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In nature, complex microbial communities catalyze cellulose hydrolysis by producing enzymes capable of breaking down cellulose polymers. Hydrolysis is a fundamental step in the carbon cycle of the industrial bioconversion of biomass to biofuels. The production of biofuels from biomass has become necessary, and the discovery of novel pretreatments is mandatory. The study of cellulose hydrolyzing bacterial communities is an essential step in achieving a sustainable future for biofuels. In this study, we propose the stabilization of an aerobic thermophilic bacterial consortium (BC) with the ability to adapt and hydrolyze cellulose-rich mixed wastes. Cellulosic decaying residues inside the Azorean hot springs were used as inoculum. Samples were enriched through twenty consecutive subcultures on a cellulosic selective medium at 60ºC. Enzymatic activity was detected using xylan and carboxymethylcellulose (CMC). Selected BC was able to hydrolyze 50% of cellulose-rich plant mix material in 4 days. Besides hydrolyzing low content lignin material as non-wood plants, newspaper and cardboard, the consortium could hydrolyze high lignin content material with lower efficiency. Cellulase and xylanase were present in BC, volatile fatty acids (VFA) and reducing sugars were higher than control. The pretreatment using BC increased the solubilization of 61% of grass mixtures, enhancing in 77% the production of methane (168 LCH4/Kg VS added). The bacterial diversity of BC was accessed by amplifying 16S rRNA hypervariable regions V3/V4, using 454 pyrosequencing and analyzed with QIIME2. The phylogenetic results showed a large diversity in the BC, with Thermobacillus representing 59% of the consortia, followed by Gemmatimonadetes 9% Symbiobacterium 7%, Thermobispora 6% Brevibacillus 4%, Limnochordaceae 3.9% and Geobacillus 0.6%. Three isolated Geobacillus species were identified; G. stearothermophilus, G.thermodenitrificans and G.debilis. Further work will comply with the improvement of this BC to improve the consortia for enhancing the efficiency to solubilize lignocellulose biomass by using the CRISPR/Cas9 system based on omics analysis of the consortium., info:eu-repo/semantics/publishedVersion

[Excerpt] Lignocellulosic plant biomass is being envisioned by biorefinery industry as an alternative to current petroleum platform because of the large scale availability, low cost and environmentally benign production. The industrial bioprocessing designed to transform lignocellulosic biomass into biofuels are harsh and the enzymatic reactions may be severely compromised reducing the production of fermentable sugars from lignocellulosic biomass. Thermophilic bacteria consortium are a potential source of cellulases and hemicellulases adapted to extreme environmental conditions, which can be exploited as a new source for the development of more robust enzymatic cocktails. (...)

A secção Biologia é coordenada pelo Professor Universitário Armindo Rodrigues. Submitted by João Tavares (tavares@uac.pt) on 2011-02-18T09:13:35Z No. of bitstreams: 1 BIOLOGIA_28nov2010.pdf: 339338 bytes, checksum: 09a258f00b0b9320329d3d44935d46ba (MD5) Made available in DSpace on 2011-02-21T16:03:01Z (GMT). No. of bitstreams: 1 BIOLOGIA_28nov2010.pdf: 339338 bytes, checksum: 09a258f00b0b9320329d3d44935d46ba (MD5) Previous issue date: 2010-11-28

Dissertação de Mestrado, Ciências Biomédicas, 25 de Maio de 2016, Universidade dos Açores. Bacillus thuringiensis (Bt), pertencente à família Bacillaceae, é classificado como gram-positiva e tem despertado elevado interesse em várias áreas, destacando-se a aplicações na agricultura e na saúde. Os isolados de Bt são caracterizados pela sua capacidade de produzir inclusões cristalinas de origem proteica durante a fase de esporulação, as quais são conhecidas como proteínas δ-endotoxinas ou proteínas pesticidas. Estas são constituídas pelas toxinas Cyt e Cry que apresentam actividade extremamente específica contra insectos, nematodes e determinadas linhagens celulares cancerígenas. Nesta tese, foram seleccionados 89 isolados de Bacillus thuringiensis pertencentes à colecção de entomopatógenos do Departamento de Biologia da Universidade dos Açores com o objetivo que realizar um screening para a identificação de genes codificantes para proteínas citotóxicas e nematicidas, nomeadamente parasporinas, Cyt e Cry nematicidas. [...]. ABSTRACT: Bacillus thuringiensis (Bt), belongs to the Bacillaceae family, is a gram-positive and has be a subject of great interest in many areas, especially for applications in agriculture and health. The Bt strains are characterized by the production of crystalline inclusions of protein origin during sporulation, which are kwon as δ-endotoxins or pesticide proteins. These crystals include Cry and Cyt toxins who presents extremely specific activity against insects, nematodes and certain cancer cells lines. In this dissertation, 89 strains of Bacillus thuringiensis from the Azorean entomopathogenic collection of the University of the Azores, were selected in order to carry out a screening for genes encoding for cytotoxic and nematicidal proteins, particularly parasporin, Cyt and Cry. [...].

Dissertação de Mestrado, Ciências Biomédicas, 13 de Maio de 2016, Universidade dos Açores. Bacillus sp. present a great diversity and a high productivity of protease. The group of Biotechnology Department of the Universidade dos Açores has a microbial isolates bank which includes about 1600 isolates of Bacillus sp. Some of the biggest potential thrombolytic enzymes are isolated from Bacillus sp. (streptokinase and natokinase). In this work we tested 79 isolates, previous tested to find a thrombolytic enzyme. The 79 isolates were tested for proteolytic activity in agar casein plate. Then the fibrinolytic activity and the thrombolytic activity of selected isolates were tested. Was determinate the group species of the bacterial isolates with higher activity and were selected 1 isolate from each group. The optimal pH and the optimal temperature of the extracellular protease produced by 11 isolates from Bacillus sp. were determinate also. A hemolytic test was made to the enzymes with a higher thrombolytic activity. After we check for fibrinolytic, thrombolytic and hemolytic activity were selected 2 isolates. These 2 isolates were tested for aPTT and PT activity, for plasminogen activator activity, fibrinogenolytic activity and euglobulin lysis time. 1 bacterial enzyme was selected for biochemical characterization and purification. From the 79 isolates with high proteolytic activity 27 were chosen by their high activity in pH 7.5 and 37˚C. From these 27 bacterial isolates 11 were spotted with fibrinolytic activity and thrombolytic activity (just 6% of the initial 79 isolates). 4 of these enzymes (S97B, S88A, S178C and 99D) belongs to Bacillus cereus group, 4 belongs to Bacillus mycoides group (S101C, S115C, S26A and S62A) and 3 belongs to Bacillus subtilis group (S157E, S122C and S150C). Were selected 2 isolates from the Bacillus mycoides group (S115C and S101C) because they didn’t present hemolytic activity. The isolate S115C didn’t interfere with aPPT test neither with PT test, otherwise S101C increase the aPPT, interfering with the normal coagulation time. Was also evidence that S101C is a plasminogen activator (t-PA) and S115C is not. Moreover, S115C presented more than 10x fibrinolytic activity then S101C, thus enzyme S115C was selected for biochemical characterization and purification. Biochemical characterization showed that S115C was a high inhibited by Benzamidine, STI, Chymostatin and PTCK indicating that S115C is a chymotrypsin-like serine protease. Suc-Ala-Ala- Pro-Phe-pNa was a specific substrate which indicates chymotrypsin activity. The activity of these enzyme was highly enhanced by Mn and slightly enhanced by Na and Ca2. This enzyme was inhibited by metal ions Mg2+, Cu+ and Ni2+. The zymogram of the purified enzyme revel a digestion band higher than 135 kDa and another band at 75kDa. SDS-page of purified fraction revel 2 bands one at 75 kDa and another at 140kDa. The SDS-page bands were cut and sent to analysis of mass spectrometry (Ms/Ms).

Dissertação de Mestrado, Mestrado em Ciências Biomédicas, 8 de Maio de 2015, Universidade dos Açores. Nesta tese, foram seleccionados 24 isolados da ilha de S. Miguel pertencentes ao banco de Bacillus spp. do grupo de entomoparasitologia e o nematode Steinernema carpocapsae pertencente à colecção entomopatogénica da Universidade dos Açores, para a realização de um screening de actividade fibrinolítica. O objectivo desta tese foi a extracção, purificação e caracterização de uma enzima fibrinolítica a partir do sobrenadante do meio de cultura do isolado que registou a maior actividade nos ensaios de screening de actividades com substratos generalistas e em placa de fibrina. Foram detectados 6 isolados bacterianos com actividade fibrinolítica: S150c, S127e, S147e, S157e, S159c e S126e. [...]. ABSTRACT: In this paper, 24 strains of S. Miguel Island from the bank of Bacillus spp, named entomoparasitology group, and the nematode Steinernema carpocapsae from the Azorean entomopathogenic collection of the University of the Azores, were selected to carry out a fibrinolytic screening test. The goal of this paper was the extraction, purification and characterization of a fibrinolytic enzyme from the culture medium of the strain with the highest activity recorded in screening tests performed with serino proteases substrates and in fibrin plate method. A total of six strains were detected with fibrinolytic activity: S150c, S127e, S147e, S157e, S159c and S126e. [...].

Dissertação de Mestrado em Ciências Biomédicas. As bactérias produzem várias substâncias antibacterianas desde antibióticos, lisozimas, exotoxinas e também bacteriocinas. As bacteriocinas são péptidos antimicrobianos de síntese ribossomal que têm suscitado um interesse crescente no combate a estirpes resistentes a antibióticos. Algumas espécies de Bacillus produzem bacteriocinas/BLIS que possuem ação contra MRSA e outras estirpes resistentes. As aplicações das bacteriocinas vão desde a Saúde Humana até à utilização como bioconservantes. O objetivo deste trabalho foi o estudo e caracterização da atividade antibacteriana de 2 isolados de Bacillus (S54c e S150c) da Coleção Açoriana de Bactérias Esporulantes, bem como das suas moléculas ativas. Os dois isolados mostraram maior crescimento e maior atividade antibacteriana em meio Brain Heart Infusion após 18 horas de incubação. Para o sobrenadante filtrado destes isolados e para as frações ativas do sobrenadante do isolado S54c foi determinada a estabilidade térmica e a sensibilidade a enzimas hidrolíticas. O resultado destes testes foi avaliado pela determinação da atividade antibacteriana pelo método de difusão em agar contra a bactéria Micrococcus luteus. A atividade dos isolados S54c e S150C contra Staphylococcus aureus e MRSA foi avaliada pelo método descrito para bactérias esporulantes e o espectro de atividade do sobrenadante do isolado S54c foi determinado. O sobrenadante liofilizado do isolado S54c foi fracionado por cromatografia de exclusão molecular, coluna Sephacryl S-200 40 ml, e as frações positivas visualizadas em SDS-PAGE. Os sobrenadantes dos isolados S54c, S150c e a fração>30kDa do sobrenadante do isolado S54c possuem estabilidade térmica até aos 100ºC, a fração entre os 3-30kDa do isolado S54c é estável até aos 121ºC. Mostrou-se que a atividade antibacteriana dos isolados S54c, S150c e a fração>30kDa (S54c) é hidrolisada pela proteinase K e quimiotripsina, enquanto a fração entre os 3-30 kDa(S54c) apenas é hidrolisada pela proteinase K. Os resultados obtidos indicam que a atividade antibacteriana é de origem proteica e permitem classificar estas moléculas como BLIS. Os isolados S54c e S150c mostraram atividade contra S.aureus e MRSA, contudo o S150c apresentou uma atividade mais reduzida. O sobrenadante filtrado do isolado S54c mostrou atividade contra M.luteus e B.cereus e o fracionamento em cromatografia de exclusão molecular resultou na identificação de uma fração ativa. A análise da sequência parcial do gene 16S mostrou que o isolado S54c é uma estirpe de B.cereus. Este trabalho permitiu identificar parcialmente BLIS produzidas por B.cereus S54c e que podem vir a ser purificadas por técnicas cromatográficas. ABSTRACT: Bacteria produce a high number of antibacterial molecules, including antibiotics, lisozimas, exotoxinas and bacteriocins. Bacteriocins are antimicrobial peptides of ribosomal synthesis that are raising a growing interest in the fighting against bacteria resistant strains. Some species of Bacillus produce bacteriocins/BLIS active against MRSA and other resistant strains. Bacteriocin applications are vast, from human health to biopreservatives. The objective of this work was the study and characterization of antibacterial activity of two Bacillus isolates (S54c and S150c) from the Azorean Sporulated Bacteria Collection. The two isolates showed improved growth and higher antibacterial activity in Brain Heart Infusion broth after 18 hours incubation. The antibacterial activity, of the cell free supernatant of both isolates and active fractions of S54c, were evaluated for thermal stability and degradation by hydrolytic enzymes. The results of these testes were evaluated by the agar well diffusion assay against Micrococcus luteus. S54c and S150c were tested against Staphylococcus aureus and MRSA by the method used for sporulating bacteria and the activity range of S54c cell free supernatant was determined. The freeze dried cell free supernatant of S54c was fractioned by Gel Filtration Chromatography (Sephacryl S-200 40 ml) and positive fractions were separated by SDS-PAGE. The antibacterial activity of S54c and S150c cell free supernatants and the S54c >30kDa fraction showed thermal stability until 100ºC. The S54c 3-30kDa fraction proved to be stable at 121ºC. The activity of S54c, S150c and S54c >30kDa fraction, is hydrolysed by proteinase K, chymotripsin while the 3-30 kDa(S54c) fraction is hydrolysed only by proteinase K. The results show that antibacterial activity is of protein nature and allow classifying these molecules as BLIS. The S54c and S150c isolates showed activity against S. aureus and MRSA, but S150s showed lower activity. The cell free supernatant of S54c showed activity against M. luteus and B. cereus and the gel filtration chromatography of the freeze dried cell free supernatant resulted in the identification of an active fraction. The analysis of the partial sequence of the 16S gene showed that S54c is a strain of B. cereus. This work allowed the partial fractioning and identification of BLIS produced by B. cereus S54c that can be purified by applying chromatographic techniques.

Dissertação de Mestrado, Biotecnologia em Controlo Biológico, 27 de Junho de 2013, Universidade dos Açores. Neste trabalho, estudaram-se os isolados S122D, S150E e S168A do banco de Bacillus spp do grupo de entomoparasitologia do Centro de Investigação de Recursos Naturais da Universidade dos Açores, nos quais se tinha previamente verificado a presença de inclusões, mas que não amplificavam genes cry: S122D, S150E e S168A. A análise dos genes 16S destes isolados apresentou uma grande homologia com isolados do grupo B.cereus. A observação detalhada deste gene revelou a presença de uma assinatura psicotrófica em todos os isolados, com uma timina e uma adenina numa posição característica da espécie Bacillus weihenstephanensis. Através da análise filogenética do gene aroE, confirmou-se que os isolados S122D, S150E e S168A pertencem à espécie B. weihenstephanensis. Testou-se a toxicidade destes isolados em Ephestia kuehniella e selecionou-se o isolado S122D que registou maior mortalidade, apresentando um LD50 próximo dos 100mg de proteína bruta, para isolamento da toxina. Este isolado contém uma inclusão associada ao esporo, apresentando uma forma trapezóide quando separado do esporo, diferente da estrutura bipiramidal dos Bacillus thuringiensis. Após a lavagem das inclusões com triton X-100 e separação dos esporos dos cristais verificou-se uma drástica redução da toxicidade dos mesmos. A maior toxicidade em E. kuehniella foi detetada na fase solúvel em triton X-100, resultante da lavagem das inclusões. A toxina presente nesta fração solúvel em triton X-100 foi fracionada e purificada através de uma sequência de cromatografias num sistema FPLC. A purificação da fração tóxica foi acompanhada de ensaios de toxicidade em E. Kuehniella, apresentando a fração semi-pura uma toxicidade de 40% em 15 µg de proteína. Numa fase seguinte à purificação, foi obtida uma única banda de 63 kDa em SDS-PAGE em condições não desnaturantes e uma banda de 42 kDa quando corrida em condições desnaturantes. Esta toxina foi identificada por espetrometria de massa como sendo uma proteína homóloga a uma Phosphate-binding protein (com um número de acesso de NCBI gi|152032648) a qual designámos Bw-PDB (Bacillus weihenstephanensis- Phosphate-binding protein) A Phosphate-binding protein é, pela primeira vez, descrita em B. weihenstephanensis e identificada como sendo tóxica em Lepidópteros. Esta proteína constitui um promissor novo biopesticida. ABSTRACT: In this paper, three strains were selected from the bank of Bacillus spp from the group of entomoparasitology of the Centre of Investigation of Natural Resources of the University of the Azores, in which the presence of inclusions had previously been verified, but that didn’t amplified cry genes: S122D, S150E and S168A. The analysis of the 16S genes from these strains showed a high homology with the Cereus group strains. A careful look over this gene indicates the presence of a psycothrophic signature in all the strains, with timine and adenine motifs in a characteristic position of the Bacillus weihenstephanensis species. Throughout the phylogenetical analysis of aroE gene, it was confirmed that the S122D, S150E and S168A strains belong to the B. Weihenstephanensis species. The toxicity of these strains was tested in Ephestia kuehniella and the strain S122D, which manifested a higher mortality, was selected, showing a LD50 near the 100 mg of total protein, in order to isolate the toxin. This strain contains an inclusion associated to the spore, with a trapezoid form, different from the bipirimidal structure of the Bacillus thuringiensis, when separated from the spore. The higher toxicity in E. Kuehniella was noticed in the soluble fase on triton X-100, after the crystal (with the spore) washing. After this washing and after isolating the spores and the crystal, a low toxicity of the spores was detected. The toxin contained in this soluble fraction in triton X-100 was fractionated and purified through a sequence of chromotographies in a FPLC system. The purification of the toxic fraction was followed by toxicity tests in E. Kuehniella. The semi-pure fraction demonstrated a toxicity of 40% in 15 µg of protein. Following the purification, a unique band of 63 kDa in SDS-PAGE was obtained in non-desnaturating conditions and a band of 42kDA in desnaturating conditions. This toxin was identified by mass spectroscopy as a homologous protein to a Phosphate-binding protein (with an access number of NCBI gi|152032648) with an assurance of 1280 (limit of 84, P < 0.05) and a coverage sequence of 64%, without knowing the organism that produces it. For the first time, this Phosphate-binding protein is described in B. Weihenstephanensis and identified as being toxic in Leptidoptera. It constitutes a promising new biopesticide.