Dye 50

Biosurfactants were dissolved in Muller Hinton broth (MHB) at twice the concentration of the final test, with pH adjusted to 7. 100 µl of the biosurfactant/MHB broth was dispensed in each well of Column 1, while Columns 2-10 contained 50 µl of MHB broth only. Column 11 contained 100 µl of diluted standardised inoculum, and Column 12 contained 100 µl of the medium broth (as a control to monitor sterility), as shown in processed plate Fig. 2. A multichannel pipette was then used to transfer and mix biosurfactants from column 1–10, resulting in 50 µl biosurfactant per well. The tested concentrations of the different biosursurfants achieved through double serial dilutions from columns 10–1 were as follows; 25–0.05 mg ml⁻¹ rhamnolipids (JBR325), 50–0.01 mg ml⁻¹ rhamnolipids from Burkholderia thailandensis E264, 12.5–0.025 mg ml⁻¹ of lactonic sophorolipids, 100–0.02 mg ml⁻¹ of acidic sophorolipids and 1–0.002 µg ml⁻¹ of polymyxin. The standardised microorganism suspension was then diluted by 1:100 in MHB broth. 50 µl of the adjusted OD₆₀₀ bacterial suspension was then added to all wells containing biosurfactant and to the control wells, resulting in approx. 5 x10⁵ CFU ml⁻¹. The time taken to prepare and dispense the OD adjusted bacteria did not exceed 15 min. After incubation for 24 h at 37 °C, resazurin (0.015 %) was added to all wells (30 µl per well), and further incubated for 2–4 h for the observation of colour change. On completion of the incubation, columns with no colour change (blue resazurin colour remained unchanged) were scored as above the MIC value. The minimum biocidal concentration (MBC) was determined by plating directly the content of wells with concentrations higher than the MIC value, as detailed in Table 1. The MBC value was determined when there was no colony growth from the directly plated contents of the wells. In addition the contents of the wells showing indications of growth inhibition were serially diluted to quantify an end-point killing of the bacteria as detailed in the results section.Fig. 2

Determination of MIC for Rhamnolipid JBR325 against Streptococcus mutans (DSM-20523). After the period of incubation, resazurin dye was added. Column 12 confirms no contamination occurred while preparing the plate. Column 11, a negative control shows a change of resazurin natural colour (blue/purple) to the reduced form (red-colourless). The highest concentration incorporated into the plate is 25 mg ml⁻¹ and the lowest achieved through double serial dilution is 0.05 mg ml⁻¹. Column 7 shows no colour changes therefore concentration of biosurfactant in that column was taken as the MIC value. The range of biosurfactant concentration in the wells was 25–0.05 mg ml⁻¹

Table 1

Determination of the MIC by Resazurin aided microdilution method of two antibiotics against two standard strains

Bacteria	Antibiotic	MIC reported in this study (µg ml−1)	MIC recommended by CLSI (µg ml−1)
Escherichia coli ATCC 25922	Tetracycline	2	0.5–2
Staphylococcus aureus ATCC 29213	Tetracycline	0.5	0.12–1
Escherichia coli ATCC 25922	Gentamicin	1	0.25–1
Staphylococcus aureus ATCC 29213	Gentamicin	0.5	0.12–1

Values obtained were compared with those recommended by the CLSI

Existing data can only yield limited new insights into the effectiveness of a DNA-based identification system for birds. Two mitochondrial genes, cyt b and COI, are rivals for the largest number of animal sequence records greater than 600 bp in GenBank (4,791 and 3,009 species, respectively). However, COI coverage for birds is modest; 173 species share COI sequences with 600-bp overlap. As these records derive from a global avifauna of 10,000 species, they provide a limited basis to evaluate the utility of a COI-based identification system for any continental fauna, impelling us to gather new sequences.
We employed a stratified sampling design to gain an overview of the patterns of COI sequence divergence among North American birds. The initial level of sampling examined a single individual from each of 260 species to ascertain COI divergences among species. These species were selected on the basis of accessibility without regard to known taxonomic issues. The second level of sampling examined one to three additional individuals from 130 of these species to provide a general sense of intraspecific sequence divergences, as well as a preliminary indication of variation in each species. When possible, these individuals were obtained from widely separated localities in North America. The third level of our analysis involved sequencing four to eight more individuals for the few species where the second level detected more than 2% sequence divergence among individuals. Our studies examined specimens collected over the last 20 years; 98% were obtained from the tissue bank at the Royal Ontario Museum, Toronto, Canada. Collection localities and other specimen information are available in the “Birds of North America” file in the Completed Projects section of the Barcode of Life website (http://www.barcodinglife.com). Taxonomic assignments follow the latest North American checklist (AOU 1998 ) and its recent supplements (Banks et al. 2000 , 2002 , 2003 ).
Mitochondrial pseudogenes can complicate PCR-based studies of mitochondrial gene diversity (Bensasson et al. 2001 (link); Thalmann et al. 2004 (link)). We used protocols to reduce pseudogene impacts that included extracting DNA from tissues rich in mitochondria (Sorenson and Quinn 1998 ), employing primers with high universality (Sorenson and Quinn 1998 ), and amplifying a relatively long PCR product because most pseudogenes are short (Pereira and Baker 2004 (link)). DNA extracts were prepared from small samples of muscle using the GeneElute DNA miniprep Kit (Sigma, St. Louis, Missouri, United States), following the manufacturer's protocols. DNA extracts were resuspended in 10 μl of H₂O, and a 749-bp region near the 5′ terminus of the COI gene was amplified using primers (BirdF1-TTCTCCAACCACAAAGACATTGGCAC and BirdR1-ACGTGGGAGATAATTCCAAATCCTG). In cases where this primer pair failed, an alternate reverse primer (BirdR2-ACTACATGTGAGATGATTCCGAATCCAG) was generally combined with BirdF1 to generate a 751-bp product, but a third reverse primer (BirdR3-AGGAGTTTGCTAGTACGATGCC) was used for two species of Falco. The 50-μl PCR reaction mixes included 40 μl of ultrapure water, 1.0 U of Taq polymerase, 2.5 μl of MgCl₂, 4.5 μl of 10× PCR buffer, 0.5 μl of each primer (0.1 mM), 0.25 μl of each dNTP (0.05 mM), and 0.5–3.0 μl of DNA. The amplification regime consisted of 1 min at 94 °C followed by 5 cycles of 1 min at 94 °C, 1.5 min at 45 °C, and 1.5 min at 72 °C, followed in turn by 30 cycles of 1 min at 4 °C, 1.5 min at 51 °C, and 1.5 min at 72 °C, and a final 5 min at 72 °C. PCR products were visualized in a 1.2% agarose gel. All PCR reactions that generated a single, circa 750-bp, product were then cycle sequenced, while gel purification was used to recover the target gene product in cases where more than one band was present. Sequencing reactions, carried out using Big Dye v3.1 and the BirdF1 primer, were analyzed on an ABI 377 sequencer. The electropherogram and sequence for each specimen are in the “Birds of North America” file, but all sequences have also been deposited in GenBank (see Supporting Information). COI sequences were recovered from all 260 bird species and did not contain insertions, deletions, nonsense, or stop codons, supporting the absence of nuclear pseudogene amplification (Pereira and Baker 2004 (link)). In addition to 429 newly collected sequences, nine GenBank sequences from five species were included (these were the only full-length COI sequences corresponding to species in this study).
Sequence divergences were calculated using the K2P distance model (Kimura 1980 (link)). A NJ tree of K2P distances was created to provide a graphic representation of the patterning of divergences among species (Saitou and Nei 1987 (link)).

Nylon sheet of 20% by mass based on fiber waste weight was employed as adsorbent material of anionic dye, Congo red (CR). Congo red dye solution have been prepared with concentration ranging from 100 to 700 mg/L. Then 10 ml of the different dye solutions is added to a predetermined weight of NS in a 50 ml glass flask. Then 50 rpm automatic shaker was used for variant times. The final concentration after adsorption was assessed using UV-Lambda 35 Perkin Elmer at λ_max = 495 nm.
The adsorption capacity q (mg g⁻¹) for Congo red dye can be evaluated using the following Eq. (3)³⁵ . $$q_e=\left(C_0,-,C_e\right)\times V/m$$ where C₀ and C_e in (mg/L), are the initial and equilibrium dye concentration, respectively, V in (L) is the volume of CR dye and m in (g) is the weight of NS adsorbent.
The removal efficiency for certain dye concentration at equilibrium can be explored by the next Eq. (4)^{36 (link)}. $$\%R=\frac{\left(C_0,-,C_e\right)}{C_0}\times 100$$
Effects of pH and regeneration studies on adsorption capacity of NS for Congo red were studied.

The affecting factors of dose (5–30 g/L), contact time (3–18 h), temperature (20–50 °C), pH (3–11), and concentration (30–90 mg/L) with the control condition of initial DR28 dye concentration of 50 mg/L, a sample volume of 100 mL, and a shaking speed of 150 rpm by using an incubator shaker (New Brunswick, Innova 42, USA)^{8 (link),9 (link),20 (link)} on DR28 dye removal efficiencies of SBB, SBBT, SBBM, SBBA, and SBBZ were investigated through a series of batch experiments which referred from the previous study of Praipipat et al.^{18 (link)} Their optimum conditions were chosen from the lowest dose or contact time or temperature or pH or concentration with obtaining the highest DR28 dye removal efficiencies^{9 (link)}. UV–VIS Spectrophotometer (UH5300, Hitachi, Japan) with a wavelength of 497 nm was used for analyzing dye concentrations, and the triplicate experiments were investigated to verify the results and report the average value. Dye removal efficiency in the percentage and dye adsorption capacity is calculated following Eqs. (1)–(2): $$\text{Dye removal efficiency}\left(\%\right)=\left(\left(C_0-C_{\text{e}}\right)/C_0\right)\times 100$$ $$\text{Dye adsorption capacity}\left(q_e\right)=(C_0-C_{\text{e}})V/m$$ where C_e is the dye concentration at equilibrium (mg/L), C₀ is the initial dye concentration (mg/L), q_e is the capacity of dye adsorption on adsorbent material at equilibrium (mg/g), V is the sample volume (L), and m is the amount of adsorbent material (g).