate and nitrite to produce molecular nitrogen. The Griess reaction has more recently been employed to detect nitrite and nitrate as products of nitric oxide synthase in bacterial

Nitrate reduction medium (per liter of distilled water): beef (meat) extract 3.0 g; KNO3 1.0 g;

Nitrite reduction medium (per liter of distilled water): beef (meat) extract 3.0 g; KNO2 1.0 g;

For either broth substrate, carefully weigh the ingredients and heat gently into solution. Dispense into test tubes and add inverted Durham tubes. Autoclave for 15 min at 121°C.

–

as a terminal electron acceptor for

For either substrate, NO3 – or NO2 – , inoculate the medium with a heavy inoculum from wellisolated colonies of the test organism. Incubate at 28°C for 18–24 h, some actinobacteria need 7–14 days. When sufficient growth is observed in the tube, test the broth for reduction of the substrate.

For NO3 – substrate: Observe for gas production in the Durham tube. Mix two drops each of reagents A and B in a small test tube. Add approximately 1 ml of the broth culture to the test tube and mix well. If the test organism has reduced the NO3 – to NO2 – , a red color will usually appear within 2 min, indicating the presence of NO2 – in the tube. If no color change is seen within 2 min, there are several possible reasons. Either the organism (i) was unable to reduce NO3 – at all, (ii) was capable of reducing NO2 – , or (iii) reduced NO3 – directly to molecular nitrogen. Zinc is a powerful reducing agent. If there is any NO3 – remaining in the tube (option (i) above), a small amount of zinc dust will rapidly reduce it to NO2 – . Therefore, the appearance of a red color after the addition of zinc dust to a colorless reaction tube indicates a negative reaction, i.e., the organism has failed to reduce NO3 – . Zinc is added to the tube by dipping a wooden applicator stick in nitrate- and nitrite-free zinc powder, just enough to get the stick dirty, and then dropping it into the tube containing the culture broth and the reagents. If too much zinc is added, the color reaction may fade rapidly. If the broth remains colorless after the addition of zinc, the organism has also reduced the NO2 – , intermediate product to N2 gas or some other nitrogenous product. N2 gas is usually visible in the Durham tube. In the absence of gas, the product is assumed to be other than N2 gas.

Occasionally, a lighter pink color will appear after the addition of zinc dust because of partial reduction, i.e., some of the primary NO3 – substrate remains in the tube. The original tube may be reincubated and retested the following day.

For NO2– substrate: Observe for gas production on the surface and in the Durham tube. Mix two drops each of reagents A and B in a small test tube. Add approximately 1 ml of the broth culture to the test tube and mix well. If the test organism has reduced the NO2 – , there will be no color change, indicating that all of the original NO2 – is gone, i.e., reduced. Reduction is often confirmed by the presence of N2 gas in the Durham tube or on the surface of the broth, but other nitrogenous products may be produced. Therefore, the absence of gas does not rule out reduction of NO2 – . If a red color appears, it indicates the presence of NO2 – , and therefore a negative reaction. Occasionally, a lighter pink color will appear because of partial reduction, i.e., some of the primary NO2 – substrate remains in the tube. The original tube may be reincubated and retested the following day. There is no need to add zinc dust to this reaction. Note:


### **3.7. Metabolic products**

Thousands of characterization tests have been described in the microbiological identification. Those that follow are designed for detection of metabolic products and they are useful for physiological characterization beyond the more general features of an actinobacterial genus or species.

**a.** MR test (Methyl red test)

A type of fermentation called the mixed acid fermentation results in the formation of formic acid, acetic acid, lactic acid, succinic acid, ethanol, CO2, and H2 in a buffered medium. The combination of acids in the mixed acid fermentation usually lowers the pH of the culture below 4.2. The test is used mainly in the differentiation of enteric bacteria. The organism being tested must be capable of catabolizing glucose [21].

Prepare MR-VP medium containing the following (per liter of distilled water): peptone 7.0 g; K2HPO4 5.0 g; glucose, 5.0 g; pH 7.5. Dispense 2–3 ml portions into narrow tubes and sterilize by autoclaving. Inoculate the tubes lightly and incubate for 4 days at the optimum temperature for the organism. Add one drop of methyl red reagent (0.25 g methyl red dissolved in 100 ml of ethanol). Look for a red colour (MR positive). A weakly positive test is red orange and a yellow or orange color indicates a negative test.

**b.** V-P test (Voges-Proskauer test)

Some fermentative organism catabolizes glucose by the butanediol pathway, in which acetoin (acetylmethylcarbinol) occurs as an intermediate in the formation of 2, 3-butanediol. In the presence of KOH and O2, the acetoin is oxidized to diacetyl, which in turn reacts with the guanidine group associated with arginine and other molecules contributed by peptone in the medium to form a pink- to red-colored product. The α-naphthol intensifies this color [21].

Prepare MR-VP medium containing the following (per liter of distilled water): peptone 7.0 g; K2HPO4 5.0 g; glucose, 5.0 g; pH 7.5. Make reagent A by dissolving 5.0 g of α-naphthol in 100 ml of absolute (100%) ethanol; the reagent must not be darker than straw color.

Prepare reagent B by dissolving 40.0 g of KOH in 100 ml of distilled water. Inoculate the tubes lightly and incubate for 2 days (routine test) and for 4 days (standard test) at the optimum temperature for the organism being tested.

Add 0.6 ml of reagent A and agitate to aerate the medium. Add 0.2 ml of reagent B and again agitate the medium. Slant the tube to increase the aeration. Allow to stand for 15–60 min. Look for development of a strong cherry red color at the surface of the medium. A negative reaction shows no color or a faint pink to copper color.

**c.** Tryptophan decomposition (indole production)

Note:

**2.** Because reduction of NO3

100 Actinobacteria - Basics and Biotechnological Applications

the reaction to take place.

**3.7. Metabolic products**

**a.** MR test (Methyl red test)

must be capable of catabolizing glucose [21].

yellow or orange color indicates a negative test.

temperature for the organism being tested.

**b.** V-P test (Voges-Proskauer test)

or species.

**1.** Be sure to run a negative control, uninoculated broth, to illustrate that the remaining

Thousands of characterization tests have been described in the microbiological identification. Those that follow are designed for detection of metabolic products and they are useful for physiological characterization beyond the more general features of an actinobacterial genus

A type of fermentation called the mixed acid fermentation results in the formation of formic acid, acetic acid, lactic acid, succinic acid, ethanol, CO2, and H2 in a buffered medium. The combination of acids in the mixed acid fermentation usually lowers the pH of the culture below 4.2. The test is used mainly in the differentiation of enteric bacteria. The organism being tested

Prepare MR-VP medium containing the following (per liter of distilled water): peptone 7.0 g; K2HPO4 5.0 g; glucose, 5.0 g; pH 7.5. Dispense 2–3 ml portions into narrow tubes and sterilize by autoclaving. Inoculate the tubes lightly and incubate for 4 days at the optimum temperature for the organism. Add one drop of methyl red reagent (0.25 g methyl red dissolved in 100 ml of ethanol). Look for a red colour (MR positive). A weakly positive test is red orange and a

Some fermentative organism catabolizes glucose by the butanediol pathway, in which acetoin (acetylmethylcarbinol) occurs as an intermediate in the formation of 2, 3-butanediol. In the presence of KOH and O2, the acetoin is oxidized to diacetyl, which in turn reacts with the guanidine group associated with arginine and other molecules contributed by peptone in the medium to form a pink- to red-colored product. The α-naphthol intensifies this color [21].

Prepare MR-VP medium containing the following (per liter of distilled water): peptone 7.0 g; K2HPO4 5.0 g; glucose, 5.0 g; pH 7.5. Make reagent A by dissolving 5.0 g of α-naphthol in 100

Prepare reagent B by dissolving 40.0 g of KOH in 100 ml of distilled water. Inoculate the tubes lightly and incubate for 2 days (routine test) and for 4 days (standard test) at the optimum

ml of absolute (100%) ethanol; the reagent must not be darker than straw color.

that the medium needs to be anaerobic or deep enough to support an anaerobic process. However, later experiments have shown that the metabolism on the surface of the broth for most organisms that grow well in the broth will reduce enough dissolving oxygen for

– is assumed to be anaerobic, many published procedures warn

NO2 will be reduced by zinc dust, producing a red color.

Organisms that possess tryptophanase can carry out the following reaction: L-tryptophan → indole + pyruvic acid + NH3. The indole can be detected by its ability to react with p-dimethy‐ laminobenzaldehyde to form a quinoidal red-violet condensation compound [21].

Xylene extraction test is more sensitive than the conventional test. Grow the test organism in a suitable culture medium supplemented with 0.1–1.0% tryptophan. Avoid using media containing carbohydrates, nitrate or nitrite, as these may interfere with the test. Distribute in 2–3 ml portions and sterilize by autoclaving. When cool, inoculate with the organism to be tested and incubate for up to 3 days.

Prepare Ehrlich's reagent as follows: 1.0 g of p-dimethylaminobenzaldehyde, 95 ml of 95% ethanol and 20 ml of HCl.

Add 1 ml of xylene to the broth culture, shake vigorously, and allow the mixture to stand for about 2 min. Then add 0.5 ml Ehrlich's reagent slowly down the side of the tube so as to form a layer between the medium and the xylene. Do not shake the tube after addition of the Ehrlich's reagent. Look for development of a pink or red ring below the xylene layer.

**d.** Hydrogen sulphide production

Some anaerobic and facultatively anaerobic actinobacteria can produce abundant H2S by the anaerobic reduction of S2O3 2–. The H2S can be detected by its reaction with iron salts contained in the medium, which form a black precipitate of FeS. A different type of H2S production is based on the ability of some organisms to form low levels of H2S from sulfur-containing amino acids (cysteine, cystine, and/or methionine) by means of amino acid desulfurases. The gaseous H2S so produced is detected by its reaction with lead acetate strips suspended above the surface of the medium.

Thiosulfate iron H2S test [22]: Prepare peptone–iron agar medium (Tresner medium) as follows (per liter of distilled water): peptone 10.0 g; ferric ammonium citrate 0.5 g; agar 15 g. To achieve more satisfactory results, inoculums from actively growing cultures were used to streak the surface of the agar slants. After a short incubation period (15 to 20 h) at 28°C, the slants were observed. A pronounced bluish-black discoloration of the medium surrounding the colonies effected no change and indicated the production of hydrogen sulfide, whereas those organisms not producing H2S in the medium emitted only faint tints of other colors.

Paper strip method [21]: Inoculate a suitable semisolid (0.2% agar) growth medium that contains a peptone or other source of sulfur amino acids. Suspend a strip of sterile, lead acetateimpregnated paper about a centimeter above the surface of the culture, fold the upper end over the lip of the tube, and hold it in place with the screw cap or cotton plug. During growth of the organisms, H2S gas reacts with the lead acetate to form black PbS, beginning at the lower part of the strip. Lead acetate strips can be prepared by soaking 5-cm strips of filter paper in a 5% aqueous solution of lead acetate, sterilizing them separately in tubes by autoclaving and drying them in an oven.

#### **3.8. Relation to oxygen**

Aerobes use O2 as a terminal electron acceptor for an electron transport system, can tolerate a level of O2 equivalent to or higher than that present in an air atmosphere (21% O2), and have a strictly respiratory type of metabolism. Anaerobes are incapable of O2-dependent growth and cannot grow in the presence of 21% O2. Facultative anaerobes can grow both in the absence of O2 and in the presence of 21% O2. Microaerophiles respire with O2 but cannot grow, or grow very poorly, under 21% O2. They grow best at low O2 levels; some require levels as low as 1%. Some microaerophiles can also respire anaerobically with electron acceptors other than O2.

Semisolid agar method [23]: Autoclave a narrow culture tube that has been filled to 60% of its capacity with an appropriate culture medium containing 0.2% agar. After the medium has cooled to 45°C, add the inoculum, mix to distribute the organisms uniformly and then allow the agar to solidify. Alternatively, inoculate the medium by stabbing with an inoculating needle after the agar has gelled; this avoids the mixing that otherwise might add dissolved O2 to the medium. Growth occurring only at the surface of the medium suggests that the organism is aerobic. However, a fermentable substrate should be present in the medium, because the organism might be a facultative anaerobe that not only respires with O2 but also grows anaerobically by fermentation. Growth occurring only in the bottom region of the tube suggests that the organism is anaerobic. However, some extremely oxygen-intolerant anae‐ robes may not be able to grow even in the lowest region of the medium, because of the presence of small amounts of O2 dissolved in the medium during the addition of the inoculum. Growth occurring throughout the tube suggests that the organism is a facultative anaerobe. It is important that no potential terminal electron acceptors other than O2 should be present, as some aerobes can respire anaerobically. Growth occurring only in a disc several millimeters below the surface of the medium suggests that the organism is a microaerophile. Motile microaerophiles usually exhibit negative or positive aerotaxis, which results in their migration to a zone where the rate at which O2 is diffusing to them matches the rate it is used by the organisms.

### **3.9. Susceptibility to antibiotics**

Antibiotic sensitivity is the susceptibility of actinobacteria to antibiotics. Antibiotic suscepti‐ bility testing (AST) is usually carried out to determine which antibiotic [Table 3] will be most successful in treating a bacterial infection in organism.

Testing for antibiotic sensitivity is often done by the Kirby-Bauer method [24]: wafers con‐ taining antibiotics are placed on an appropriate agar plate where actinobacteria have been placed, and the plate is left to incubate. If an antibiotic stops the actinobacteria from growing or kills the actinobacteria, there will be an area around the wafer where the bacteria have not grown enough to be visible. This is called a zone of inhibition. The size of this zone depends on how effective the antibiotic is at stopping the growth of the bacterium. A stronger antibiotic will create a larger zone, because a lower concentration of the antibiotic is enough to stop growth.


**Table 3.** Common antibiotics and the suitable concentration

#### **3.10. Antibacterial activity detection**

part of the strip. Lead acetate strips can be prepared by soaking 5-cm strips of filter paper in a 5% aqueous solution of lead acetate, sterilizing them separately in tubes by autoclaving and

Aerobes use O2 as a terminal electron acceptor for an electron transport system, can tolerate a level of O2 equivalent to or higher than that present in an air atmosphere (21% O2), and have a strictly respiratory type of metabolism. Anaerobes are incapable of O2-dependent growth and cannot grow in the presence of 21% O2. Facultative anaerobes can grow both in the absence of O2 and in the presence of 21% O2. Microaerophiles respire with O2 but cannot grow, or grow very poorly, under 21% O2. They grow best at low O2 levels; some require levels as low as 1%. Some microaerophiles can also respire anaerobically with electron acceptors other than O2.

Semisolid agar method [23]: Autoclave a narrow culture tube that has been filled to 60% of its capacity with an appropriate culture medium containing 0.2% agar. After the medium has cooled to 45°C, add the inoculum, mix to distribute the organisms uniformly and then allow the agar to solidify. Alternatively, inoculate the medium by stabbing with an inoculating needle after the agar has gelled; this avoids the mixing that otherwise might add dissolved O2 to the medium. Growth occurring only at the surface of the medium suggests that the organism is aerobic. However, a fermentable substrate should be present in the medium, because the organism might be a facultative anaerobe that not only respires with O2 but also grows anaerobically by fermentation. Growth occurring only in the bottom region of the tube suggests that the organism is anaerobic. However, some extremely oxygen-intolerant anae‐ robes may not be able to grow even in the lowest region of the medium, because of the presence of small amounts of O2 dissolved in the medium during the addition of the inoculum. Growth occurring throughout the tube suggests that the organism is a facultative anaerobe. It is important that no potential terminal electron acceptors other than O2 should be present, as some aerobes can respire anaerobically. Growth occurring only in a disc several millimeters below the surface of the medium suggests that the organism is a microaerophile. Motile microaerophiles usually exhibit negative or positive aerotaxis, which results in their migration to a zone where the rate at which O2 is diffusing to them matches the rate it is used by the

Antibiotic sensitivity is the susceptibility of actinobacteria to antibiotics. Antibiotic suscepti‐ bility testing (AST) is usually carried out to determine which antibiotic [Table 3] will be most

Testing for antibiotic sensitivity is often done by the Kirby-Bauer method [24]: wafers con‐ taining antibiotics are placed on an appropriate agar plate where actinobacteria have been placed, and the plate is left to incubate. If an antibiotic stops the actinobacteria from growing or kills the actinobacteria, there will be an area around the wafer where the bacteria have not grown enough to be visible. This is called a zone of inhibition. The size of this zone depends

drying them in an oven.

102 Actinobacteria - Basics and Biotechnological Applications

**3.8. Relation to oxygen**

organisms.

**3.9. Susceptibility to antibiotics**

successful in treating a bacterial infection in organism.

Actinobacteria are the most economically and biotechnologically valuable prokaryotes. They are responsible for the production of the discovered bioactive secondary metabolites, notably antibiotics, antitumor agents, immunosuppressive agents, and enzymes. Because of the excellent track record of actinobacteria in this regard, it is necessary to preliminarily screen antibacterial activity of isolated actinomycetes.

Common test strains: *Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Mycobacterium tuberculosis avium, Candida albicans, Aspergillus niger.* Also test strains can be selected according to necessity.

Cross streak method [25]: The actinomycetes isolates were inoculated at the centre of the sterile agar plates and the plates were incubated at 28°C for 5–7 days. After incubation, the nutrient broth (peptone 5 g; beef extract 3 g; NaCl 5 g; distilled water 1000 ml) cultures of test bacteria were streaked perpendicular to the growth of the actinomycete isolates. The plates were incubated at 37°C for 24–48 h and the extent of growth inhibition of the test bacteria was observed. The absence of growth or a less dense growth of test bacteria near the growth of actinomycete isolate was considered positive for the production and secretion of antibacterial metabolite.

Double-layer agar diffusion method [4]: Prepare double-layer agar plates; the lower is water agar (0.8–1% agar). Cool the basal medium (YIM 38 agar medium or LB agar medium) to 45– 50°C, add the suspension of test strains, mix and spread on the water agar layer. Inoculate tested actinobacteria for 7 days at 28°C. Or pour fermentation broth of tested actinobacteria into sterilized steel rim or holes on medium and inoculate for 24–48 h at 37°C. Or inoculate the filter paper containing fermentation broth on the plates for 24-48 h at 37°C. Observe and record the diameter of inhibition zone. Results: no antibacterial activity (no zone of inhibition); weak antibacterial activity (diameter of zone, 6–15 mm), strong antibacterial activity (diameter of zone, >15 mm).

### **4. Commercial multitest system for identification of actinobacteria**

In recent 40 years, with the development of microelectronics, computer, molecular biology, physics, chemistry, and subject crossing universality, a significant contribution of scientific and technological development to the clinical microbiology was the development of minia‐ turized identification systems based on classical method. Several systems are commercially available [Table 4], and new systems are being developed continually. These systems were mostly based on modifications of classical methods and were improved by the incorporation of highly sophisticated, computer-generated identification databases tailored for each system [26, 27]. Each manufacturer provides charts, tables, coding systems, and characterization profiles for use with the particular multi-test system being offered. These systems offer the advantages of miniaturization and are usually used in conjunction with a computerized system for identification of the organisms. As mentioned earlier, the use of these systems can increase standardization among various laboratories because of the high degree of quality control exercised over the media and reagents. Now actinobacteria is defined as a phylum of Grampositive bacteria with high G+C content in their DNA. Although classical actinobacteria have the largest and most complex bacterial cells, some groups of actinobacteria possess the small and simple cell. For these simple (rod, cocci-shaped, without hyphae differentiated) actino‐ bacteria, physiological and biochemical experiments are more important. Their physiological and biochemical tests can be carried by the automatic identification systems like the common bacteria.


Cross streak method [25]: The actinomycetes isolates were inoculated at the centre of the sterile agar plates and the plates were incubated at 28°C for 5–7 days. After incubation, the nutrient broth (peptone 5 g; beef extract 3 g; NaCl 5 g; distilled water 1000 ml) cultures of test bacteria were streaked perpendicular to the growth of the actinomycete isolates. The plates were incubated at 37°C for 24–48 h and the extent of growth inhibition of the test bacteria was observed. The absence of growth or a less dense growth of test bacteria near the growth of actinomycete isolate was considered positive for the production and secretion of antibacterial

Double-layer agar diffusion method [4]: Prepare double-layer agar plates; the lower is water agar (0.8–1% agar). Cool the basal medium (YIM 38 agar medium or LB agar medium) to 45– 50°C, add the suspension of test strains, mix and spread on the water agar layer. Inoculate tested actinobacteria for 7 days at 28°C. Or pour fermentation broth of tested actinobacteria into sterilized steel rim or holes on medium and inoculate for 24–48 h at 37°C. Or inoculate the filter paper containing fermentation broth on the plates for 24-48 h at 37°C. Observe and record the diameter of inhibition zone. Results: no antibacterial activity (no zone of inhibition); weak antibacterial activity (diameter of zone, 6–15 mm), strong antibacterial activity (diameter of

**4. Commercial multitest system for identification of actinobacteria**

In recent 40 years, with the development of microelectronics, computer, molecular biology, physics, chemistry, and subject crossing universality, a significant contribution of scientific and technological development to the clinical microbiology was the development of minia‐ turized identification systems based on classical method. Several systems are commercially available [Table 4], and new systems are being developed continually. These systems were mostly based on modifications of classical methods and were improved by the incorporation of highly sophisticated, computer-generated identification databases tailored for each system [26, 27]. Each manufacturer provides charts, tables, coding systems, and characterization profiles for use with the particular multi-test system being offered. These systems offer the advantages of miniaturization and are usually used in conjunction with a computerized system for identification of the organisms. As mentioned earlier, the use of these systems can increase standardization among various laboratories because of the high degree of quality control exercised over the media and reagents. Now actinobacteria is defined as a phylum of Grampositive bacteria with high G+C content in their DNA. Although classical actinobacteria have the largest and most complex bacterial cells, some groups of actinobacteria possess the small and simple cell. For these simple (rod, cocci-shaped, without hyphae differentiated) actino‐ bacteria, physiological and biochemical experiments are more important. Their physiological and biochemical tests can be carried by the automatic identification systems like the common

metabolite.

104 Actinobacteria - Basics and Biotechnological Applications

zone, >15 mm).

bacteria.


The products information of manufacturer come from:

bioMérieux, Marcy l'Etoile, France (http:// www.biomerieux.com/servlet/srt/bio/portail/home);

Biolog, Hayward, CA. (http://www.biolog.com);

BD Diagnostic Systems, Franklin Lakes, NJ (http://www.bd.com);

Dade Behring, Inc., MicroScan Inc., West Sacramento, CA (now owned by Siemens Medical Solutions, Henkestraße 127, Erlangen 91052, Germany) (http://www.medical.siemens.com/webapp/wcs/stores/servlet/SMBridgeBq\_catalo‐ gIdBe\_-999B a\_catTreeBe\_100001Ba\_langIdBe\_-999Ba\_storeIdBe\_10001.htm);

Remel, Lenexa, Kansas (http://www.remel.com/clinical/ microbiology.aspx);

Trek Diagnostic Systems, Ltd., East Grinstead, West Sussex, UK (http://www.trekds.com).

**Table 4.** Some commercial multitest systems for prokaryote identification

#### **4.1. API Numerical identification system**

API (analytical profile index) is a classification of bacteria based on experiments, allowing fast identification. This system is developed for quick identification of clinically relevant bacteria. Because of this, only known bacteria can be identified. It was invented in the 1970s in the United States by Pierre Janin of Analytab Products, Inc. Presently, the API test system is manufactured by bioMérieux [28]. The API range introduced a standardized, miniaturized version of existing techniques, which up until then were complicated to perform and difficult to read.

API systems can determinate simultaneously more than 20 items of biochemical indicators. Choose appropriate API strip according to different bacterial groups. The API strip consists of more than 20 microtubes containing dehydrated substrates. The conventional tests are inoculated with a saline bacterial suspension which reconstitutes the media. During incuba‐ tion, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. The assimilation tests are inoculated with a minimal medium and the bacteria grow if they are capable of utilizing the corresponding substrate. The reactions are read according to the Reading Table and the identification is obtained by referring to the Analytical Profile Index or using the identification software. Such as API 20NE is an identifi‐ cation system for non-fastidious, non-enteric Gram-negative rods [Figure 2].

**Figure 2.** API 20NE operational flowchart

**Manufacturer Test system Designed for Number of**

bioMérieux, Marcy l'Etoile, France (http:// www.biomerieux.com/servlet/srt/bio/portail/home);

Trek Diagnostic Systems

The products information of manufacturer come from:

106 Actinobacteria - Basics and Biotechnological Applications

BD Diagnostic Systems, Franklin Lakes, NJ (http://www.bd.com);

gIdBe\_-999B a\_catTreeBe\_100001Ba\_langIdBe\_-999Ba\_storeIdBe\_10001.htm);

Remel, Lenexa, Kansas (http://www.remel.com/clinical/ microbiology.aspx);

**Table 4.** Some commercial multitest systems for prokaryote identification

**4.1. API Numerical identification system**

Trek Diagnostic Systems, Ltd., East Grinstead, West Sussex, UK (http://www.trekds.com).

Biolog, Hayward, CA. (http://www.biolog.com);

RapidID POS ID *Streptococci* and *enterococci* 34 RapidID STR *Enterococci* and *streptococci* 14

Sensititre AP80 Enterobacteriaceae and non-enteric Gram-negative rods 32 Sensititre AP90 *Enterococci* and *streptococci* 32

Dade Behring, Inc., MicroScan Inc., West Sacramento, CA (now owned by Siemens Medical Solutions, Henkestraße 127, Erlangen 91052, Germany) (http://www.medical.siemens.com/webapp/wcs/stores/servlet/SMBridgeBq\_catalo‐

API (analytical profile index) is a classification of bacteria based on experiments, allowing fast identification. This system is developed for quick identification of clinically relevant bacteria. Because of this, only known bacteria can be identified. It was invented in the 1970s in the United States by Pierre Janin of Analytab Products, Inc. Presently, the API test system is manufactured by bioMérieux [28]. The API range introduced a standardized, miniaturized version of existing

API systems can determinate simultaneously more than 20 items of biochemical indicators. Choose appropriate API strip according to different bacterial groups. The API strip consists of more than 20 microtubes containing dehydrated substrates. The conventional tests are inoculated with a saline bacterial suspension which reconstitutes the media. During incuba‐ tion, metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. The assimilation tests are inoculated with a minimal medium and the bacteria grow if they are capable of utilizing the corresponding substrate. The reactions are read according to the Reading Table and the identification is obtained by referring to the Analytical Profile Index or using the identification software. Such as API 20NE is an identifi‐

techniques, which up until then were complicated to perform and difficult to read.

cation system for non-fastidious, non-enteric Gram-negative rods [Figure 2].

**tests**

#### **4.2. Biolog automatic bacterial identification system**

The Biolog Microbial ID System (Biolog, Inc., Hayward, Calif.) can rapidly identify over 2,500 species of aerobic and anaerobic bacteria, yeasts, and fungi. These easy-to-use systems provide reference laboratory quality identifications. Biolog Systems do this without the labor-intensive requirements of conventional strips or panels [29, 30]. Biolog's latest generation redox chemistry enables testing and microbial identification of aerobic Gram-negative and Grampositive bacteria in the same test panel. Gram stain and other pre-tests are no longer needed. A simple 1-min setup protocol and microbial samples are ready to be analyzed. Expanded GEN III database is designed to meet the needs of Biolog's broad customer base covering diverse disciplines of microbiology. All Biolog Microbial Identification Systems (manual, semiautomated, or fully automated) use the powerful new GENIII MicroPlate, allowing users to determine the most appropriate system to fit their current budget and level of throughput [Figure 3].

**Figure 3.** A common procedure of Biolog Microbial ID System [http://www.biolog.com]

It is important to realize that most such systems are designed for the identification of particular taxa and not for determining the physiological features of other taxa or new taxa. Indeed, a particular system may not even be applicable to other taxa. With these precautions in mind, multitest systems can provide useful information about the physiological characteristics of other organisms. For describing new taxa, the characterization systems that are used, as well as the inoculum age and size and the incubation temperature, must always be stated because reactions may not always agree with the results from classical characterization tests or with the results with other multitest systems.

### **Acknowledgements**

This project was supported by the National Natural Science Foundation of China (No. 31270001, and N0. 31460005), Yunnan Provincial Society Development Project (2014BC006), National Institutes of Health USA (1P 41GM 086184 -01A 1). We are grateful to Ms. Chun-hua Yang and Mr. Yong Li for their excellent technical assistance.

### **Author details**

Qinyuan Li1 , Xiu Chen1,2, Yi Jiang1\* and Chenglin Jiang1

\*Address all correspondence to: jiangyi@ynu.edu.cn

1 Yunnan Institute of Microbiology, School of Life Science, Yunnan University, Kunming, P. R. China

2 Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern University, Shenyang, P. R. China

### **References**


[3] Krige NR, Padgtt PJ. Phenotypic and physiological characterization methods. In: Rainey F, Oren A. (Eds.) Methods in Microbiology, Vol. 38, pp. 15–60. Academic Press, New York. 2011. DOI: 10.1016/B978-0-12-387730-7.00003-6

It is important to realize that most such systems are designed for the identification of particular taxa and not for determining the physiological features of other taxa or new taxa. Indeed, a particular system may not even be applicable to other taxa. With these precautions in mind, multitest systems can provide useful information about the physiological characteristics of other organisms. For describing new taxa, the characterization systems that are used, as well as the inoculum age and size and the incubation temperature, must always be stated because reactions may not always agree with the results from classical characterization tests or with

This project was supported by the National Natural Science Foundation of China (No. 31270001, and N0. 31460005), Yunnan Provincial Society Development Project (2014BC006), National Institutes of Health USA (1P 41GM 086184 -01A 1). We are grateful to Ms. Chun-hua

1 Yunnan Institute of Microbiology, School of Life Science, Yunnan University, Kunming, P.

2 Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern

[1] Vandamme P, Pot B, Gillis M, de Vos P, Kersters K, Swings J. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 1996;60:407–38. PMC:

[2] Ludwig W, Klenk HP. Overview: a phylogenetic backbone and taxonomic frame‐ work for prokaryotic systematics. In: Garrrity GM, Brenner DJ, Krieg NR, Staley JT. (Eds.) Bergey's Manual of Systematic Bacteriology, 2nd edition., 2005; vol. 2, Part A,

the results with other multitest systems.

108 Actinobacteria - Basics and Biotechnological Applications

Yang and Mr. Yong Li for their excellent technical assistance.

, Xiu Chen1,2, Yi Jiang1\* and Chenglin Jiang1

\*Address all correspondence to: jiangyi@ynu.edu.cn

pp. 49–65. Springer-Verlag, New York.

University, Shenyang, P. R. China

**Acknowledgements**

**Author details**

Qinyuan Li1

R. China

**References**

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[30] Holmes B, Costas M, Ganner M, On SL, Steven M. Evaluation of Biolog system for identification of some gram-negative bacteria of clinical importance. J Clin Microbiol 1994;32(8):1970–75. PMCID: PMC263912

[17] Medina P, Baresi L. Rapid identification of gelatin and casein hydrolysis using TCA.

[18] Teather RM, Wood PJ. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen Appl Environ Mi‐

[19] Volokita M, Abeliovich A, Soares MIM. Detection of microorganisms with overall cellulolytic activity. Curr Microbiol 2000;40:136–136. DOI: 10.1007/s002849910027

[20] Granger DL, Taintor RR, Boockvar KS, Hibbs JR. Measurement of nitrate and nitrite in biological samples using nitrate reductase and Griess reaction. Methods Enzymol

[21] Lányi, B. Classical and rapid identification methods for medically important bacteria. In: Colwell RR, Grigorova R. (Eds.) Methods in Microbiology, Vol. 19, pp. 1–67.1987.

[22] Levine M, Epstein SS, Vaughn RH. Differential reactions in the colon group of bacte‐ ria. Am J Public Health Nations Health 1934;24:505–10. PMCID: PMC1558757

[23] Smibert RM, Krieg NR. Phenotypic characterization. In: Gerhardt P, Murray RGE, Wood WA, Krieg NR. (Eds.) Methods for general and molecular bacteriology, Ameri‐

[24] Esser VM, Elefson DE. Experiences with the Kirby-Bauer method of antibiotic sus‐

[25] Williston EH, Zia-Walrath P, Youmans GP. Plate methods for testing antibiotic activ‐ ity of actinomycetes against virulent human type tubercle bacilli. J Bacteriol

[26] D'Amato EE, Taylor RH, Blannon JC, Reasoner DJ. Substrate profile systems for the identification of bacteria and yeasts by rapid and automated approaches. In: Balows A, Hausler WJJ, Herrmann KL, Isenberg HD, Shadomy H. (Eds.) Manual of Clinical Microbioloy, 1911. pp. 128–36. American Society for Microbiology, Washington, DC.

[27] O'Hara CM. Manual and Automated Instrumentation for Identification of *Enterobac‐ teriaceae* and Other Aerobic Gram-Negative *Bacilli*. Clin Microbiol Rev 2005;18:147–

[28] Washington II J A, Yu PKW, Martin WJ. Evaluation of accuracy of multitest micro‐ method system for identification of *Enterobacteriaceae.* Appl Microbiol 1971;22:267–9.

[29] Washington II J A, Yu PKW and Martin WJ. Evaluation of accuracy of multitest mi‐ cromethod system for identification of *Enterobacteriaceae.* Appl Microbiol. 1971;

can Society for Microbiology, Washington, D.C., 1994. pp. 607–654.

ceptibility testing. Am J Clin Pathol 1970;54:193–9. PMID: 4916518

J Microbiol Meth 2007;69:391–3. DOI: 10.1016/j.mimet.2007.01.005

crobiol 1982;43(4):777–80. PMCID: PMC241917.

1996;268:142–52. DOI: 10.1016/S0076-6879(96)68016-1

Academic Press, New York.

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PMCID: PMC376296

22:267-269. PMCID: PMC376296

### **Chapter 5**

## **Chemotaxonomy of Actinobacteria**

### Yongxia Wang and Yi Jiang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61482

#### **Abstract**

Actinobacterial classification was originally based largely on morphological observation; it is not adequate in itself to differentiate between many genera, because some are so sim‐ ilar morphologically, but differ from their diagnostic chemical composition. In search of reliable classification methods reflecting phylogenetic relationships, at least to the genus level, it has been demonstrated that the analyses of chemotaxonomic markers fulfill these requirements. Chemotaxonomy of actinobacteria is concerned with the distribution of specific chemicals of the cell envelope such as amino acid, sugar, polar lipids, menaqui‐ nones, and fatty acid. For some coryneform genera of actinobacteria, analysis of mycolic acid composition is required specially. In this chapter, we will introduce the methods of chemotaxonomy including the extraction, fractionation, purification, and analysis of the target compounds.

**Keywords:** Chemotaxonomy, Amino acid, Sugar, Polar lipids, Menaquinones, Fatty acid, Mycolic acid

### **1. Introduction**

Chemotaxonomy is the study of the chemical variation in microbial cell and the use of chemical characteristics in the classification and identification of bacteria including actinobacteria. In search of reliable classification methods reflecting phylogenetic relationships, it has been demonstrated that the analyses of chemotaxonomic markers fulfill these requirements [1]. Therefore, chemotaxonomy is an essential tool in the modern classification of bacteria; it has been recommended in a polyphasic approach to apply to the species, genus, and higher taxa level [2, 3]. Chemotaxonomy of actinobacteria is concerned with the distribution of specific chemicals of the actinobacteria cell envelope such as amino acid, sugar, polar lipids, mena‐ quinones, mycolic acid, and fatty acid (Table 1) by using chemical techniques, including the extraction, fractionation, purification, and resolution of the target compounds [4].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**Table 1.** Chemotaxonomic markers applied in polyphasic approach of Actinobacteria

### **2. Amino acid of cell wall**

Actinobacteria could be separated into broad groups at the generic level on the basis of morphology and cell wall composition [5]. For such grouping, the compositions of cell wall diaminopimeric acid isomers and whole cell sugars have become widely accepted as the taxonomic markers [6, 7]. Cell wall of actinobacteria consists of a single 20–80 nm thick homogeneous peptidoglycan and frequently represents more than 20% of the cell dry weight. Peptidoglycan constitutes between 40 % and 80 % of the wall weight, while the remainder is made up largely of other macromolecules (lipids, teichoic acids, and acidic polysaccharides and proteins) covalently linked either directly to peptidoglycan or to one another. The structure of peptidoglycan is very stable; it is an enormous polymer com‐ posed of many identical subunits; the polymer contains β1–4 linked disaccharides, Nacetylglucosamine and N-acetylmuramic acid, and several different amino acids. The backbone of this polymer is composed of alternating β1–4 linked disaccharides of Nacetylglucosamine and N-acetylmuramic acid. A peptide chain of four alternating D- and L-amino acids is connected to the carboxyl group of N-acetylmuramic acid. Chains of linked peptidoglycan subunits are joined by cross-links between the peptides. Often, the carbox‐ yl group of the terminal D-alanine is connected directly or through a peptide inter-bridge to the amino group of diaminopimelic acid (Figure 1).

The differences in the amino acid sequence of the peptide chains, the mode of cross-links between the chains, and the diaminoacids present give important information for the classi‐ fication of actinobacteria and have been used for the description of peptidoglycan type [9]. Detection of the presence of diaminoacids at position 3 of the peptide chain is useful for the classification of actinobacteria. *Meso*- and LL-diaminopimelic acid, L-ornithine, L-lysine, and L-diaminobutyric acid are found present at position 3 of the peptide chain [9–11].

A method for analysis of the diaminoacids of peptidoglycan from whole cells has been described by Staneck and Roberts, Lechevalier, Hasegawa et al., Bousfield et al., and Busse et al. [1, 12–15]; this method is rapid, simple, inexpensive equipment and requires only small amount of biomass. The procedure for rapid determination of the diaminoacids present in the

Glu

Meso‐DAP

DD‐DAP

**Figure 5.1. Peptidoglycan structure (a) and peptidoglycan cross‐links type (b) (from Prescott Figure 1.** Peptidoglycan structure (a) and peptidoglycan cross-links type (b) (from Prescott et al. [8])

cell, described by Hasegawa et al. [14] with the solvent system of thin-layer chromatography [12], is quite suitable for separation of diaminoacids (Table 2 and Figure 2). However, for analysis of the amino acid in the peptide chains or inter-peptide bridge of the peptidoglycan, the cell wall extraction is required. Detailed cell wall extraction was described by Schleifer and Hancock [16, 17]. Here, we describe a method of cell wall extraction cited from the library of Yunnan Institution of Microbiology, Yunnan University (YIM) (Table 3). *et al.***, [8])** Ala

#### **2.1. Extraction of whole cell amino acid**

Samples Standard Samples

**Categories Site in cell Composition**

**Table 1.** Chemotaxonomic markers applied in polyphasic approach of Actinobacteria

to the amino group of diaminopimelic acid (Figure 1).

**2. Amino acid of cell wall**

Cell wall Amino acid Plasma membranes Polar lipids Plasma membranes Menaquinones Plasma membranes Fatty acids Plasma membranes Mycolic acids

Actinobacteria could be separated into broad groups at the generic level on the basis of morphology and cell wall composition [5]. For such grouping, the compositions of cell wall diaminopimeric acid isomers and whole cell sugars have become widely accepted as the taxonomic markers [6, 7]. Cell wall of actinobacteria consists of a single 20–80 nm thick homogeneous peptidoglycan and frequently represents more than 20% of the cell dry weight. Peptidoglycan constitutes between 40 % and 80 % of the wall weight, while the remainder is made up largely of other macromolecules (lipids, teichoic acids, and acidic polysaccharides and proteins) covalently linked either directly to peptidoglycan or to one another. The structure of peptidoglycan is very stable; it is an enormous polymer com‐ posed of many identical subunits; the polymer contains β1–4 linked disaccharides, Nacetylglucosamine and N-acetylmuramic acid, and several different amino acids. The backbone of this polymer is composed of alternating β1–4 linked disaccharides of Nacetylglucosamine and N-acetylmuramic acid. A peptide chain of four alternating D- and L-amino acids is connected to the carboxyl group of N-acetylmuramic acid. Chains of linked peptidoglycan subunits are joined by cross-links between the peptides. Often, the carbox‐ yl group of the terminal D-alanine is connected directly or through a peptide inter-bridge

The differences in the amino acid sequence of the peptide chains, the mode of cross-links between the chains, and the diaminoacids present give important information for the classi‐ fication of actinobacteria and have been used for the description of peptidoglycan type [9]. Detection of the presence of diaminoacids at position 3 of the peptide chain is useful for the classification of actinobacteria. *Meso*- and LL-diaminopimelic acid, L-ornithine, L-lysine, and

A method for analysis of the diaminoacids of peptidoglycan from whole cells has been described by Staneck and Roberts, Lechevalier, Hasegawa et al., Bousfield et al., and Busse et al. [1, 12–15]; this method is rapid, simple, inexpensive equipment and requires only small amount of biomass. The procedure for rapid determination of the diaminoacids present in the

L-diaminobutyric acid are found present at position 3 of the peptide chain [9–11].

Chemotaxonomic cell Sugars

114 Actinobacteria - Basics and Biotechnological Applications

**Table 2.** Method of extraction and analysis of whole cell amino acids (modified from [12, 14])

<sup>1.</sup> A loop of cell mass is added into an ampule, add 0.2 ml of 6 N HCl into the ampule, seal and sand bath to hydrolyze for 16 h at 121<sup>ο</sup> C. Gly

<sup>2.</sup> Spot 1 µl to the bottom of a 10 × 20 cm of thin-layer plate coated with cellulose.

<sup>3.</sup> Spot 1 µl of 0.01 M DL-A2pm containing both LL-and meso-A2pm on the same plate as a standard.

<sup>4.</sup> Develop with methanol–water–6 N HCl–pyridine (80:26:4:10, v/v) for 3 h and dry the plates in a fume cupboard. Asp

<sup>5.</sup> Repeat the fourth step once.

<sup>6.</sup> Spray the plate very lightly with 0.4% of ninhydrin and heated at 100<sup>ο</sup> C for 2 min to reveal the spots; amino acids are shown as pink spots. LL‐DAP

*et al.***, [8])**

Pentaglycine interbridge

Peptide chain

**Chapter 5 Figures and Tables**

**Figure 2.** Separation of A2pm isomers from the hydrolysate of whole cell by thin-layer chromatography

#### **2.2. Preparation of cell wall amino acid**

1. Add 1 g of freshly harvested or 0.3 g lyophilized cell mass into 10 ml screw cap test tube; add 1.5 ml of 1 % NaCl (w/v) into the test tube, mix , cap tightly, and stand for 10 min.

2. Add 7 ml of 0.05 mol/l PBS (pH7.6) into the test tube; sonicate for 40 min to lysis the cells (46 w, treatment 5 sec and standing 8 sec, total 40 min).

3. Centrifugate for 15 min at 4,000 rpm; remove the supernatant into a new 10 ml screw cap test tube and discard the precipitate.

4. Centrifugate for 40 min at 12,000 rpm, remove, and discard the supernatant.

5. Add 1 ml of 4 % SDS into the test tube containing precipitate, boiling water bath for 15 min or at room temperature overnight, centrifugate for 30 min at 12,000 rpm, and discard the supernatant.

6. Add 1 ml deionized distilled water, mix and centrifugate for 30 min at 12,000 rpm, and discard supernatant. The addition of deionized distilled water is repeated once.

7. The final insoluble pellet (precipitate) is dried at 65<sup>ο</sup> C; add 200 µl of 6 N HCl into the test tube, mix until the dried pellet dissolved completely, and transfer the solution into the ampule, seal and sand bath overnight at 100<sup>ο</sup> C.

8. Neutralize with 0.2 M NaOH to pH 7.0 and add three volumes sodium borate and mix.

Filter the mixture solution by using 0.45 µm fiber membrane, place the filtered solution into the sample bottle for detecting the amino acid composition by HPLC.

**Table 3.** Preparation method of cell wall amino acid

#### **2.3. Detection of cell wall amino acid**

Amino acids in cell wall hydrolysates were analyzed by precolumn derivatization with ophthalaldehyde (OPA): ten amino acids standards (10 ml, 0.2 mM) and 10 ml hydrolyzed purified cell wall were dissolved in 0.1 M (30 ml) borax buffer, and 10 ml OPA was added and allowed to react for 50 sec at room temperature and analyzed by high-performance liquid chromatography (HPLC). The elution time of 10 amino acids standards by HPLC is shown in Figure 3.

### **High-performance liquid chromatography (HPLC):**

Agilent 1100, HPLC system equipped with an Agilent four-unit pump, a 7125 injector, a G1314A UV detector

Columns: ZORBAX Eclipse-AAA (4.6 × 150 mm, 3.5 µm; Agilent)

Columns temperature: 40<sup>ο</sup> C

**Chapter 5 Figures and Tables**

**Figure 5.1. Peptidoglycan structure (a) and peptidoglycan cross‐links type (b) (from Prescott**

Samples Standard Samples

1. Add 1 g of freshly harvested or 0.3 g lyophilized cell mass into 10 ml screw cap test tube; add 1.5 ml of 1 % NaCl

2. Add 7 ml of 0.05 mol/l PBS (pH7.6) into the test tube; sonicate for 40 min to lysis the cells (46 w, treatment 5 sec and

3. Centrifugate for 15 min at 4,000 rpm; remove the supernatant into a new 10 ml screw cap test tube and discard the

5. Add 1 ml of 4 % SDS into the test tube containing precipitate, boiling water bath for 15 min or at room temperature

6. Add 1 ml deionized distilled water, mix and centrifugate for 30 min at 12,000 rpm, and discard supernatant. The

pellet dissolved completely, and transfer the solution into the ampule, seal and sand bath overnight at 100<sup>ο</sup>

Filter the mixture solution by using 0.45 µm fiber membrane, place the filtered solution into the sample bottle for

Amino acids in cell wall hydrolysates were analyzed by precolumn derivatization with ophthalaldehyde (OPA): ten amino acids standards (10 ml, 0.2 mM) and 10 ml hydrolyzed

8. Neutralize with 0.2 M NaOH to pH 7.0 and add three volumes sodium borate and mix.

**Figure 2.** Separation of A2pm isomers from the hydrolysate of whole cell by thin-layer chromatography

Ala

Glu

Gly

Asp

LL‐DAP

Meso‐DAP

DD‐DAP

C; add 200 µl of 6 N HCl into the test tube, mix until the dried

C.

*et al.***, [8])**

116 Actinobacteria - Basics and Biotechnological Applications

**2.2. Preparation of cell wall amino acid**

addition of deionized distilled water is repeated once. 7. The final insoluble pellet (precipitate) is dried at 65<sup>ο</sup>

detecting the amino acid composition by HPLC.

**Table 3.** Preparation method of cell wall amino acid

**2.3. Detection of cell wall amino acid**

standing 8 sec, total 40 min).

precipitate.

(w/v) into the test tube, mix , cap tightly, and stand for 10 min.

4. Centrifugate for 40 min at 12,000 rpm, remove, and discard the supernatant.

overnight, centrifugate for 30 min at 12,000 rpm, and discard the supernatant.

Pentaglycine interbridge

Peptide chain

UV detect wavelength: 338 nm

Mobile phase A: 0.05 mol l–1 CH3COONa and 0.3 % tetrahydrofuran

Mobile phase B: acetonitrile/methanol (1:1, v/v)

Gradient elution: 0–50–50 % buffer B by a linear increase from 0 to 25 to 30

Elution flow rate: 1.0 ml min–1

Injection volume: 20 µl.

**Figure 3.** The elution time of 10 amino acids standards by HPLC

The presented method of cell wall amino acids analysis by HPLC will not separate well the LL-, meso-, and dd-A2pm. So, analysis of the cell wall type of actinobacteria to the genus level requires a combination of HPLC and TLC.

### **3. Sugar of whole cell hydrolytes**

For the classification and identification of actinobacteria, the analysis of sugars from whole cells is needed [10, 11, 19–23]. For discrimination of meso-diaminopimelic acid containing actinomycetes, five whole cell sugar patterns have been recognized [24], based on the presence of distinct sugars (A: arabinose and galactose; B: madurose; C: no diagnostic sugars; D: arabinose and xylose; E: rhamnose). The combination of the characteristic diaminoacid and some amino acids used cell wall sugars to describe eight wall chemotypes to distinguish actinomycetes [13] (Table 4).


**Table 4.** Chemotypes of cell wall [13]

The analysis of diaminoacids and sugars from whole cell preparations is less time-consuming and often allows an allocation to the correct wall chemotype, but the resulting pattern may be contaminated by non-peptidoglycan-linked saccharides from the cytoplasm, capsules, or slimes. Different methods have been described for whole cell preparations [14, 19, 25], cell wall preparations [9, 26, 27], as well as analysis of sugars [12, 27, 28].

As the methods used to prepare whole cell extracts are similar, the procedure of extraction and analysis of whole cell sugar reported by Hasegawa et al. [14] are briefly described. Although the procedure from Staneck and Roberts [12] for thin-layer chromatography of diagnostic sugar on cellulose plates works reasonably well, it is not able to separate the mannose and arabinose. We described a modified method by changing the developed solvent to separate the mannose and arabinose (Table 5 and Figure 4).

4. Develop with ethyl acetate–pyridine–acetic acid–water (8:5:1:5, v/v) for 3 h and dry the plates in a fume cupboard.

5. Repeat the fourth step once.

#### **Table 5.** Extraction and analysis of whole cell sugars (modified from [12, 14])

<sup>1.</sup> Add a loop of cell mass into an ampule, add 0.1 ml of 0.25 N HCl into the ampule, seal and sand bath to hydrolyze for 15 min at 121<sup>ο</sup> C.

<sup>2.</sup> Spot 2 µl to the bottom of a 10 × 20 cm of thin-layer plate coated with cellulose.

<sup>3.</sup> Spot 1 µl of standard solution 1 containing rhamnose, xylose, and mannose, and standard solution 2 containing ribose, madurose, arabinose, and glucose on the same plate, respectively.

<sup>6.</sup> Spray the plate very lightly with acid aniline phthalate and heated at 100<sup>ο</sup> C for 4 min to reveal the spots.

Besides the procedure of TLC [12, 14], a better procedure to analyze whole cell sugars has been described in our laboratory [18]. It described a method to extract sugars of whole cell and a procedure for preparation of sugar sample for HPLC analysis (Table 6). **Figure 5.3. The elution time of 10 amino acids standards by HPLC** 

**Figure 5.2. Separation of A2pm isomers from hydrolysate of whole cell by thin‐layer**

**chromatography**

**Figure 5.4. Separation of whole cell sugars from whole cell hydrolysate by thin‐layer chromatography** (rha=rhamnose, xyl=xylose, man=mannose, rib=ribose, mad=madurose, ara=arabinose, glu=glucose) **Figure 4.** Separation of whole cell sugars from whole cell hydrolysate by thin-layer chromatography (rha = rhamnose, xyl = xylose, man = mannose, rib = ribose, mad = madurose, ara = arabinose, glu = glucose)

#### **3.1. Extraction and preparation of whole cell sugar**

actinomycetes, five whole cell sugar patterns have been recognized [24], based on the presence of distinct sugars (A: arabinose and galactose; B: madurose; C: no diagnostic sugars; D: arabinose and xylose; E: rhamnose). The combination of the characteristic diaminoacid and some amino acids used cell wall sugars to describe eight wall chemotypes to distinguish

> *meso*-Diaminopimelic acid, various amino acids *meso*-Diaminopimelic acid, L-Diaminopimelic acid

The analysis of diaminoacids and sugars from whole cell preparations is less time-consuming and often allows an allocation to the correct wall chemotype, but the resulting pattern may be contaminated by non-peptidoglycan-linked saccharides from the cytoplasm, capsules, or slimes. Different methods have been described for whole cell preparations [14, 19, 25], cell wall

As the methods used to prepare whole cell extracts are similar, the procedure of extraction and analysis of whole cell sugar reported by Hasegawa et al. [14] are briefly described. Although the procedure from Staneck and Roberts [12] for thin-layer chromatography of diagnostic sugar on cellulose plates works reasonably well, it is not able to separate the mannose and arabinose. We described a modified method by changing the developed solvent to separate

1. Add a loop of cell mass into an ampule, add 0.1 ml of 0.25 N HCl into the ampule, seal and sand bath to hydrolyze

3. Spot 1 µl of standard solution 1 containing rhamnose, xylose, and mannose, and standard solution 2 containing

4. Develop with ethyl acetate–pyridine–acetic acid–water (8:5:1:5, v/v) for 3 h and dry the plates in a fume cupboard.

C for 4 min to reveal the spots.

actinomycetes [13] (Table 4).

118 Actinobacteria - Basics and Biotechnological Applications

**Table 4.** Chemotypes of cell wall [13]

VIII IX X

for 15 min at 121<sup>ο</sup>

C.

5. Repeat the fourth step once.

**Cell wall chemotype Characteristic cell wall components** I L-Diaminopimelic acid, glycine II *meso*-Diaminopimelic acid, glycine III *meso*-Diaminopimelic acid

V Lysine and ornithine

VII Diaminobutyric acid, glycine

IV *meso*-Diaminopimelic acid, arabinose, galactose

VI Variable presence of aspartic acid and galactose

Ornithine

preparations [9, 26, 27], as well as analysis of sugars [12, 27, 28].

2. Spot 2 µl to the bottom of a 10 × 20 cm of thin-layer plate coated with cellulose.

ribose, madurose, arabinose, and glucose on the same plate, respectively.

6. Spray the plate very lightly with acid aniline phthalate and heated at 100<sup>ο</sup>

**Table 5.** Extraction and analysis of whole cell sugars (modified from [12, 14])

the mannose and arabinose (Table 5 and Figure 4).

1. Add 1 g of freshly harvested or 0.3 g lyophilized cell mass into ampule; add 0.5 ml of 0.5 N HCl into the ampule, seal and sand bath for 2 h.

2. Unseal the ampule, 80 µl hydrolysed whole cell solution and 80 µl 0.25 M methanol solution of 1-phenyl-3-methyl-5 pyrazolone (PMP) and 80 µl 0.2 M NaOH were mixed.

3. Mixture was allowed to react for 30 min at 70<sup>ο</sup> C, cooled to room temperature and neutralized with 80 ml 0.2 M NaOH to pH 7.0, and extracted with isoamyl acetate.

4. After vigorous shaking and centrifugation, the organic phase was carefully discarded to remove the excess reagents.

5. The extraction process was repeated three times, using chloroform instead of isoamyl acetate for the third process; the aqueous layer was then collected and 10 ml was taken for HPLC analysis.

**Table 6.** Method for extraction and preparation of whole cell sugar [14, 29]

#### **3.2. Analysis of sugar of whole cell hydrolytes**

The sugar of whole cell hydrolytes was analyzed by high-performance liquid chromatography (HPLC). The elution time of nine sugar standards by HPLC is shown in Figure 5.

Agilent 1100, HPLC system equipped with an Agilent four-unit pump, a 7125 injector, a G1314A UV detector

Columns: ZORBAX Eclipse XDB-C18 (4.6 × 150 mm, 5 µm; Agilent)

Columns temperature: 40<sup>ο</sup> C

UV detect wavelength: 250 nm

Mobile phase A: acetonitrile

Mobile phase B: 0.05 M sodium acetate (pH 6.9)

Elution: A : B = 17 : 83 (v/v).

Elution flow rate: 1.0 ml min–1

Injection volume: 10 µl

**Figure 5.** The elution time of nine sugar standards by HPLC (from YIM library) (Man = mannose, Rib = ribose, Rha = rhamnose, GlcN = glucosamine hydrochloride, Glc = glucuronic acid, Gal = galactose, Xyl = xylose, Ara = arabinose, Fuc = fucose)

### **4. Polar lipids**

Polar lipids are important components of bacterial plasma membranes. Bacterial plasma membranes are composed of amphipathic polar lipids associated with specific membrane proteins. Amphipathic polar lipids consist of hydrophilic head groups usually linked to two hydrophobic fatty acid chains. Phospholipids are the most common polar lipids, including phosphatidyglycerol, diphosphatidyglycerol, phosphatidylcholine, phosphatidylethanola‐ mine, phosphatidylserine, phosphatidyliositol, and other phosphatidylglycolipids. In addi‐ tion, glycolipids and acylated ornithine or lysine amides also fall into this category. For the description and differentiation of actinobacteria, five phospholipid types (PI–PV) have been recognized (Table 7) [30, 31].


**Table 7.** Phospholipid types according to Lechevalier et al. [31]

In taxonomic studies, polar lipids have largely been analyzed by one- or two-dimensional thinlayer chromatography.

#### **4.1. Extraction of polar lipids**

Columns: ZORBAX Eclipse XDB-C18 (4.6 × 150 mm, 5 µm; Agilent)

11.381

Man

14.085

14.914

Rib Rham

15.286

0 5 10 15 20 25 30 min

**Figure 5.** The elution time of nine sugar standards by HPLC (from YIM library) (Man = mannose, Rib = ribose, Rha = rhamnose, GlcN = glucosamine hydrochloride, Glc = glucuronic acid, Gal = galactose, Xyl = xylose, Ara = arabinose,

Polar lipids are important components of bacterial plasma membranes. Bacterial plasma membranes are composed of amphipathic polar lipids associated with specific membrane proteins. Amphipathic polar lipids consist of hydrophilic head groups usually linked to two hydrophobic fatty acid chains. Phospholipids are the most common polar lipids, including phosphatidyglycerol, diphosphatidyglycerol, phosphatidylcholine, phosphatidylethanola‐ mine, phosphatidylserine, phosphatidyliositol, and other phosphatidylglycolipids. In addi‐

19.047

GlcN Glc

21.893

24.690

25.937

Gal Xyl Ara

26.941

31.038

Fuc

C

Mobile phase B: 0.05 M sodium acetate (pH 6.9)

Columns temperature: 40<sup>ο</sup>

UV detect wavelength: 250 nm

120 Actinobacteria - Basics and Biotechnological Applications

Mobile phase A: acetonitrile

Elution: A : B = 17 : 83 (v/v).

Injection volume: 10 µl

mAU

0

Fuc = fucose)

**4. Polar lipids**

20

40

60

80

100

120

140

Elution flow rate: 1.0 ml min–1

VWD1 A, =250 nm (TANG\2011-05-0600010.D)

The classic method of polar lipid extraction [13] is a time-consuming process taking at least 13 days from start to finish. Subsequently, more rapid procedures have been proposed by Minnikin et al. and Tindall [32, 33]. This utilizes a monophasic methanol for polar lipids extraction; the addition of more chloroform and water forces a phase separation. The lower, mainly chloroform, layer contains the polar lipids, whereas non-lipid components remain in the upper aqueous phase. Minnikin et al. [34] introduced a modified procedure, in which an initial extraction with hexane removes non-polar components such as isoprenoid quinones; in this way menaquinones and polar lipids can be extracted from a single sample of biomass. In this section, a modified procedure for polar lipids extraction is described (Table 8).

**Table 8.** Extraction of polar lipids (modified by Minnikin et al. and Tindall [32, 33])

#### **4.2. Two-dimensional thin-layer chromatography**

Separation of the mixture of polar lipids is performed by two-dimensional TLC (Table 9) on silica gel GF254 plate.

<sup>1.</sup> Place approximately 100~200mg of dried cell mass into a 50 ml tube with Teflon-lined screw cap.

<sup>2.</sup> Add 2 ml of 0.85% aqueous NaCl, followed by 15 ml methanol.

<sup>3.</sup> Heat for 10 min at 100<sup>ο</sup> C in a boiling bath and cool to room temperature.

<sup>4.</sup> Add 10 ml chloroform and 6 ml 0.85% aqueous NaCl , then shake for 10 min.

<sup>5.</sup> Centrifuge at 8,000 rpm for 10 min, collect the lower layer.

<sup>6.</sup> The lower layer in a flask is evaporated to dryness under reduced pressure at 40<sup>ο</sup> C on a rotary evaporator.

1. Dissolve the dried polar lipids in 100 µl of petroleum ether (boiling point: 70-90<sup>ο</sup> C). Spot 10 µl to the bottom of a 10 × 10cm of thin-layer plate coated with silica gel (Merck F254). Develop with chloroform–methanol–water (65:25:4, v/v) in the first dimension and dry the plates overnight in a fume cupboard.

2. Develop with chloroform–acetic acid–methanol–water (80:18:12:5, v/v) in the second dimension. Dry the plates in a fume cupboard.

**Table 9.** Two-dimensional thin-layer chromatography (modified by Minnikin et al. [35])

#### **4.3. Identification of polar lipid component**

Identification of the various lipids is carried out by comparison of their Rf values in the plates and staining behavior (Table 10) with references.

**Molybdophosphoric acid for total lipids [36]**

1. Dissolve 10% (w/v) molybdophosphoric acid in 95% (v/v) ethanol.

2. Spray the TLC plate and heat at 150<sup>ο</sup> C for at least 10 min. Lipids show as dark spots on a light-green background.

**Ninhydrin reagent for lipids containing free amino groups (modified by Consden and Gordon, [37])**

1. Dissolve ninhydrin (0.1%, w/v) in acetone.


#### **α-Naphthol reagent for containing sugar groups [38]**

1. Dissolve 15 g α-naphthol in 100 ml 95% (v/v) ethanol.

2. Mix 10.5 ml of this solution with 6.5 ml H2SO4, 40.5 ml ethanol, and 4 ml water to make a working solution.

3. Spray the plate lightly and heat a 100<sup>ο</sup> C for 10 min. Glycolipids appear as purple-brown or brown spot.

#### **Dragendorff reagent for lipids containing quaternary nitrogen groups**

1. Add bismuth nitrate (1.7 g) to 100 ml of 20 % acetic acid (solution A).

2. Add potassium iodide (40 g) to 100 ml water (solution B).

3. Mix solution A (3.5 ml) and solution B (5 ml) with acetic acid (20 ml) and water (50 ml) to make a working solution.

4. Spray the plate lightly at room temperature; lipids containing quaternary nitrogen shown as orange-red spots. Mark the orange-red spots with a soft pencil to prevent them from fading on storage.

5. The same plate can be used for the detection of lipid phosphorus using molybdenum reagent.

#### **Zinzadze reagent for phosphorus-containing lipids [39]**

1. Add molybdenum trioxide (40.11 g) to 1 L of 25 N H2SO4 and boil gently in a fume cupboard until all the residue dissolves (solution A).

2. Add powdered molybdenum (1.78 g) to 500 ml of solution A, and boil the mixture gently for 15 min and leave a cool (solution B).

3. Mix equal volumes of solutions A and B and dilute with two volumes of distilled water to make a working solution.

4. Spray the plate very lightly at room temperature, lipids containing phosphorus shown as blue spots.

### **5. Menaquinones**

1. Dissolve the dried polar lipids in 100 µl of petroleum ether (boiling point: 70-90<sup>ο</sup>

**Table 9.** Two-dimensional thin-layer chromatography (modified by Minnikin et al. [35])

the first dimension and dry the plates overnight in a fume cupboard.

**4.3. Identification of polar lipid component**

122 Actinobacteria - Basics and Biotechnological Applications

**Molybdophosphoric acid for total lipids [36]**

1. Dissolve ninhydrin (0.1%, w/v) in acetone.

3. Spray the plate lightly and heat a 100<sup>ο</sup>

2. Spray the TLC plate and heat at 150<sup>ο</sup>

2. Spray plate and heat at 100<sup>ο</sup>

dissolves (solution A).

(solution B).

and staining behavior (Table 10) with references.

1. Dissolve 10% (w/v) molybdophosphoric acid in 95% (v/v) ethanol.

spots with a soft pencil to prevent them from fading on storage.

**Dragendorff reagent for lipids containing quaternary nitrogen groups** 1. Add bismuth nitrate (1.7 g) to 100 ml of 20 % acetic acid (solution A).

the orange-red spots with a soft pencil to prevent them from fading on storage.

5. The same plate can be used for the detection of lipid phosphorus using molybdenum reagent.

2. Add potassium iodide (40 g) to 100 ml water (solution B).

**Zinzadze reagent for phosphorus-containing lipids [39]**

**Table 10.** Spray reagents for identification of individual components

**α-Naphthol reagent for containing sugar groups [38]** 1. Dissolve 15 g α-naphthol in 100 ml 95% (v/v) ethanol.

fume cupboard.

10cm of thin-layer plate coated with silica gel (Merck F254). Develop with chloroform–methanol–water (65:25:4, v/v) in

2. Develop with chloroform–acetic acid–methanol–water (80:18:12:5, v/v) in the second dimension. Dry the plates in a

Identification of the various lipids is carried out by comparison of their Rf values in the plates

**Ninhydrin reagent for lipids containing free amino groups (modified by Consden and Gordon, [37])**

2. Mix 10.5 ml of this solution with 6.5 ml H2SO4, 40.5 ml ethanol, and 4 ml water to make a working solution.

3. Mix solution A (3.5 ml) and solution B (5 ml) with acetic acid (20 ml) and water (50 ml) to make a working solution. 4. Spray the plate lightly at room temperature; lipids containing quaternary nitrogen shown as orange-red spots. Mark

1. Add molybdenum trioxide (40.11 g) to 1 L of 25 N H2SO4 and boil gently in a fume cupboard until all the residue

2. Add powdered molybdenum (1.78 g) to 500 ml of solution A, and boil the mixture gently for 15 min and leave a cool

3. Mix equal volumes of solutions A and B and dilute with two volumes of distilled water to make a working solution.

4. Spray the plate very lightly at room temperature, lipids containing phosphorus shown as blue spots.

3. The same plate can be used for the detection of lipid phosphorus using molybdenum reagent

C for at least 10 min. Lipids show as dark spots on a light-green background.

C for 5 min to reveal lipids which contain amino groups as pink spots. Mark the pink

C for 10 min. Glycolipids appear as purple-brown or brown spot.

C). Spot 10 µl to the bottom of a 10 ×

Respiratory isoprenoid quinones are constituents of the bacterial cytoplasmic membrane as well as the mitochondrial membrane where they play an important role in the electron transport chain. The potential of analyzing the quinone system for the characterization of bacteria is based on the different types of quinones (e.g., ubiquinones, menaquinones and their derivatives dihydromenaquinone, demethylmenaquinone, and rhodoquinone), the length of isoprenoid side chain, and the number of saturated isoprenoid units. To date, menaquinones are the only type of respiratory isoprenoid quinones found in actinobacteria, and the variations in the number of isoprene units and hydrogenated double bonds make these membrane constituents of considerable chemotaxonomic value [40].

### **5.1. Extraction and purification of menaquinones**

Menaquinones are free lipids that can be readily extracted from freeze-dried cells. Different methods have been described for the extraction of menaquinones [13, 33, 34, 41]. Menaqui‐ nones are normally extracted with organic solvents or with their mixture such as acetone, chloroform, and hexane (Table 11). However, they are susceptible to strong acid or alkaline, and photo-oxidation in the presence of oxygen and strong light conditions. But it is not necessary to work in a nitrogen atmosphere or dim light [42]. The menaquinones in these extracts are purified by preparative thin-layer chromatography (TLC), and analysis is then performed by HPLC.

1. Approximately 100 of lyophilized cells are extracted with a volume (40 ml) of chloroform–methanol (2:1 v/v) for approximately 1 h or overnight using a magnetic stirrer.

2. The cell/solvent mixture is passed through filter paper to remove cell debris.

3. The eluate is collected in a flask and evaporated to dryness under reduced pressure at 40<sup>ο</sup> C on a rotary evaporator.

**Table 11.** Extraction of menaquinones (from Collins et al. [43, 44]).

The menaquinones can be readily purified from extracts by thin-layer chromatographic procedures using silica gel with hexane–diethylether as the developing solvent [43, 44]. Purified menaquinones are revealed by using UV light at 254 nm. In this section, we describe a new developing solvent to purify the menaquinones (Table 12).

4. Allow plate to dry in a fume cupboard (~5 min), view menaquinones by brief irradiation with ultraviolet light at 254 nm. The menaquinones appear as dark-brown/purple bands on a green fluorescent background, Rf ~0.7.

#### **Table 12.** Purification of menaquinones by thin-layer chromatography

<sup>1.</sup> Dissolve the dried menaquinones in 800 µl of acetone.

<sup>2.</sup> Apply extract (with 200 µl pipette) as a uniform streak (5 cm long) to a silica gel F254 sheet.

<sup>3.</sup> Develop the plate in methylbenzene, developing time ~20 min.

<sup>5.</sup> Scrape gel containing menaquinones from the plate with spatula, dissolve scraped gel in 500 µl of methanol and elute through syringe and 0.45 µm filter membrane.

### **5.2. High-Performance Liquid Chromatography (HPLC) analyzing the menaquinone component**

TLC techniques generate only qualitative data. In contrast, high-performance liquid chroma‐ tography can be used to generate quantitative data. The resolving power and sensitivity of HPLC are also superior to that of thin-layer chromatographic techniques. The purified menaquinones are rapidly analyzed by reverse-phase high-performance liquid chromatogra‐ phy (rpHPLC) [41, 45].

Menaquinones series is analyzed by HPLC with a UV detector, a C18 column, and an online computer integrator. A large number of different mobile phases have been described [44, 45, 46]. Here, we prefer methanol/isopropanol mixtures (65:35, v/v) as the mobile phase to analyze the menaquinones. The column should be maintained at a constant temperature (40<sup>ο</sup> C).

High-performance liquid chromatography (HPLC):

Agilent 1100, HPLC system equipped with an Agilent four-unit pump, a 7125 injector, a G1314A UV detector

Columns: Zorbax Eclipse XDB-C18 (4.6 × 250 mm, 5 µm; Agilent)

Columns temperature: 40<sup>ο</sup> C

UV detect wavelength: 269 nm

Mobile phase A: methanol

Mobile phase B: isopropanol

Elution: A: B = 65:35 (v/v).

Elution flow rate: 1.0 ml min–1

Injection volume: 20 µl.

### **6. Mycolic acid**

Mycolic acids are high molecular-weight long-chain (up to 90 carbon atoms) 2-alkyl 3-hydroxy fatty acids found in representative of *Corynebacterium, Dietzia, Gordona, Myobacterium, Nocardia, Rhodococcus, Turicella*, and *Tsukumurellu* [47–53]. For the extraction and analysis of mycolic acids, different methods have been described based on TLC, GC, or HPLC [54–59].

### **6.1. Extraction of mycolic acids from whole cell**

In this section, we describe the extraction and analysis of mycolic acids based on TLC [55].


### **6.2. Analysis of mycolic acids from whole cell**

ThemycolicacidsofwholecellswereanalyzedaccordingtothedescribedbyMinnikinetal.[55].


The evaluation of the presence of mycolic acid is only advisable, if other results (e.g., coryne‐ form morphology) allocate the isolate to be identified to the group of coryneform bacteria, but the detection of mycolic acids strongly reduces the number of possible relatives. Further identification to the genus level is often possible by additional application of a few of the other described chemotaxonomic methods (quinones, fatty acids, polar lipids, and/or sugars).

### **7. Fatty acids**

**5.2. High-Performance Liquid Chromatography (HPLC) analyzing the menaquinone**

TLC techniques generate only qualitative data. In contrast, high-performance liquid chroma‐ tography can be used to generate quantitative data. The resolving power and sensitivity of HPLC are also superior to that of thin-layer chromatographic techniques. The purified menaquinones are rapidly analyzed by reverse-phase high-performance liquid chromatogra‐

Menaquinones series is analyzed by HPLC with a UV detector, a C18 column, and an online computer integrator. A large number of different mobile phases have been described [44, 45, 46]. Here, we prefer methanol/isopropanol mixtures (65:35, v/v) as the mobile phase to analyze

Agilent 1100, HPLC system equipped with an Agilent four-unit pump, a 7125 injector, a

Mycolic acids are high molecular-weight long-chain (up to 90 carbon atoms) 2-alkyl 3-hydroxy fatty acids found in representative of *Corynebacterium, Dietzia, Gordona, Myobacterium, Nocardia, Rhodococcus, Turicella*, and *Tsukumurellu* [47–53]. For the extraction and analysis of mycolic

In this section, we describe the extraction and analysis of mycolic acids based on TLC [55].

**2.** Add 3 ml mixture solvent of methanol, toluene, and conc. sulfuric acid (30:15:1) into the

acids, different methods have been described based on TLC, GC, or HPLC [54–59].

C).

the menaquinones. The column should be maintained at a constant temperature (40<sup>ο</sup>

High-performance liquid chromatography (HPLC):

C

**6.1. Extraction of mycolic acids from whole cell**

test tube and tightly seal the test tube.

**1.** Add 50–100 mg freeze-drying cells into a clean, dry test tube.

Columns: Zorbax Eclipse XDB-C18 (4.6 × 250 mm, 5 µm; Agilent)

**component**

phy (rpHPLC) [41, 45].

124 Actinobacteria - Basics and Biotechnological Applications

G1314A UV detector

Columns temperature: 40<sup>ο</sup>

Mobile phase A: methanol Mobile phase B: isopropanol

Elution: A: B = 65:35 (v/v).

Injection volume: 20 µl.

**6. Mycolic acid**

Elution flow rate: 1.0 ml min–1

UV detect wavelength: 269 nm

Fatty acid profiles analysis is well introduced for chemotaxonomy of bacteria. Fatty acids most commonly found in the cytoplasmic membrane and lipopolysaccharides of the outer mem‐ brane of Gram-negative bacteria as well as lipoteichoic acids in Gram-positive bacteria are relatively simple in structure and possess between 8 and 20 carbon atoms. The variation of carbon chain length, presence of saturated and unsaturated, occurrence of methyl groups fatty acids (iso-, anteiso-, and methylated within the molecule), occurrence of cyclopropane fatty acid (cyclo 17:0, cyclo 19:0), and occurrence of hydroxyl-fatty acid with an OH-group at position 2 or 3 of the molecule all have a taxonomic utility.

Commonly, different bacteria can have different fatty acids. Some fatty acids have a restricted distribution and may be diagnostic for particular groups. Branched fatty acids of the iso and/ or anteiso type are important constituents of the *Flavobacterium*/*Cytophaga*/*Bacteroides* [60–62]. Cyclohexyl and cycloheptyl fatty acids are characteristic components of some acidothermo‐ philic bacilli [63–65]. Cyclopropane fatty acids are often found in Campylobacter and Lacto‐ bacillus [66–68]. 10-Methyloctadecanoic acid and its homologs distribute in many actinomycetes [46, 69].

As the fatty acid composition of bacteria is dependent on the growth phase, temperature, and growth medium, preparing the biomass for analysis of the fatty acids should be taken to ensure that bacteria are grown under standardized conditions. The extraction of fatty acids can be performed with biomass (approximately 40 mg wet weight harvested from agar plates). For most actinomycetes, the reader can select the trypticase soy agar as the growth medium, but for the actinomycetes from extreme environment, the reader should select the optimum growth medium, as well as the possible media should omit material containing fatty acids, such as Tweens and serum.

### **7.1. Methods for analyzing fatty acids**

Different methods have been described involving acid or base [55, 62, 70–72]. In taxonomic studies, it is important to use a consistent method. Here, we introduce a method for preparation of fatty acid methyl esters from whole wet cell material, which is developed by Sasser [73].

### **7.2. Preparation of reagents**

Four reagents are required to liberate, esterify, and extract the fatty acids from living cells.

#### **Reagent 1 Saponification Reagent**

Sodium hydroxide (Certified ACS) 45 g

Methanol (reagent Grade) 150 ml

Deionized distilled water 150 ml

Add water and methanol to NaOH pellets in bottle. Stir until NaOH pellets have dissolved.

#### **Reagent 2 Methylation Reagent**

12 N hydrochloric acid 195 ml

Methanol (reagent Grade) 275 ml

Deionized distilled water 130 ml

Add acid to water, then to methanol while stirring

### **Reagent 3 Extraction Solvent**

Hexane (HPLC Grade) 200 ml

Methyl-tert-Butyl-ether (HPLC Grade) 200 ml

Add MTBE to hexane and stir

### **Reagent 4 Base Wash**

Sodium hydroxide (Certified ACS) 10.8 g

Deionized distilled water 900 ml

Add water to NaOH pellets in bottle. Stir until NaOH pellets have dissolved.

### **Warning**

Commonly, different bacteria can have different fatty acids. Some fatty acids have a restricted distribution and may be diagnostic for particular groups. Branched fatty acids of the iso and/ or anteiso type are important constituents of the *Flavobacterium*/*Cytophaga*/*Bacteroides* [60–62]. Cyclohexyl and cycloheptyl fatty acids are characteristic components of some acidothermo‐ philic bacilli [63–65]. Cyclopropane fatty acids are often found in Campylobacter and Lacto‐ bacillus [66–68]. 10-Methyloctadecanoic acid and its homologs distribute in many

As the fatty acid composition of bacteria is dependent on the growth phase, temperature, and growth medium, preparing the biomass for analysis of the fatty acids should be taken to ensure that bacteria are grown under standardized conditions. The extraction of fatty acids can be performed with biomass (approximately 40 mg wet weight harvested from agar plates). For most actinomycetes, the reader can select the trypticase soy agar as the growth medium, but for the actinomycetes from extreme environment, the reader should select the optimum growth medium, as well as the possible media should omit material containing fatty acids, such as

Different methods have been described involving acid or base [55, 62, 70–72]. In taxonomic studies, it is important to use a consistent method. Here, we introduce a method for preparation of fatty acid methyl esters from whole wet cell material, which is developed by Sasser [73].

Four reagents are required to liberate, esterify, and extract the fatty acids from living cells.

Add water and methanol to NaOH pellets in bottle. Stir until NaOH pellets have dissolved.

actinomycetes [46, 69].

Tweens and serum.

**7.1. Methods for analyzing fatty acids**

126 Actinobacteria - Basics and Biotechnological Applications

**7.2. Preparation of reagents**

**Reagent 1 Saponification Reagent**

Methanol (reagent Grade) 150 ml Deionized distilled water 150 ml

**Reagent 2 Methylation Reagent** 12 N hydrochloric acid 195 ml

Methanol (reagent Grade) 275 ml Deionized distilled water 130 ml

**Reagent 3 Extraction Solvent** Hexane (HPLC Grade) 200 ml

Add acid to water, then to methanol while stirring

Methyl-tert-Butyl-ether (HPLC Grade) 200 ml

Sodium hydroxide (Certified ACS) 45 g

Reagent 1 and 2 are caustic, wear safety glasses and gloves.

Methyl-tert-Butyl-ether is extremely flammable. Extinguish all flames and heat sources before use.

Handle in a chemical fume hood.

### **7.3. Extraction of fatty acids.**

Five steps involved in extraction of fatty acids from biomass [73] (see Figure 6):


**Figure 6.** Five steps involved in extraction of fatty acids

#### **Warning:**

In the methylation step, excess time or excess temperature of the water bath can degrade some fatty acids.

### **7.4. Identification of fatty acids**

For identification of the fatty acid profiles at the species level, integrated system, including a gas chromatography apparatus with identification software (the Sherlock Microbial Identifi‐ cation System) are required.

Gas chromatographic conditions

Gas chromatographic: Agilent 7890

Agilent 7890, column, flame ionization detector (FID)

Columns:

**Figure 6.** Five steps involved in extraction of fatty acids

128 Actinobacteria - Basics and Biotechnological Applications

**7.4. Identification of fatty acids**

cation System) are required.

Gas chromatographic conditions Gas chromatographic: Agilent 7890

Agilent 7890, column, flame ionization detector (FID)

In the methylation step, excess time or excess temperature of the water bath can degrade some

For identification of the fatty acid profiles at the species level, integrated system, including a gas chromatography apparatus with identification software (the Sherlock Microbial Identifi‐

**Warning:**

fatty acids.

Agilent 19091B-102, 25 × 200 m × 0.33 µm Oven temperature 170<sup>ο</sup> C Injector 250<sup>ο</sup> C Detector 300<sup>ο</sup> C Carries gas (hydrogen) 1 ml/min Inlet pressure 9.000 psi Electrometer setting 4 × 1012amps Splitting 1:20 0.4bar Hydrogen (for flame) 30 ml/min Synthetic air (for flame) 400 ml/min Septum purge 10 ml/min Auxiliary gas (N2) 30 ml/min Sample 2 µl

### **8. Gas chromatography of fatty acids**

In general, >5% of fatty acids as "major fatty acid" should be recorded. Fatty acid component of strain YIM 47672 analyzed with gas chromatography, as an example, is showed in Table 13 and Figure 7.




**RT Response Ar/Ht RFact ECL Peak Name Percent Comment1 Comment2**

ECL deviates 0.001

ECL deviates 0.000

ECL deviates -0.009

ECL deviates -0.004

ECL deviates -0.001

ECL deviates 0.008

ECL deviates 0.000

ECL deviates 0.000

ECL deviates -0.002

0.000

ECL deviates -0.001

ECL deviates 0.002

Reference -0.002

Reference -0.002

Reference -0.005

13:0 3OH/15:1 i H

Reference -0.001

Reference -0.001

Reference 0.001

3.134 837 0.044 ---- 9.783 ---- 3.285 453 0.025 ---- 10.053 ---- 4.676 795 0.032 ---- 11.754 ----

130 Actinobacteria - Basics and Biotechnological Applications

4.910 221 0.024 1.051 12.001 12:0 0.03

5.135 567 0.035 ---- 12.192 ----

6.953 881 0.042 0.994 13.619 14:0 iso 0.13

7.334 1399 0.044 0.986 13.892 14:1 w5c 0.21

7.479 6652 0.047 0.984 13.996 14:0 0.98

7.555 4193 0.055 ---- 14.045 ---- 7.674 3021 0.051 ---- 14.121 ---- 7.768 1640 0.038 ---- 14.181 ---- 7.868 1764 0.054 ---- 14.245 ---- 7.976 1064 0.040 ---- 14.314 ---- 8.049 2496 0.059 ---- 14.361 ---- 8.156 2643 0.043 ---- 14.429 ----

8.231 1716 0.044 0.972 14.477 Sum In Feature 1 0.25

8.322 1111 0.044 0.971 14.535 15:1 anteiso A 0.16

8.459 7432 0.037 0.969 14.623 15:0 iso 1.08

8.600 5278 0.037 0.967 14.713 15:0 anteiso 0.77

8.821 533 0.039 0.964 14.854 15:1 w6c 0.08

9.824 3366 0.043 0.952 15.460 16:1 iso H 0.48

**10.109 216646 0.041 0.949 15.629 16:0 iso 30.90**

9.049 <sup>39473</sup> 0.041 0.961 15.000 15:0 ---- ECL deviates


Volume: DATA; File: E068264.61A; Samp Ctr: 5; ID Number: 1107; Type: Samp; Bottle: 3

Method: TSBA6; Created: 5/26/2015 12:39:52 PM; Sample ID: **YIM 47672**

ECL Deviation: 0.003; Reference ECL Shift: 0.002; Number Reference Peaks: 13

Total Response: 756936; Total Named: 708097; Percent Named: 93.55%; Total Amount: 703700; \*\*\* No Matches found in TSBA6

**Table 13.** Fatty acid component of strain YIM 47672 with gas chromatography

**Figure 7.** Gas chromatography of fatty acid of strain YIM 47672

### **Acknowledgements**

**RT Response Ar/Ht RFact ECL Peak Name Percent Comment1 Comment2**


ECL deviates -0.002

ECL deviates 0.001

ECL deviates

ECL deviates 0.000

ECL deviates 0.000

15:1 iso H/13:0 3OH

18:2 w6,9c/18:0 ante

19:1 w11c/19:1 w9c

un 18.846/19:1

19w6

Reference -0.004

19:1 w11c/19:1 w9c

Reference -0.003

13:0 3OH/15:1 i H

18:0 ante/18:2 w6,9c

19:1 w9c/19:1 w11c

w6c 19:1 w6c/.846/19cy

0.004 19:0 cyclo w10c/19w6

14.248 8805 0.046 0.924 17.998 18:0 1.22

132 Actinobacteria - Basics and Biotechnological Applications

14.601 1763 0.079 ---- 18.199 ---- 14.742 1095 0.043 ---- 18.279 ---- 15.507 2979 0.061 ---- 18.714 ----

15.583 1055 0.047 0.920 18.757 Sum In Feature 6 0.15

15.784 3100 0.047 0.919 18.871 Sum In Feature 7 0.43

16.010 573 0.038 0.919 19.000 19:0 0.08

17.223 6351 0.051 ---- 19.699 ----

17.347 440 0.030 0.916 19.770 20:1 w9c 0.06


**----** 69473 --- ---- ---- Summed Feature 5 9.66




Volume: DATA; File: E068264.61A; Samp Ctr: 5; ID Number: 1107; Type: Samp; Bottle: 3

Method: TSBA6; Created: 5/26/2015 12:39:52 PM; Sample ID: **YIM 47672** ECL Deviation: 0.003; Reference ECL Shift: 0.002; Number Reference Peaks: 13

**Table 13.** Fatty acid component of strain YIM 47672 with gas chromatography

TSBA6


Total Response: 756936; Total Named: 708097; Percent Named: 93.55%; Total Amount: 703700; \*\*\* No Matches found in

This work was supported by grants from the Ministry of Environmental Protection of China (National Key Sciences and Technology Program forWater Solutions, grant 2012ZX07102-003), the National Natural Science Foundation of China (NSFC) (30860013, 31200138, 31160123, 31270001 and 31460005), Yunnan Provincial Society Development Project (2014BC006). We are grateful to Mr. Xiao-Long Cui and Wei Xiao for their help during writing.

### **Author details**

Yongxia Wang and Yi Jiang

\*Address all correspondence to: jiangyi@ynu.edu.cn

Yunnan Institute of Microbiology, School of Life Science, Yunnan University, Kunming, Yunnan, P. R. China

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