VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY LE PHUONG THAO UNDERGRADUATE THESIS TITLE: MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF SOME ACID-TOLERANT FUNGI SPECIES Hanoi, March 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY UNDERGRADUATE THESIS TITLE: MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF SOME ACID-TOLERANT FUNGI SPECIES Student name : Le Phuong Thao Class : K62CNSHE Faculty : Biotechnology Supervisors : Nguyen Thi Thuy Hanh, PhD. Vu Nguyen Thanh, Assoc. Hanoi, March 2022 COMMITMENT I hereby declare that: This is my study, which was conducted under the guidance of the supervisors; All data provided are true and accurate; All published data and information have been duly cited. Hanoi, March 2022 Student Le Phuong Thao i ACKNOWLEDGEMENTS Firstly, I would like to express my gratitude to Assoc.
Vu Nguyen Thanh – who has given me all support, guidance, all the necessary information that made me complete the thesis. He allowed me to do the necessary research work and use the lab equipment needed in his lab – Center for industrial microbiology, Food Industries Research Institute. In addition, I would like to extend our sincere esteems to all members in laboratory for their timely support. Secondly, I owe my deep gratitude to Dr.
Nguyen Thi Thuy Hanh for an opportunity to conduct my thesis at Food Industries Research Institute and her invaluable guidance during the past time. It is also giving my thankfulness to friends and relatives for sharing my difficulties and giving me various used advices during the process of learning and studying. Finally, I would like to thanks the leaders of Vietnam National University of Agriculture, Department of Biotechnology and Food Industries Research Institute for creating conditions for me to complete my thesis well. Thank you so much! Le Phuong Thao ii TABLE OF CONTENTS COMMITMENT.
ii TABLE OF CONTENTS. iii LIST OF TABLES. v LIST OF FIGURES. Introduction of acid-tolerant fungi.
Origin and characteristics of acid-tolerant fungi. Some representative group of acid-tolerant fungi. Introduction of the genus Penicillium. Research on acid-tolerant fungi in the world and in Vietnam.
Research on acid-tolerant fungi in the world. Research on acid-tolerant fungi in Vietnam. DNA barcoding for identification for phylogenetic species recognition. Methods to evaluate the biochemical and physiological characteristics of fungi.
MATERIALS AND METHODS. Chemicals, equipment and media. Observation of colonies, cells. Purification and maintenance of strains.
Method for biochemical and physiological test. DNA extraction and purification method for fungi cells. Method to classify based on DNA barcoding. Staining the gel and reading the result.
RESULTS AND DISCUSSION. Observation of colonies and cells. Classification of strains based on rDNA sequence. Classification of strains based on β-tubulin and calmodulin sequence.
The growth of strains on different environmental conditions. CONCLUSION AND PROPOSAL. 41 iv LIST OF TABLES Table 2. The list of the acid-tolerant fungi, the fungi originally described as indigenous inhabitants of highly acidic habitats (pH < 3).
(Hujslová et al. Primers used for amplification and sequencing. (Visagie et al. Thermal cycle programs used for amplification.
(Visagie et al. Origin of isolates of low pH acid-tolerant fungi strains. Primers used for amplification and sequencing. The colony diameter at the widest part of the colony after 7 days of cultivation.
36 v LIST OF FIGURES Figure 2. Extreme acidic environments. Morphological features of the Acidomyces acidophilus WKC-1. Microscopy of Hortaea acidophila, CBS 113389.
Morphological characteristics of colonies and cells on PDA medium and Malt 2Bx medium pH 1.0 of representative strains. Neighbour-joining phylogram depicting the relationships between strains and neighbouring taxa based on ITS sequences. Neighbour-joining phylogram depicting the relationships between Amplistroma and neighbouring taxa based on D1/D2 sequences. Neighbour-joining phylogram depicting the relationships between Penicillum and neighbouring taxa based on CaM sequences.
Neighbour-joining phylogram depicting the relationships between Penicillum and neighbouring taxa based on BenA sequences. Colonies after 7 days of cultivation on different media. 35 vi ABBREVIATIONS DNA Deoxyribonucleic Acid rDNA Recombinant Deoxyribonucleic Acid dNTPs Deoxyribonucleotide triphosphates PCR Polymerase Chain Reaction TAE Tris-acetate-EDTA ITS Internal Transcribed Spacer BenA β-tubulin CaM Calmodulin RPB2 RNA polymerase II second largest subunit vii ABSTRACT Life in natural and man-made environments with extremely low pH can be very diverse, in which microbial studies are particularly interesting. It simply because the importance in biotechnology and their potential solutions to environmental pollution.
This study was carried out with 19 strains that have been identified with the ability to grow on low pH environment were isolated in Vietnam. The results of this study have identified 8 new species including: Amplistroma sp. The above species have also been studied in detail through DNA barcoding gene sequencing include: Internal Transcribed Spacer (ITS), β-tubulin (BenA), Calmodulin (CaM), RNA polymerase II second largest subunit (RPB2). Morphological and physiological information was also clarified by culturing the strains on different media.
INTRODUCTION The biodiversity of microorganisms living in extreme environments has been studied since the last century. An environment characterized by a high degree of acidity belongs to this environment. Fungi able to tolerate acidic conditions are frequently encountered in nature, and several species are capable of growing at very low pH levels. There is unclear demarcation between acid-tolerant and acidophilic fungi, but it is often assumed that acid-tolerant fungi are those that can grow at pH 1.0 and have optimum growth at pH 3.
Studies on acid-tolerant fungi have been discovered and published since the beginning of the last century. In 1943, a strain of Acontium velatum and a “Fungus D” were shown to be capable of growing in a glucose medium containing 1.25M sulphuric acid at pH 0. Unfortunately, the strain of Acontium velatum appears to have been lost since the initial publication, but “Fungus D” is now believed to be a strain of Acidomyces acidophilus which is commonly found in extremely acidic environments. According to Thanh et al (2019), acidophility has been shown for only 6 fungal species, including Acidomyces acidophilus (=Scytalidium acidophilum = Acidomyces richmondensis = Fungus D), Acidomyces acidothermus, Acidothrix acidophila, Acidea extrema, Acontium velatum (no living specimen available) and Hortaea acidophila (=Neohortaea acidophila).
Phylogenetically, all acid-tolerant species are Ascomycota, and the teleomorphic state is known only for Acidomyces acidothermus (described as Teratosphaeria acidotherma). However, studies on acid-tolerant species have not been published much, and the confirmation of which fungal species can grow under these conditions is still an open question. Acid-tolerant fungi have received considerable attention, as their thermostable enzymes can be employed in industrial processes at elevated temperatures. Increasing the process temperature can have advantages, for example, increasing the rate of chemical reactions, decreasing the viscosity of substrates and reducing the risk of contamination by mesophilic microorganisms.
For example, the strain Bispora sp. 1 MEY-1, well-known for the production of a range of thermophilic and acid-tolerant lignocellulolytic enzymes. In recent studies on acid-tolerant fungi at the Food Industry Research Institute, many new species capable of growing in low pH conditions have been discovered. Interestingly, most of the fungal strains identified as this new species are quite different from previously published studies.
To study this difference more clearly, we conducted the topic: “Morphological and physiological characteristics of some acid- tolerant fungi species”. This study attempts to clarify the morphology and physiology of strains that lead to purification, identification, and practical application of some new characteristics. Objective: Description of morphological and physiological characteristics of some acid- tolerant fungi strains isolated in Vietnam. Requirements: - Observation of colony and cell morphology of acid-tolerant strains - Classification of acid-tolerant strains 2 II.
Introduction of acid-tolerant fungi 2. Origin and characteristics of acid-tolerant fungi Extreme environments usually possess various factors incompatible with most life forms. Thus, certain environmental conditions such as low water availability in hyperarid deserts or high temperatures seem to be close to the limit of biological activity (Schulze-Makuch, Airo and Schirmack, 2017). However, despite the apparent hostility of these extreme habitats, they contain a higher level of biodiversity than expected.
The number of different organisms known to reside and thrive in these environmentally extreme conditions has grown rapidly in recent years. For example, robust microbial communities at high-temperature ranges, i., the hot springs acid- tolerant algae (Cyanidiaceae) grow at 45–56 °C (Skorupa et al., 2013) while the hyper thermophilic archaea tolerate a temperature range above the boiling point (>100 °C) (Kambura et al. Similarly, there are microbes living in very alkaline environments (as high as pH 12) (Kambura et al. On the other end of the pH scale there are the acid-tolerant archaea (i., Thermoplasma acidophilum) or the unicellular alga Cyanidium caldarium thriving in very acidic habitats (pH ranges from 0–4).
Furthermore, they can survive exposure to such conditions for weeks, months, years, or even centuries (Aguilera et al. Eukaryotic organisms are exceedingly adaptable, and they are present in all the extreme environments reported until now. In this regard, acid-tolerant environments are not an exception. Although it is usually assumed that high metal concentrations in acidic habitats limit eukaryotic growth and diversity due to their toxicity, most of these extreme environments showed an unexpectedly high degree of eukaryotic diversity.
Extreme acidic ecosystems usually include as well different abiotic extremes than low pH (Rothschild and Mancinelli, 2001); (Tiquia-Arashiro and Rodrigues, 2016). Thus, eukaryotes thriving at these habitats are often also exposed to low nutrient levels (Brake and Hasiotis, 2010), high concentrations of toxic metals (Aguilera et al., 2007), 3 and/or extreme temperature (González-Toril et al. Additionally, several studies have revealed representatives from multiple evolutionary eukaryotic lineages, suggesting that the ability to adapt to pH extremes may be widespread (Amaral Zettler et al. Extreme acidic environments.
Acid-tolerant fungi have been reported from various acidic environments. Hitherto, five fungal species isolated from acidic environments are known to be able to grow in extremely acidic conditions. Acontium velatum Morgan was isolated from a solution containing 4% copper sulfate (pH 0. Capnodialean anamorphic fungi were also isolated from acidic environments.
Acidomyces acidophilus was reported as an acid-tolerant species and has been isolated from the soil (pH 1.5) adjacent to a sulphur pilefield from a natural gas purification plant as Scytalidium acidophilum (Sigler and Carmichael, 1974) and acid mine drainage 4 (pH 0.38) as „Acidomyces richmondensis‟ (nom.) (Baker et al. Hortaea acidophila Ho¨lker et al. was also isolated from brown coal (pH 0.6) containing humic and fulvic acids (Hölker et al. These latter two species were reported to be able to grow even at pH 1 (Sigler and Carmichael 1974; Baker et al.
2004; Hölker et al., 2004; Selbmann et al. Interestingly, these acid- tolerant fungi mentioned above are all anamorphic fungi, and no teleomorphic species have been reported from such highly acidic environments. The list of the acid-tolerant fungi, the fungi originally described as indigenous inhabitants of highly acidic habitats (pH < 3). (Hujslová et al., 2019) Species Isolated from Highly acidic soil (Czech Republic) Acidea extrema Biofilm from the highly acidic river (Spain) Highly acidic soil (Czech Republic) Biofilm from the highly acidic river Acidiella bohemica (Spain) Abandoned mine (Japan) Highly acidic oil shale by-products (Brazil) Highly acidic water from uranium mine (Australia) Acidiella uranophilac Highly acidic river water and sediment (Spain)d Acid-tolerant algae, acid drainage (Germany) Soil near sulfur pile (Canada) Sulfuric acid (Denmark) Volcanic soil Acidomyces acidophilus (Iceland) Acidic industrial water (The Netherlands) Highly acidic soil (Czech Republic) Highly acidic hot springs (Japan) Highly acidic water from uranium mine (Australia) Acidic waste water of Acidomyces acidothermus uranium mine (China) Highly acidic soil (Czech Republic, Iceland) Acid mine drainage biofilm (USA) Acid transfer pipeline (India) Highly acidic soil (Czech Republic) Acidothrix acidophila Enrichment culture of archaeal Richmond mine Acid-tolerant Nanoorganisms from biofilms of mine (Germany) Highly acidic water from uranium mine (Australia) Coniochaeta fodinicola Acidic waste water from uranium mine (China) Highly acidic soil (Czech Republic) Neohortaea acidophila Extract of brown coal with humic and fulvic acids, pH 0.
Some representative group of acid-tolerant fungi Acidomyces acidophilus Acidomyces acidophilus is a fungus first described by Sigler & J.