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- Biosynthesis of antioxidant xanthan gum by Xanthomonas campestris using substrates added with moist olive pomacePublication . Crugeira, Pedro Jorge Louro; Almeida, Heloísa H.S.; Marcet, Ismael; Rendueles, Manuel; Pires, Marcella Golini; Rafael, Helder Martins; Rodrigues, Ana Isabel G.; Santamaria-Echart, Arantzazu; Rafael, Helder; Barreiro, M.F.; Golini Pires, MarcellaMoist olive pomace (MOP) is a high moisture content by-product of the olive oil industry. Managing this recalcitrant residue (transport, storage, and drying) is a priority demanding investment in finding alternative valorisation routes. In this context, the biosynthesis of xanthan gum (XG) incorporating MOP in the substrate (0.0 %, 5.0 %, 10.0 %, 15.0 %, 20.0 %, 25.0 %, 30.0 % and 50.0 %) to induce bacterial stress was attempted. XG biosynthesis yield was quantified, and the product was characterised by structural analysis (FTIR), thermal behaviour (TG), rheology and antioxidant capacity. Relative to the control (sample with no added MOP), a significant increase in XG biosynthesis was found for concentrations up to 30.0 % MOP. In particular, for XG produced with 15 % MOP, a 50.91 % (p < 0.0001) increase was achieved, together with 395.78 % for viscosity. In general, XG produced with MOP presence showed antioxidant activity, a value-added property, especially for applications in the food, pharmaceutical and cosmetic areas. The results indicated that the stress imposed by the MOP induced a microbial response leading to XG production increase, structural and viscosity modifications, and antioxidant properties incorporation. Overall, this work points out a new MOP application contributing to the sustainability of the olive oil productive chain from a biobased circular economy perspective.
- Patente Nacional Nº 118006 - Método de produção de goma xantana com bagaço húmido da azeitona e aumento do biopolímero produzidoPublication . Crugeira, Pedro Jorge Louro; Barreiro, M.F.; Almeida, Heloísa Helena Scorsato de; Pires, Marcella Golini; Rafael, Helder Martins; Golini Pires, MarcellaO presente invento refere-se a um processo de produção de goma xantana, a partir da cepa de Xanthomonas campestris atcc 33913, com o uso do bagaço húmido da azeitona, proveniente da extração do azeite em sistema contínuo de duas fases. O método utiliza o bagaço húmido de azeitona pasteurizado até uma concentração de 45% suplementado com 2,0% de sacarose para meio de produção. Os polifenóis, poliálcoois e ácidos orgânicos existentes no resíduo induzem o estresse microbiano e consequente aumento da produção do exopolissacarídeo, como resposta à adaptabilidade celular e proteção às condições desfavoráveis. O dissacarídeo funciona como fonte de carbono, conseguindo-se uma relação aproximada de carbono/nitrogénio de 10:1, favorável a produção. A reutilização do bagaço húmido, resíduo recalcitrante, insere-se no processo sustentável e de inclusão no sistema de economia circular, reduzindo o custo do bioprocesso. O presente invento é aplicável, por exemplo, nas indústrias alimentares, farmacêuticas, agrícolas e petrolíferas.
- Influence of Produced Water and Light Irradiation on the Composition of Exopolysaccharide Produced by L. amnigena Evaluated by Raman SpectroscopyPublication . Silva, Anna Paula Lima Teixeira da; Soares, Luiz Guilherme Pinheiro; Gulberg, Lars Duarte; Crugeira, Pedro; Almeida, Paulo Fernando de; Fernandes, Adjaci Uchoa; Silveira, Landulfo; Pinheiro, Antonio Luiz BarbosaThis study aimed to compare the changes in the composition of the exopolysaccharide (EPS) produced by Lelliottia amnigena in culture medium containing distilled water (DW) and dialyzed produced water (DPW) irradiated by either Laser (λ660 nm, 8.0 J/cm2) or LED (λ630 nm, 12.0 J/cm2) during bacterial growth using Raman spectroscopy at 1064 nm. The cultures of L. amnigena were irradiated at 9‐ and 12‐h, and the EPS obtained from different production protocols were analyzed dehydrated. Raman spectra showed peaks assigned to saccharides from EPS polymer, and principal component analysis revealed differences in the composition of the EPS produced depending on the water used in production and the light source used for irradiation. Remarkably, the presence of acyl groups (acetyl and pyruvyl) in the mannose residues at the group DW and mannose without evidence of acetyl in the irradiated groups; the irradiated groups also presented evidence of carboxylate (succinyl).
- Inactivation of Staphylococcus aureus by antimicrobial photodynamic therapy using 1,9-Dimethyl-Methylene Blue: in vitro and in vivo studiesPublication . Brito Júnior, Anildo Alves de; Crugeira, Pedro; Barbosa, Andressa Vollono; Costa, Wellington Luis Reis; Cangussu, Maria Cristina Teixeira; Oliveira, Susana Carla Pires Sampaio de; Pinheiro, Antônio Luiz Barbosa; Melo, Amanda Inês Vieira de; Pinheiro, Antônio Luiz Barbosa; Azevedo, Juliana MonteiroThe efficiency of antibiotics in terms of their bacterial inhibition is well known. However, studies show that its overuse, underuse, and misuse induce antimicrobial resistance, promoting the need to work with alternative methods. In this sense, antimicrobial photodynamic therapy (aPDT) is a promising selective method demonstrating excellent response. This study aimed to evaluate the antimicrobial action promoted in Staphylococcus aureus using 1,9 dimethyl methylene blue dye (DMMB) combined with red LED (λ 630 ± 20 nm, CW, 125 mW, 12 J/ cm², 192 s) in planktonic culture and rats skin wounds contaminated with staphylococcal bacteria. The experimental in vitro and in vivo groups were Control, LED, DMMB, and LED + DMMB; after aPDT, the triplicate samples for each dilution were incubated for 24 h, and the number of bacteria was determined by counting the colony-forming units, and the logarithm (CFU/mL log). Based on in vitro data obtained, the LED + DMMB group, when compared to the Control, showed a reduction in microbial load of 99.943% (p < 0.0001), with decimal reduction (RD = 3). Whereas in vivo results, the same comparing groups demonstrated a reduc- tion in microbial load, reaching 99.994% (RD = 4). In this research, the aPDT was a unique treatment, and it is possible to repeat it to obtain higher microbial reduction, providing an alternative therapeutic that can be clinically validated to combat infections caused by S. aureus.
- Photoinactivation of sulfate-reducing bacteria using 1,9-dimethyl-methylene blue – DMMB and laser lightPublication . Santos, Gustavo Vital dos; Moura, Hesrom Fernandes Serra; Pedro Jorge Louro Crugeira; Anna Paula Lima Teixeira da Silva; Isabele Cardoso Vieira de Castro; Wellington Luís Reis Costa; Paulo Fernando de Almeida; Antonio Luiz Barbosa Pinheiro; Crugeira, PedroAnnually, the oil and gas industry faces equipment losses and product quality degradation due to the presence of sulfate-reducing bacteria (SRB). Given the negative impact of SRB, this study evaluates the use of photoinactivation (PI) with zinc chloride double salt of 1,9-Dimethyl-Methylene Blue (DMMB) as a photosensitizer (PS) in varying concentrations and combined with Laser light at different exposures in an SRB consortium. For culture growth, a modified Postgate C medium (without ferrous sulfate) was used, and cell quantification was performed on 100 μL aliquots of the consortium, read on a spectrophotometer (λ600 nm) in an oxygen- and light-free environment at room temperature. Statistical analyses included two-way ANOVA and ANOVA with interaction to separately and jointly evaluate the effects of PS and light in PI. Results indicated microbial activity in all groups, with an antimicrobial inhibition rate exceeding 50 % (p < 0.05) for concentrations above 1.5 μg/mL of DMMB. PI efficacy significantly depended on DMMB concentration and light density, achieving a 70.58 % (55.73–70.58, with a mean of 66.71 %) reduction (p < 0.05) with 1.5 μg/mL of DMMB and a 70.15 % (65–70.15, with a mean of 68.21 %) reduction with 2.0 μg/mL at an intensity of 21.6 J/cm2. In conclusion, PI presents a promising alternative to biocides in the oil and gas industry, offering easy application, avoiding bacterial resistance, being environmentally safe, and compatible with other SRB population control techniques.
- Bacterial cellulose biosynthesis in the presence of raw moist olive pomace: A green sustainable approach that enhances biopolymer production and propertiesPublication . Crugeira, Pedro; Khelifa, Halima; Barreira, Luísa; Halla, Noureddine; Barreiro, Filomena; Rodrigues, Paula; Peres, António M.; Schreiner, Tatiana B.; Schreiner, Tatiana B.In this study, the biosynthesis of bacterial cellulose (BC) by Komagataeibacter intermedius strain isolated from Kombucha tea in the presence of raw moist olive pomace (MOP) (concentration up to 40 % in the fermentation media) was studied. The BC membranes were characterized by their antioxidant activity, structural character- istics, crystallinity, thermal stability, and mechanical behavior. Using raw MOP activated the inherent activity of the phenolic compounds, leading to cellular adaptation under unfavorable conditions and increased BC pro- duction for all tested MOP concentrations (p < 0.0001). This led to a 166.61 % increase for the 20 % MOP group compared to the control (HS medium). For this sample, enhanced antioxidant activity (~40-fold higher than the control) was found, which might be associated with the molecular interactions established between hydroxyls of BC and phenolic compounds. Moreover, an increase of 603.03 % in strain capacity, and a 376.01 % improvement in stress at break compared to the control was observed. The study confirmed that BC can be synthesized using MOP in its natural state, supporting a sustainable circular economy while enhancing the biosynthesis of a value- added product. By reducing synthetic media and utilizing MOP, a greener bioprocess can be achieved, and BC’s applicability can be expanded.
- Up‐recycling oil produced water as the media‐base for the production of xanthan gumPublication . Sampaio, Igor Carvalho Fontes; Crugeira, Pedro Jorge Louro; Ferreira, Joalene de Azevedo Santos; Santos, Jacson Nunes; Matos, Josilene Borges Torres Lima; Pinheiro, Antonio Luiz Barbosa; Chinalia, Fabio Alexandre; Almeida, Paulo Fernando deProduced water (PW) and crude glycerin (CG) are compounds overproduced by the oil and biodiesel industry and significant scientific efforts are being applied for properly recycling them. The aim of this research is to combine such industrial byproducts for sustaining the production of xanthan by Xanthomonas campestris. Xanthan yields and viscosity on distinct PW ratios (0, 10, 15, 25, 50, 100) and on 100% dialyzed PW (DPW) in shaker batch testing identified DPW treatment as the best approach for further bioreactor experiments. Such experiments showed a xanthan yield of 17.3 g/L within 54 h and a viscosity of 512 mPa s. Physical-chemical characterization (energy dispersive X-ray spectroscopy, scanning electron microscopy and Raman spectroscopy) showed similarities between the produced gum and the experimental control. This research shows a clear alternative for upcycling high salinity PW and CG for the generation of a valued bioproduct for the oil industry.
- Photodynamic antimicrobial therapy (A-PDT) using 1,9-Dimethyl-Methylene Blue zinc chloride double salt - DMMB and λ640 ± 5ηm LED light in patients undertaking orthodontic treatmentPublication . Soares, Luiz G.P.; Almeida, Cristiane G. de; Crugeira, Pedro Jorge Louro; Nunes, Iago P.F.; Silva, Anna P.L.T. da; Almeida, Jeovana A.; Cangussu, Maria C.T.; Almeida, Paulo Fernando de; Habib, Fernando A.L.; Pinheiro, Antonio Luiz BarbosaOrthodontic treatment involves the use of apparatuses that impairs oral hygiene making patients susceptible to periodontal diseases and caries. To prevent increased antimicrobial resistance A-PDT has shown itself a feasible option. The aim of this investigation was to assess the efficiency of A-PDT employing 1,9-Dimethyl-Methylene Blue zinc chloride double salt -DMMB as a photosensitizing agent combined with red LED irradiation (lambda 640 +/- 5 eta m) against oral biofilm of patients undertaking orthodontic treatment. Twenty-one patients agreed to participate. Four biofilm collections were carried out on brackets and gingiva around inferior central incisors; first was carried out before any treatment (Control); second followed five minutes of pre-irradiation, the third was immediately after the first AmPDT, and the last after a second AmPDT. Then, a microbiological routine for microorganism growth was carried out and, after 24-h, CFU counting was performed. There was significant difference between all groups. No significant difference was seen between Control and Photosensitizer and AmpDT1 and AmPDT2 groups. Significant differences were observed between Control and AmPDT1 and AmPDT2 groups, Photosensitizer and AmPDT1 and AmPDT2 groups. It was concluded that double AmPDT using DMBB in nano concentration and red LED was capable to meaningfully decrease the number of CFUs in or-thodontic patients.
- Oilfield carbonated produced water recycling coupled to exopolysaccharide transformation by lelliottia amnigenaPublication . Sampaio, Igor Carvalho Fontes; Ferreira, Joalene de Azevedo Santos; Crugeira, Pedro Jorge Louro; Oliveira, Ian M. da Silva; Santos, Jacson Nunes; Matos, Josilene Borges Torres Lima; Pinheiro, Antonio Luiz Barbosa; Almeida, Paulo Fernando deIt was tested the production of a viscous exopolysaccharide (EPS) by Lelliottia amnigena using different concentrations (10, 15, 25, 50 and 100%) of produced water (PW) and Dialyzed PW (DPW) at 100% concentration, with a nutrient medium based on glycerin and sucrose as carbon sources in 44 h batches. There was an increase in EPS recovery when used PW up to a concentration of 25%. The viscosity of the EPS solutions was maintained with DPW and up to 15% of PW, such a condition resulted in 9.34 gL− 1 of EPS, representing an increase of 84.9% compared to the control (P < 0.0001). The PW 15% increased the viscosity per produced liter by 114.3% (P < 0.0001). Scanning electron microscopy and Energy-dispersive X-ray spectroscopy analysis showed that EPS adsorbed mineral constituents of the substrate and its characterization by Fourier Transform Infrared Spectroscopy was similar to an external control of a commercial EPS produced by an Enterobacter A47 strain. PW at 15% becomes an alternative to increase the EPS production process and can contribute to the mitigation of environmental damage through sustainable PW management in the oilfield chain.
- Disclosing the potential of Cupressus leylandii A.B. Jacks & Dallim, Eucalyptus globulus Labill., Aloysia citrodora Paláu, and Melissa officinalis L. hydrosols as eco-friendly antimicrobial agentsPublication . Almeida, Heloísa H.S.; Crugeira, Pedro Jorge Louro; Amaral, Joana S.; Rodrigues, Alírio; Barreiro, M.F.Antimicrobial resistance is a major global health concern, threatening the effective prevention and treatment of infections caused by microorganisms. These factors boosted the study of safe and green alternatives, with hydrosols, the by-products of essential oils extraction, emerging as promising natural antimicrobial agents. In this context, four hydrosols obtained from Cupressus leylandii A.B. Jacks & Dallim, Eucalyptus globulus Labill., Aloysia citrodora Paláu and Melissa officinalis L. were studied. Their chemical composition comprises neral, geranial, 1,8-cineole, terpinen- 4-ol, and oplopanonyl acetate, compounds with recognised antimicrobial activity. Concerning antimicrobial activity, significant differences were found using different hydrosol concentrations (10–20% v/v) in comparison to a control (without hydrosol), showing the potential of the tested hydrosols to inhibit the microbial growth of Escherichia coli, Staphylococcus aureus, and Candida albicans. A. citrodora hydrosol was the most effective one, inhibiting 90% of E. coli growth and 80% of C. albicans growth, for both hydrosol concentrations (p < 0.0001). With hydrosol concentration increase, it was possible to observe an improved antimicrobial activity with significant reductions (p < 0.0001). The findings of this work indicate the viability of reusing and valuing the hydrosols, encouraging the development of green applications for different fields (e.g., food, agriculture, pharmaceuticals, and cosmetics).