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Applied Microbiology and Biotechnology (v.79, #5)

CO₂ bio-mitigation using microalgae by Bei Wang; Yanqun Li; Nan Wu; Christopher Q. Lan (pp. 707-718).

Microalgae are a group of unicellular or simple multicellular photosynthetic microorganisms that can fix CO₂ efficiently from different sources, including the atmosphere, industrial exhaust gases, and soluble carbonate salts. Combination of CO₂ fixation, biofuel production, and wastewater treatment may provide a very promising alternative to current CO₂ mitigation strategies.

Keywords: Microalga; CO₂ mitigation; Biofuel; Biodiesel; Biomass conversion

Biomass and lutein productivity of Scenedesmus almeriensis: influence of irradiance, dilution rate and temperature by J. F. Sánchez; J. M. Fernández-Sevilla; F. G. Acién; M. C. Cerón; J. Pérez-Parra; E. Molina-Grima (pp. 719-729).

In this paper, the biomass and lutein productivity of the lutein-rich new strain Scenedesmus almeriensis is modelled versus irradiance and temperature. The results demonstrate that S. almeriensis is a mesophile microorganism with an optimal growth temperature of 35°C, and capable of withstanding up to 48°C, which caused culture death. This strain is also tolerant to high irradiances, showing no signs of photoinhibition even at the maximum irradiance essayed of 1625 μE m⁻² s⁻¹ accumulating up to 0.55% dry weight (d.wt.) of lutein. The optimal conditions that maximise the biomass productivity also favour the lutein productivity, lutein being a primary metabolite. Maximal biomass and lutein productivities of 0.87 g l⁻¹ day⁻¹ and 4.77 mg l⁻¹ day⁻¹, respectively, were measured. The analysis of light availability inside the cultures, quantified as average irradiance, demonstrates that the cultures were mainly photo-limited, although photosaturation also took place at high external irradiances. The effect of temperature was also investigated finding that the specific maximal growth rate is modified by the temperature according to the Arrhenius equation. The influence of both light availability and temperature was included in an overall growth model, which showed, as a result, capable of fitting the whole set of experimental data. An overall lutein accumulation rate model was also proposed and used in a regression analysis. Simulations performed using the proposed models show that under outdoor conditions a biomass productivity of 0.95 g l⁻¹ day⁻¹ can be expected, with a lutein productivity up to 5.31 mg l⁻¹ day⁻¹. These models may be useful to assist the design and operation optimisation of outdoor cultures of this strain.

Keywords: Scenedesmus almeriensis ; Lutein; Biomass productivity; Continuous culture; Irradiance; Temperature

Molecular cloning and heterologous expression of a laccase gene from Pleurotus eryngii in free and immobilized Saccharomyces cerevisiae cells by Gianluca Bleve; Chiara Lezzi; Giovanni Mita; Patrizia Rampino; Carla Perrotta; Luciano Villanova; Francesco Grieco (pp. 731-741).

A full length cDNA encoding an extracellular laccase was isolated by reverse transcription polymerase chain reaction from the mycelia of the mushroom Pleurotus eryngii. The isolated sequence, denoted Ery3, encodes for a mature laccase isoenzyme of 531 amino acid residues with a predicted molecular weight of 56.6 kDa. All sequence motifs, being the signature sequences used to identify the laccases, were found in the Ery3 protein sequence. The Ery3 cDNA was expressed in Saccharomyces cerevisiae and the effects of copper concentration and cultivation temperature were investigated. S. cerevisiae cells were immobilized in calcium alginate gel and the optimal immobilization parameters for the enhanced production of laccase were determined. The immobilization was most effective with 3% sodium alginate, 0.1 M calcium chloride and an initial biomass of 4.5 × 10⁸ cells. The enzyme yield obtained with immobilized cells (139 mU ml⁻¹) showed a 1.6-fold increase compared to the highest yield obtained with free cells. The alginate beads showed good stability and retained 84% capacity of enzyme production after seven repeated cycles of batch fermentation. The immobilization system proved to increase the proteolytic stability of the recombinant Ery3 protein. To our knowledge, this is the first report on S. cerevisiae whole-cell immobilization for recombinant laccase production.

Keywords: Pleurotus eryngii ; Laccase; Recombinant protein in yeast; Ca-alginate immobilization

Identification and characterization of novel poly(dl-lactic acid) depolymerases from metagenome by Daisuke Mayumi; Yukie Akutsu-Shigeno; Hiroo Uchiyama; Nobuhiko Nomura; Toshiaki Nakajima-Kambe (pp. 743-750).

Many poly(lactic acid) (PLA)-degrading microorganisms have been isolated from the natural environment by culture-based methods, but there is no study about unculturable PLA-degrading microorganisms. In this study, we constructed a metagenomic library consisting of the DNA extracted from PLA disks buried in compost. We identified three PLA-degrading genes encoding lipase or hydrolase. The purified enzymes degraded not only PLA, but also various aliphatic polyesters, tributyrin, and p-nitrophenyl esters. From their substrate specificities, the PLA depolymerases were classified into an esterase rather than a lipase. Among the PLA depolymerases, PlaM4 exhibited thermophilic properties; that is, it showed the highest activity at 70 °C and was stable even after incubation for 1 h at 50 °C. PlaM4 had absorption and degradation activities for solid PLA at 60 °C, which indicates that the enzyme can effectively degrade PLA in a high-temperature environment. On the other hand, the enzyme classification based on amino acid sequences showed that the other PLA depolymerases, PlaM7 and PlaM9, were not classified into known lipases or esterases. This is the first report on the identification and characterization of PLA depolymerase from a metagenome.

Keywords: Metagenome; Depolymerase; Esterase; Biodegradable plastic; Polylactide; Lipase

Assembly of mutations for improving thermostability of Escherichia coli AppA2 phytase by Moon-Soo Kim; Jeremy D. Weaver; Xin Gen Lei (pp. 751-758).

We previously identified a number of mutations in Escherichia coli AppA2 phytase for enhancing its thermostability. The objective of the present study was to determine if these mutations (K46E, K65E, G103S, D112N, D144N, S209G, V227A, and G344D) could be sequentially added to further improve the thermostability of AppA2. Compared with the wild-type enzyme, two variants (D144N/V227A and D144N/V227A/G344D) out of the eight resulting mutants showed 15% enhancement in thermostability (as measured by residual activity after being heated at 80°C for 10 min) and 4 to 5°C increases in the melting temperatures (T _m). Based on the structural predictions with a highly homologous AppA phytase, the substitution D144N introduces a side-chain–side-chain hydrogen bond, thereby stabilizing the loop region (Gln137–Asn144), and the V227A substitution might eliminate structural hindrance between Val222 and Val227 that face each other in the β-hairpin structure. In addition, overall catalytic efficiency (k _cat/K _m) of the two mutants was also improved (P < 0.05) compared to the wild type. However, no further improvement in thermostability was observed by adding other mutations to D144N/V227A/G344D, which might result from unfavorable electrostatic interactions or structural perturbation. In conclusion, our results underscore the potential as well as difficulty of predicting synergistic effects of multiple mutations on thermostability within phytase.

Keywords: Enzyme; Phytase; Protein engineering; Structure; Thermostability

Purification and characterization of plantaricin LR14: a novel bacteriocin produced by Lactobacillus plantarum LR/14 by Santosh Kumar Tiwari; Sheela Srivastava (pp. 759-767).

Bacteriocin produced by Lactobacillus plantarum strain LR/14 was purified to homogeneity by a multi-step protocol consisting of ammonium sulfate precipitation, cation-exchange chromatography, gel-filtration, and reverse-phase FPLC. L. plantarum LR/14 secreted a low-molecular-weight bacteriocin consisting of two peptides designated as plantaricin LR14α and -β with molecular mass of 3,012.46 and 5,605.74 Da, respectively. The purified peptides were characterized to be highly thermostable and active in acidic pH range, with a pI of >10.0. Both α and β peptides showed bactericidal mode of action against indicator strain, Micrococcus luteus and together showed a synergistic action. These peptides were differentially sensitive to a range of proteolytic enzymes, indicating differences in their composition. Amino acid sequencing revealed that the N-terminus in both the cases is blocked; thus, only a partial sequence could be obtained after CNBr digestion. These sequences, when compared with those available in the database, showed no homology with known bacteriocins, indicating it to be a novel compound.

Keywords: Bacteriocin; Purification; Cation-exchange chromatography; RP-FPLC; Characterization; Mode of action

Induction, purification and characterization of α-N-acetylgalactosaminidase from Aspergillus Niger by L. Weignerová; T. Filipi; D. Manglová; V. Křen (pp. 769-774).

A set of filamentous fungi (42 strains) was screened for α-N-acetylgalactosaminidase activity, and a series of inducers and different cultivation conditions were tested. Enzyme production by the best producer Aspergillus niger CCIM K2 was optimized and scaled up. α-N-Acetylgalactosaminidase was purified to apparent homogeneity by cation exchange chromatography, gel filtration, and chromatofocusing, and basic biochemical data of the enzyme were determined: The native molecular weight was estimated by gel filtration to be approximately 440 kDa, the molecular weight of the subunit was determined to be 76 kDa and the pI = 4.8. The K _M was 0.73 mmol/l for o-nitrophenyl 2-acetamido-2-deoxy-α-D-galactopyranoside (o-NP-α-GalNAc), and optimum enzyme activity was achieved at pH 1.8 and 55 °C. This α-N-acetylgalactosaminidase is a retaining-type glycosidase, and it was N-deglycosylated without any loss of activity.

Keywords: α-N-Acetylgalactosaminidase; α-GalNAc; Aspergillus niger ; Glycosidase library

Two groups of thermophilic amino acid aminotransferases exhibiting broad substrate specificities for the synthesis of phenylglycine derivatives by Daisuke Koma; Toshiya Sawai; Ryotaro Hara; Shigeaki Harayama; Kuniki Kino (pp. 775-784).

Thirty two thermophilic amino acid aminotransferases (AATs) were expressed in Escherichia coli as soluble and active proteins. Based on their primary structures, the 32 AATs were divided into four phylogenetic groups (classes I, II, IV, and V). The substrate specificities of these AATs were examined, and 12 AATs were found capable of synthesizing ring-substituted phenylglycine derivatives such as hydroxyl-, methoxy-, and fluorophenylglycines. Eleven out of the 12 AATs were enzymes belonging to two phylogenetic groups namely, one subgroup of the class I family and the class IV family. AATs in these two groups may thus be useful for the synthesis of a variety of ring-substituted phenylglycine derivatives.

Keywords: Aminotransferase; Thermophile; Phenylglycine; Unnatural amino acid; Nonproteinogenic amino acid

Production of novel angiotensin I-converting enzyme inhibitory peptides by fermentation of marine shrimp Acetes chinensis with Lactobacillus fermentum SM 605 by Yu-Kai Wang; Hai-Lun He; Xiu-Lan Chen; Cai-Yun Sun; Yu-Zhong Zhang; Bai-Cheng Zhou (pp. 785-791).

Acetes chinensis is an underutilized shrimp species thriving in Bo Hai Gulf of China. Its hydrolysate digested with protease SM98011 has been previously shown to have high angiotensin I-converting enzyme (ACE) inhibitory activity (He et al., J Pept Sci 12:726–733, 2006). In this article, A. chinensis were fermented by Lactobacillus fermentum SM 605 and the fermented sauce presented high ACE inhibitory activity. The minimum IC₅₀ value (3.37 ± 0.04 mg/mL) was achieved by response surface methodology with optimized process parameters such as fermentation time of 24.19 h, incubation temperature at 38.10°C, and pH 6.12. Three ACE inhibitory peptides are purified by ultrafiltration, gel filtration, and reverse-phase high performance liquid chromatography. Identified by mass spectrometry, their amino acid sequences are Asp-Pro, Gly-Thr-Gly, and Ser-Thr, with IC₅₀ values of 2.15 ± 0.02, 5.54 ± 0.09, and 4.03 ± 0.10 μM, respectively. Also, they are all novel ACE inhibitory peptides. Compared with protease digestion, fermentation is a simpler and cheaper method to produce ACE inhibitory peptides from shrimp A. chinensis.

Keywords: Angiotensin I-converting enzyme (ACE); ACE inhibitory peptides; Antihypertensive; Acetes chinensis ; Lactobacillus fermentum ; Fermentation

Conversion of the lycopene monocyclase of Myxococcus xanthus into a bicyclase by Antonio A. Iniesta; María Cervantes; Francisco J. Murillo (pp. 793-802).

Depending on the cyclized hydrocarbon backbone ends, carotenoids can be acyclic, monocyclic, or bicyclic. Lycopene cyclases are the enzymes responsible for catalyzing the formation of cyclic carotenoids from acyclic lycopene. Myxococcus xanthus is a bacterium that accumulates monocyclic carotenoids such as a glycoside ester of myxobacton. We show here that this bacterium possesses a cyclase belonging to the group of the heterodimeric cyclases CrtYc and CrtYd. These two individual proteins are encoded by crtYc and crtYd, which are located in the carotenogenic carA operon of the carB–carA gene cluster, and the presence of both is essential for the cyclization of lycopene. CrtYc and CrtYd from M. xanthus form a heterodimeric cyclase with β-monocyclic activity, which converts lycopene into monocyclic γ-carotene, but not into bicyclic β-carotene like most β-cyclases. This is an unusual case where two different proteins constitute a lycopene cyclase enzyme with monocyclic activity. We were able to convert this lycopene monocyclase into a lycopene bicyclase enzyme producing β-carotene, by fusing both proteins with an extra transmembrane domain. The chimeric protein appears to allow a proper membranal disposition of both CrtYc and CrtYd, to perform two cyclization reactions, while a hybrid without the extra transmembrane helix performs only one cyclization.

Keywords: Carotenes; Cyclization; Beta-carotene; Gamma-carotene; Heterodimeric

A new and efficient phosphate starvation inducible expression system for Lactococcus lactis by Noora Sirén; Kalle Salonen; Matti Leisola; Antti Nyyssölä (pp. 803-810).

A new expression system for Lactococcus lactis was developed. The system is based on a phosphate starvation inducible pstF promoter of L. lactis MG1363. Intracellular β-galactosidase and secreted α-amylase were produced using this tightly regulated system. No evidence of regulatory sites in regions of the 5′-end of the pstF coding sequence was found. High expression levels of the β-galactosidase gene were obtained using the original pstF RBS in a phosphate-depleted medium. The results suggested that with the phosphate starvation inducible system, it is possible to achieve expression levels comparable to the ones obtained with the widely used nisin-controlled gene expression system (NICE). A specific β-galactosidase activity of 670 μkat g⁻¹ using a phosphate-depleted medium and an α-amylase activity of 3.6 μkat l⁻¹ in a bioreactor cultivation were produced. The advantages of the current expression system include that no prior removal of phosphate from the medium in bioreactor scale is required, and no additions of inducing agents are needed. Furthermore, the system can be operated in L. lactis without introduction of regulatory genes into the host.

Keywords: Lactococcus lactis ; Gene expression; Promoter; Phosphate starvation; α-Amylase; β-Galactosidase

Composition of nifH in a wastewater treatment system reliant on N₂ fixation by T. H. Bowers; N. M. Reid; G. Lloyd-Jones (pp. 811-818).

High levels of nitrogen fixation have been observed in the wastewaters of pulp and paper mills. In this study, we show that nitrogen fixation in a model pulp and paper wastewater treatment system is supported by a high density of nifH sequences that are of low diversity. Quantitative PCR revealed a ratio of nifH to 16S rDNA of 1.14 ± 0.76 which shows that very high levels of the nifH gene were enriched to support the high rates of nitrogen fixation that occur in this wastewater. Changes in wastewater composition and dissolved oxygen levels did not affect the nifH levels and allowed stable wastewater treatment. The nifH sequences identified display a similar profile to those seen in forest soil environments where nifH sequences derived from α-proteobacteria and β-proteobacteria are also prevalent.

Keywords: Nitrogen fixation; Biorefinery; Wastewater; nifH; qPCR; DNA hybridisation

Isolation and characterization of a stress-inducible Dunaliella salina Lcy-β gene encoding a functional lycopene β-cyclase by Ana Ramos; Sacha Coesel; Ana Marques; Marta Rodrigues; Alexandra Baumgartner; João Noronha; Amélia Rauter; Bertram Brenig; João Varela (pp. 819-828).

The halotolerant green alga Dunaliella salina accumulates large amounts of β-carotene when exposed to various stress conditions. Although several studies concerning accumulation and biotechnological production of β-carotene have been published, the molecular basis and regulation of the genes involved in carotenoid biosynthesis in D. salina are still poorly known. In this paper, we report the isolation and regulation of the lycopene β-cyclase (Lcy-β) gene by abiotic stress. The function of this gene was determined by heterologous genetic complementation in E. coli. Gene expression and physiological analyses revealed that D. salina Lcy-β steady-state transcript and carotenoid levels were up-regulated in response to all stress conditions tested (salt, light and nutrient depletion). The results presented here suggest that nutrient availability is a key factor influencing carotenogenesis as well as carotenoid biosynthesis-related gene expression in D. salina.

Keywords: Carotenoid biosynthesis; Dunaliella salina ; Lycopene β-cyclase; Gene expression; Stress response regulation

Red mold rice extract represses amyloid beta peptide-induced neurotoxicity via potent synergism of anti-inflammatory and antioxidative effect by Chun-Lin Lee; Jyh-Jye Wang; Tzu-Ming Pan (pp. 829-841).

Amyloid β-peptide (Aβ), a risk of Alzheimer's disease (AD), causes cell death by inflammation and oxidative stress. Red mold rice (RMR) fermented by Monascus species is regarded as cholesterol-lowering functional food in virtue of the metabolite monacolin K identified as lovastatin. In addition, RMR is also demonstrated to express antioxidation because of multiple antioxidants. Therefore, this study focuses on the synergism of RMR against Aβ neurotoxicity and compares the effect between lovastatin and RMR including monacolin K and other functional metabolites. In this study, RE 568, an ethanol extract of RMR produced by strain Monascus purpureus NTU 568, is used to protect PC12 cell against Aβ40 neurotoxicity. All tests contain the treatments with lovastatin or RE 568 including equal monacolin K levels in order to compare the effect and investigate whether other metabolites of RE 568 provide potent assistance against Aβ40 neurotoxicity. In the results, monacolin K represses Aβ40 neurotoxicity via repressing small G-protein-mediated inflammation, and other metabolites of RE 568 also exhibit potent antioxidative ability against Aβ-induced oxidative stress. Importantly, stronger effects on repressing the Aβ40-induced cell death, inflammation, and oxidative stress are performed by RE 568 than that by the equal levels of lovastatin, which results from a potent synergism made up of monacolin K, antioxidants, and anti-inflammatory agents. The present study is the first report to demonstrate the potent synergistic protection of RMR against Aβ40 neurotoxicity, which would cause RMR to be developed as potential and novel functional food for the prophylaxis of AD pathogenesis.

Keywords: Alzheimer's disease; Amyloid; Monascus ; Monacolin K; Anti-inflammatory; Antioxidative

Potassium and sodium transporters of Pseudomonas aeruginosa regulate virulence to barley by Akihiro Ueda; Thomas K. Wood (pp. 843-858).

We investigated the role of three uncharacterized cation transporters of Pseudomonas aeruginosa PAO1 as virulence factors for barley: PA1207, PA5021, and PA2647. PAO1 displayed reduced barley virulence with inactivated PA1207, PA5021, and PA2647 as well as with one known Na⁺/H⁺ antiporter, PA1820. Using the Escherichia coli LB2003 mutant lacking three K⁺ uptake systems, the expression of the PA5021 gene repressed LB2003 growth with low K⁺, but the strain acquired tolerance to high K⁺. In contrast, the expression of the PA1207 gene enhanced growth of LB2003 with low K⁺ but repressed its growth with high K⁺; therefore, the PA5021 protein exports K⁺, while the PA1207 protein imports K⁺. The PA5021 mutant of P. aeruginosa also showed impaired growth at 400 mM KCl and at 400 mM NaCl; therefore, the PA5021 protein may also export Na⁺. The loss of the PA5021 protein also decreased production of the virulence factor pyocyanin; corroborating this result, pyocyanin production decreased in wild-type PAO1 under high salinity. Whole-genome transcriptome analysis showed that PAO1 induced more genes in barley upon infection compared to the PA5021 mutant. Additionally, PAO1 infection induced water stress-related genes in barley, which suggests that barley may undergo water deficit upon infection by this pathogen.

Keywords: Antiporter; Barley; Virulence

The influence of morphology on geldanamycin production in submerged fermentations of Streptomyces hygroscopicus var. geldanus by Lynne F. Dobson; Cormac C. O’Cleirigh; Daniel G. O’Shea (pp. 859-866).

The diverse morphology of the filamentous organism Streptomyces hygroscopicus var. geldanus was characterised by image analysis under various environmental conditions. In the presence of surfactant compounds, a significant decrease in the mean pellet diameter was observed. Cell aggregation was also influenced by spore inoculum level, with high concentrations reducing pellet size. In addition, the dispersion of pellets was found to increase with the inclusion of glass beads to submerged shake-flask cultures. In all cases, production of the secondary metabolite geldanamycin was determined to be dependent on the morphological profile of the organism, with a concomitant increase of 88% in geldanamycin yield observed as the mean pellet diameter was reduced by 70%. Thus, to maximise the yield of geldanamycin, it is necessary to limit pellet formation in Streptomyces hygroscopicus var. geldanus to an appropriate size.

Keywords: Geldanamycin; Morphology; Pellet; Streptomyces hygroscopicus

A comparative study on the formation and characterization of aerobic 4-chloroaniline-degrading granules in SBR and SABR by Liang Zhu; Xiangyang Xu; Weiguo Luo; Zhijuan Tian; Haizhuan Lin; Nini Zhang (pp. 867-874).

The formation and characterization of the aerobic 4-chloroaniline-degrading granules in the three column-type sequencing batch reactors were investigated in this paper. The granular sludge was observed since 15 days after start-up in R2 and R3 which had the high ratio of height to diameter (H/D). Since then and within the subsequent 75 days, the granulation of aerobic sludge was apparently developed by the decreased settling time and gradually increased 4-chloroaniline (4-ClA) concentration to above 400 mg·L⁻¹ in R1 to R3. The aerobic granules tended to be mature in all reactors continuously operated with 4-ClA loading rates of around 800 g·m⁻³·d⁻¹, and the removal efficiencies of chemical oxygen demand, total nitrogen, and 4-ClA were maintained above 93%, 70%, and 99.9%, respectively. Mature aerobic granules in R1 to R3 featured with the average diameter of 0.78, 1.68, and 1.25 mm, minimal settling velocity of 20.5, 70.1 and 66.6 m·h⁻¹, specific 4-ClA degradation rates of 0.14, 0.21, and 0.27 g·gVSS⁻¹·d⁻¹, and the ratio of proteins to polysaccharides of 8.2, 10.8, and 13.7 mg·mg⁻¹, respectively. This study demonstrates that the reactor with a high H/D ratio and internal circulation favors the granulation and stabilization of aerobic sludge.

Keywords: 4-Chloroaniline (4-ClA); Aerobic granule; Sequential air-lift bioreactor (SABR); SBR

Assessment of protoxin composition of Bacillus thuringiensis strains by use of polyacrylamide gel block and mass spectrometry by Zujiao Fu; Yunjun Sun; Liqiu Xia; Xuezhi Ding; Xiangtao Mo; Xiaohui Li; Kexue Huang; Youming Zhang (pp. 875-880).

Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties.

Keywords: Bacillus thuringiensis ; Polyacrylamide gel block; LC-MS/MS; Protoxin expression profile; Insecticidal activity

An effective method of DNA extraction for bioleaching bacteria from acid mine drainage by Leping Zeng; Jufang Huang; Yanfei Zhang; Guanzhou Qiu; Jianbin Tong; Dan Chen; Jin Zhou; Xuegang Luo (pp. 881-888).

An effective and versatile method for microorganism lysis and direct extraction of DNA from bioleaching bacteria was developed using pure cultures and an acid mine drainage (AMD) sediment sample. In the described method, microorganisms are treated at three different incubation temperatures: boiling water incubation for 6–10min, followed by 60 ± 5°C for 30min, then 72°C for 30min. The extracted DNA is purified using a phenol/chloroform/alcohol mixture and precipitated in absolute alcohol. The 16S ribosomal RNA (rRNA) and gyrB genes of the pure cultures were amplified using the polymerase chain reaction (PCR) and differentiated using repetitive intergenic DNA sequences amplification (Rep-PCR). For the AMD sediment sample, the 16S rRNA and gyrB genes of the amplicons were digested with Hin6I and MspI, and the restriction fragment length polymorphism analysis patterns were used as a fingerprint to discern community diversity. The results indicated that this method is a versatile, reproducible, effective, and rapid technique for routine DNA extraction from bioleaching bacteria. The low cost of this method also makes it attractive for large-scale studies.

Keywords: Bioleaching bacteria; Acid mine drainage; DNA extraction; Rep-PCR; PCR–RFLP

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Applied Microbiology and Biotechnology (v.79, #5)

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Applied Microbiology and Biotechnology (v.79, #5)