To study the underlying mechanisms for microbial cellulose biosythesis, a novel compound, CBI28, was used as an inhibitor along with classical genetics and EMS mutagenesis. An EZ-Link Biotin Hydrazide Kit was used to create a CBI28-Biotin conjugate for further studies. Gluconacetobacter xylinus cells were exposed to 10 uM CBI28 to induce cellulose biosythesis inhibition, lysed and small hydrophobic molecules were extracted using methanol and Waters Oasis HLB SPE-Paks. Samples were separated and detected using the Ultra Performance Liquid Chromatograph-Mass Spectrometer/Photo Diode Array. Putative mutants were isolated but did not survive for further study. An ion with the expected mass of a CBI28-Biotin conjugate (552 m/z) was detected but not in sufficiently high concentrations for characterization. Metabolite studies revealed putative metabolites derived from the HLB SPE and methanol extractions with no significant difference in extraction methods. Potential metabolites with masses of ~281.77 m/z and ~79 m/z were detected in CBI28 exposed cells. Further analysis needs to be performed to determine if CBI28 metabolites prevent cellulose production. / UOIT

La celulosa es un polisacárido que se encuentra presente en la naturaleza, el cual es de importancia mundial, debido a sus propiedades únicas que lo hacen fundamental en aplicaciones industriales tan diversas, como lo son en la producción del papel, en la industria biomédica, en el vestuario, cosméticos, entre otros. No sólo los organismos vegetales son capaces de producir celulosa, sino que también microorganismos como las bacterias Acetobacter, entre ellas la bacteria Gluconacetobacter xylinus (G. xylinus), la cual es el organismo más estudiado por el rendimiento de biopelícula producida, como también por la fuente de celulosa limpia obtenida sin necesidad de purificación. Sin embargo, este rendimiento varía según las condiciones en las que crece la bacteria, como lo son la fuente de glucosa utilizada y la oxigenación que necesita al ser un microorganismo aeróbico estricto. Se ha utilizado esta bacteria a nivel industrial de producción, no obstante, el alto costo de mantenimiento por las necesidades antes mencionadas, no ha logrado concretar el potencial que esta fuente entrega. Es por esto, que en el presente estudio se buscó una fuente natural de oxigenación, como lo es la microalga Chlorella vulgaris (C.vulgaris), la cual es de fácil manejo y económicamente rentable de mantener en laboratorio por sus amplios usos, como también su capacidad de producir oxigeno bajo luminosidad constante como fotoperiodo. Así es como en este trabajo de investigación se caracterizó el crecimiento de G. xylinus y C. vulgaris. Luego, se realizaron co-cultivos (CC) y cultivos separados (CS) entre ambos microorganismos. El objetivo de esta investigación fue estudiar la capacidad de optimizar la producción de celulosa bacteriana, entregando oxigenación controlada xv desde las microalgas fotosintéticamente activas (bajo fotoperiodo y luz constante) hacia el cultivo bacteriano. Nuestros resultados indican que, mediante la metodología de CS y bajo luminosidad constante al sexto día de cultivo estático, se produjo un rendimiento de celulosa bacteriana, dos veces mayor al producido por los controles, siendo el oxígeno producido por las microalgas esencial para esta producción. Estos resultados sugieren que sería más eficiente producir CS por sólo seis días que dejar cultivos por doce días estáticos, siendo factible producir más de dos cultivos para producir un mayor rendimiento que uno por más días. / Cellulose is a polysaccharide that is present in nature, which is of global importance, due to its unique properties that make it essential in industrial applications as diverse as in paper production in the biomedical industry, clothes, cosmetics, among others. Not only are plants capable of producing cellulose, but also microorganisms such as the bacteria Acetobacter, including the bacteria Gluconacetobacter xylinus (G. xylinus), which is the organism most studied for the biofilm yield produced, as well as by the source of clean cellulose obtained without purification. However, this yield varies depending on the conditions in which the bacteria grow, such as the source of glucose used and the oxygenation it needs as a strict aerobic microorganism. This bacteria has been used at the industrial production level, however, the high cost of maintenance because of the aforementioned needs, has failed to realize the potential that this source delivers.For this reason, in the present study, a natural source of oxygenation was sought, as is the microalga Chlorella vulgaris (C.vulgaris), which is easy to use and economically profitable to maintain in the laboratory for its wide uses, as well as its ability to produce oxygen under constant light as photoperiod. Therefore,the growth of G. xylinus and C. vulgaris was characterized in this research work, by performing co-cultures (CC) and separate cultures (CS) between both microorganisms. The objective of this research was to study the ability to optimize the production of bacterial cellulose, by delivering controlled oxygenation from photosynthetically active microalgae (under photoperiod and constant light) to the bacterial culture. xvii Our results indicate that, through the CS methodology and under constant luminosity at the sixth day of static cultivation, a yield of bacterial cellulose, was produced twice as high as that produced by the controls, the oxygen produced by the microalgae being essential for this production. These results suggest that it would be more efficient to produce CS for only six days than to leave cultures for twelve static days, being feasible to produce more than two cultures to produce a higher yield than one for more days. / Programa de Estímulo a la Excelencia Institucional (PEEI) de la Universidad de Chile

Seminario de Título entregado a la Universidad de Chile en cumplimiento parcial de los requisitos para optar al Título de Ingeniero en Biotecnología Molecular / La celulosa es un polisacárido que se encuentra presente en la naturaleza, el cual es de importancia mundial, debido a sus propiedades únicas que lo hacen fundamental en aplicaciones industriales tan diversas, como lo son en la producción del papel, en la industria biomédica, en el vestuario, cosméticos, entre otros. No sólo los organismos vegetales son capaces de producir celulosa, sino que también microorganismos como las bacterias Acetobacter, entre ellas la bacteria Gluconacetobacter xylinus (G. xylinus), la cual es el organismo más estudiado por el rendimiento de biopelícula producida, como también por la fuente de celulosa limpia obtenida sin necesidad de purificación. Sin embargo, este rendimiento varía según las condiciones en las que crece la bacteria, como lo son la fuente de glucosa utilizada y la oxigenación que necesita al ser un microorganismo aeróbico estricto. Se ha utilizado esta bacteria a nivel industrial de producción, no obstante, el alto costo de mantenimiento por las necesidades antes mencionadas, no ha logrado concretar el potencial que esta fuente entrega. Es por esto, que en el presente estudio se buscó una fuente natural de oxigenación, como lo es la microalga Chlorella vulgaris (C.vulgaris), la cual es de fácil manejo y económicamente rentable de mantener en laboratorio por sus amplios usos, como también su capacidad de producir oxigeno bajo luminosidad constante como fotoperiodo. Así es como en este trabajo de investigación se caracterizó el crecimiento de G. xylinus y C. vulgaris. Luego, se realizaron co-cultivos (CC) y cultivos separados (CS) entre ambos microorganismos. El objetivo de esta investigación fue estudiar la capacidad de optimizar la producción de celulosa bacteriana, entregando oxigenación controlada xv desde las microalgas fotosintéticamente activas (bajo fotoperiodo y luz constante) hacia el cultivo bacteriano. Nuestros resultados indican que, mediante la metodología de CS y bajo luminosidad constante al sexto día de cultivo estático, se produjo un rendimiento de celulosa bacteriana, dos veces mayor al producido por los controles, siendo el oxígeno producido por las microalgas esencial para esta producción. Estos resultados sugieren que sería más eficiente producir CS por sólo seis días que dejar cultivos por doce días estáticos, siendo factible producir más de dos cultivos para producir un mayor rendimiento que uno por más días. / Cellulose is a polysaccharide that is present in nature, which is of global importance, due to its unique properties that make it essential in industrial applications as diverse as in paper production in the biomedical industry, clothes, cosmetics, among others. Not only are plants capable of producing cellulose, but also microorganisms such as the bacteria Acetobacter, including the bacteria Gluconacetobacter xylinus (G. xylinus), which is the organism most studied for the biofilm yield produced, as well as by the source of clean cellulose obtained without purification. However, this yield varies depending on the conditions in which the bacteria grow, such as the source of glucose used and the oxygenation it needs as a strict aerobic microorganism. This bacteria has been used at the industrial production level, however, the high cost of maintenance because of the aforementioned needs, has failed to realize the potential that this source delivers.For this reason, in the present study, a natural source of oxygenation was sought, as is the microalga Chlorella vulgaris (C.vulgaris), which is easy to use and economically profitable to maintain in the laboratory for its wide uses, as well as its ability to produce oxygen under constant light as photoperiod. Therefore,the growth of G. xylinus and C. vulgaris was characterized in this research work, by performing co-cultures (CC) and separate cultures (CS) between both microorganisms. The objective of this research was to study the ability to optimize the production of bacterial cellulose, by delivering controlled oxygenation from photosynthetically active microalgae (under photoperiod and constant light) to the bacterial culture. xvii Our results indicate that, through the CS methodology and under constant luminosity at the sixth day of static cultivation, a yield of bacterial cellulose, was produced twice as high as that produced by the controls, the oxygen produced by the microalgae being essential for this production. These results suggest that it would be more efficient to produce CS for only six days than to leave cultures for twelve static days, being feasible to produce more than two cultures to produce a higher yield than one for more days. / Programa de Estímulo a la Excelencia Institucional (PEEI) de la Universidad de Chile

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-07-19T13:51:41Z No. of bitstreams: 3 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Cynthia Coimbra BC.pdf: 5378162 bytes, checksum: c76cbf49b8ae27ba5d421eba30de2290 (MD5) Tese Cynthia Coimbra BC.pdf: 5378162 bytes, checksum: c76cbf49b8ae27ba5d421eba30de2290 (MD5) / Made available in DSpace on 2016-07-19T13:51:41Z (GMT). No. of bitstreams: 3 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Cynthia Coimbra BC.pdf: 5378162 bytes, checksum: c76cbf49b8ae27ba5d421eba30de2290 (MD5) Tese Cynthia Coimbra BC.pdf: 5378162 bytes, checksum: c76cbf49b8ae27ba5d421eba30de2290 (MD5) Previous issue date: 2015-10-28 / Celulose bacteriana é um biopolímero flexível, constituído por fibras mais finas e poros menores do que as da celulose vegetal. Muitas aplicações já são conhecidas, mas ainda são necessárias informações para a viabilização de sua produção em escala industrial. Este trabalho visou contribuir com informações sobre os fatores que influenciam sua produção, sua purificação e elaboração de filmes para uso na área farmacêutica e de alimentos, bem como a interação entre estes fatores. Para tanto, foi realizado um estudo em três etapas: (1) avaliação da interação entre diversas fontes de carbono (glicose, xilose, sacarose, frutose e glicerol) e a velocidade de agitação sobre crescimento da biomassa, produção de celulose, sua capacidade de retenção de água e produção de acetana; (2) estudo do tratamento da celulose bacteriana, interrelacionando diferentes concentrações de NaOH e tempos de aquecimento em forno de micro-ondas de acordo com a efetividade de purificação e a possíveis alterações estruturais; e (3) aproveitamento da celulose produzida sob forma de filmes comestíveis, pela associação com glicerol e manitol em diferentes proporções e a caracteriação dos mesmos. Identificou-se, para as condições investigadas, que: (1) a maior produção de celulose (1,08 ± 0,07gL-1) é obtida com a mistura de glicose e frutose, em cultivo estático, já que a modificação das velocidades de agitação propiciou aumento da biomassa, sem acarretar maior produção de celulose e que as melhores condições de produção de celulose diferem das para a produção de acetana; (2) as condições ideais para a purificação da celulose é com NaOH a 0,85 mol.L-1 e 3,3 min de aquecimento em forno de micro-ondas; (3) o aquecimento a partir de 7 min promoveu modificações estruturais nos domínios cristalinos da celulose e que a partir de 12,5 min obtém-se a mercerização completa da celulose; e (4) é possível determinar as características finais dos filmes produzidos manipulando-se as proporções de manitol e glicerol associados à celulose, de forma a adequá-lo à aplicação desejada. Tais resultados conduziram à conclusão de que as determinações e os tratamentos realizados em cada uma das etapas possibilitou o conhecimento de que dois importantes fatores influenciam juntos a produção de celulose e de acetana por Gluconacetobacter xylinus, bem como a purificação de membranas de celulose e a elaboação de filmes comestíveis com possibilidade de manipulação de suas características para ampliação dos usos deste biopolímero na área farmacêutica. / Bacterial cellulose is a polymer flexible, consisting of thinner fibers and smaller pores than plant cellulose. Many applications are already known, but are still necessary more information about its production for the viability of its production on an industrial scale. This work contributed with information on the factors that influence its production, purification and preparation of films for use in pharmaceuticals and food, as well as the interaction between these factors. To this end, this thesis was prepared in three stages: (1) evaluation of the interaction of various carbon sources (glucose, xylose, sucrose, fructose and glycerol), and the stirring rate on biomass growth, cellulose production, water retention capacity and acetan production; (2) study of the treatment of the bacterial cellulose, cross correlating different concentrations of NaOH and heating time in a microwave oven according to the effectiveness of the purification and the possible structural changes; and (3) use of the cellulose produced in the form of edible films, the combination with glycerol and mannitol in different proportions and characterization thereof. It was identified that (1) higher production of pulp (1.08 ± 0.07 gL-1) was obtained with the mixture of glucose and fructose in static culture, since the modification of agitation rates resulted in an increase of the biomass, without causing larger cellulose production and the best cellulose producing conditions different from those for the production of acetan; (2) optimum conditions for the purification of cellulose is with NaOH 0.85 mol L -1 and 3.3 min of heating in microwave oven; (3) the heating from 7 min promoted structural modifications on cellulose crystalline domains and heating times from the 12.5 min cause complete mercerizing cellulose; and (4) it can determine the final characteristics of the films produced by manipulating the proportions of mannitol and glycerol associated with the cellulose in order to adjust it to the desired application. These results led to the conclusion that the proposed aims have been achieved, since the requirements and treatments carried out in each of steps allowed the understanding of how two important factors influencing together, the synthesis of cellulose and acetan by Gluconacetobacter xylinus and the purification of cellulose membranes and the preparation of edible films with the possibility of handling characteristics to expand the uses of this biopolymer in pharmaceutical area.

The acsD gene is a unique gene present in the cellulose biosynthesis operon in G. xylinus. With the use of homologous recombination, the acsD gene disruption mutation was created in the G. xylinus genome. Phenotypic characterization of the acsD gene mutant was investigated with the assistance of light and electron microscopy observations, carboxymethyl cellulose alterations, and lower temperature incubation. The microscopic analysis of the cellulose ribbons secreted from the acsD gene mutant shows that the polymerization and the crystallization components in mutant cells were functional. Observations of the mutant cells after incubation with carboxymethyl cellulose and temperature changes indicate that the arrangements of the pores on the cell surface have been altered. These arrangements led to decreased cellulose secretion capacity of the mutant cells. Successful complementation was achieved by using gene expression plasmids with green fluorescence protein tag in the acsD mutant background. Anti-GFP antibodies were used to determine the in vitro localization of the protein. Using subcellular fractionation and western blotting, the AcsD protein was found to be localized in the periplasm of the cells. Taking all these results together, a new model for bacterial cellulose biosynthesis has been suggested and discussed. / text

A biomembrana, que é uma membrana de celulose bacteriana (C6H10O5)n formada na superfície do meio de cultivo durante a fermentação acética, foi obtida através do cultivo associado de Gluconacetobacter xylinus (formalmente Acetobacter xylinum) e Saccharomyces cerevisiae em meio de folhas de chá verde, resíduos de frutas (abacaxi, mamão, laranja), resíduos de vegetais (beterraba), vinho e colágeno em condições estáticas a 28 ± 2°C de 7 a 30 dias de cultivo. Foi incorporado à biomembrana, extrato hidroalcoólico de Calendula officinalis, devido as suas propriedades anti-inflamatórias, antioxidantes e cicatrizantes. A espessura, o diâmetro e o peso da biomembrana foram mensurados e foram calculados a produtividade, bem como o fator de conversão de açúcar em celulose. A caracterização da biomembrana foi realizada por Differential Scanning Calorimetric, espectroscopia infravermelho, Brunauer-Emmett-Teller, resistência à tração e alongamento, microscopia eletrônica (Escola Politécnica - USP) e difração de raio-X. Através destas análises verificou-se que a biomembrana obtida nos diferentes meios de cultivo é composta por celulose, o tamanho médio dos poros variou de 517,9 a 1582,0 nm, a resistência à tração variou de 0,76 a 4,32 kN/m e o índice de cristalinidade entre 75% e 91%, a espessura da biomembranas variou de 0,16 a 6,38 mm. Foram realizados 576 experimentos, a maior produtividade (8,23 g de celulose/dia) foi atingida no meio de mamão com suco de laranja (suco de mamão: 50% v/v e suco de laranja: 19% v/v) em 7 dias de cultivo. O maior fator de conversão (2,36 g celulose/g de açúcar) foi obtido no meio de chá verde em 25 dias de cultivo. A adição de 1,5% p/v de colágeno ao meio de chá verde dobrou a massa da biomembrana. A incorporação do extrato de calêndula aumentou a flexibilidade, a cristalinidade e as propriedades mecânicas da biomembrana de chá verde. / The biomembrane, cellulose membrane (C6H10O5)n formed in medium surface, was obtained from an associate culture Gluconacetobacter xylinus (formally Acetobacter xylinum) and Saccharomyces cerevisiae in green tea leaves, fruit residues (pineapple, papaya, orange), vegetables residues (beet), wine and collagen media in static condition , at 28 ± 2 ºC in 7 - 30 days cultivation. The Calendula officinalis hydroalcoholic extract was incorporated in the Biomembrane, due to its anti-inflammatory, anti-oxidants and cicatrizing proprieties. The biomembrane thickness, diameter and weight were measured. The productivity and conversion factor from cellulose to sugar were calculated. The biomembrane caracterization was performed by Differential Scanning Calorimetric, infrared spectroscopy, Brunauer-Emmett-Teller, resistance to tension, elongation, eletrocnic microscopy and raio-X difraction. In these analyses were verified that biomembrane obtained in different media were composed by cellulose, average porous size varied from 517.9 to 1582.0 nm, the resistance to tension varied from 0.76 to 4.32 kN/m and cristalinity index varied from 75% to 91%. The biomembrane thickness varied from 0,16 to 6,38 mm. It was performed 596 tests, the highest bacterial cellulose yield (8.23 ± 0.58 g cellulose/day) was obtained in papaya with orange (papaya juice: 50% v/v and orange juice: 19% v/v) in 7 cultivation days. The highest conversion factor (2,36 g cellulose/g sugar) was obtained in green tea medium in 25 days. The addition of 1.5% w/v collagen to the green tea media increased 2 times the biomembrane weight. The biomembrane absorption capacity for water and Marigold hydroalcoholic extract (1:1), were from 1.73 to 22 and, from 1.75 to 24 times dry weight, respectively. The Marigold extract improved the green tea biomembrane flexibility, cristalinity, and physical proprieties.

A biomembrana, que é uma membrana de celulose bacteriana (C6H10O5)n formada na superfície do meio de cultivo durante a fermentação acética, foi obtida através do cultivo associado de Gluconacetobacter xylinus (formalmente Acetobacter xylinum) e Saccharomyces cerevisiae em meio de folhas de chá verde, resíduos de frutas (abacaxi, mamão, laranja), resíduos de vegetais (beterraba), vinho e colágeno em condições estáticas a 28 ± 2°C de 7 a 30 dias de cultivo. Foi incorporado à biomembrana, extrato hidroalcoólico de Calendula officinalis, devido as suas propriedades anti-inflamatórias, antioxidantes e cicatrizantes. A espessura, o diâmetro e o peso da biomembrana foram mensurados e foram calculados a produtividade, bem como o fator de conversão de açúcar em celulose. A caracterização da biomembrana foi realizada por Differential Scanning Calorimetric, espectroscopia infravermelho, Brunauer-Emmett-Teller, resistência à tração e alongamento, microscopia eletrônica (Escola Politécnica - USP) e difração de raio-X. Através destas análises verificou-se que a biomembrana obtida nos diferentes meios de cultivo é composta por celulose, o tamanho médio dos poros variou de 517,9 a 1582,0 nm, a resistência à tração variou de 0,76 a 4,32 kN/m e o índice de cristalinidade entre 75% e 91%, a espessura da biomembranas variou de 0,16 a 6,38 mm. Foram realizados 576 experimentos, a maior produtividade (8,23 g de celulose/dia) foi atingida no meio de mamão com suco de laranja (suco de mamão: 50% v/v e suco de laranja: 19% v/v) em 7 dias de cultivo. O maior fator de conversão (2,36 g celulose/g de açúcar) foi obtido no meio de chá verde em 25 dias de cultivo. A adição de 1,5% p/v de colágeno ao meio de chá verde dobrou a massa da biomembrana. A incorporação do extrato de calêndula aumentou a flexibilidade, a cristalinidade e as propriedades mecânicas da biomembrana de chá verde. / The biomembrane, cellulose membrane (C6H10O5)n formed in medium surface, was obtained from an associate culture Gluconacetobacter xylinus (formally Acetobacter xylinum) and Saccharomyces cerevisiae in green tea leaves, fruit residues (pineapple, papaya, orange), vegetables residues (beet), wine and collagen media in static condition , at 28 ± 2 ºC in 7 - 30 days cultivation. The Calendula officinalis hydroalcoholic extract was incorporated in the Biomembrane, due to its anti-inflammatory, anti-oxidants and cicatrizing proprieties. The biomembrane thickness, diameter and weight were measured. The productivity and conversion factor from cellulose to sugar were calculated. The biomembrane caracterization was performed by Differential Scanning Calorimetric, infrared spectroscopy, Brunauer-Emmett-Teller, resistance to tension, elongation, eletrocnic microscopy and raio-X difraction. In these analyses were verified that biomembrane obtained in different media were composed by cellulose, average porous size varied from 517.9 to 1582.0 nm, the resistance to tension varied from 0.76 to 4.32 kN/m and cristalinity index varied from 75% to 91%. The biomembrane thickness varied from 0,16 to 6,38 mm. It was performed 596 tests, the highest bacterial cellulose yield (8.23 ± 0.58 g cellulose/day) was obtained in papaya with orange (papaya juice: 50% v/v and orange juice: 19% v/v) in 7 cultivation days. The highest conversion factor (2,36 g cellulose/g sugar) was obtained in green tea medium in 25 days. The addition of 1.5% w/v collagen to the green tea media increased 2 times the biomembrane weight. The biomembrane absorption capacity for water and Marigold hydroalcoholic extract (1:1), were from 1.73 to 22 and, from 1.75 to 24 times dry weight, respectively. The Marigold extract improved the green tea biomembrane flexibility, cristalinity, and physical proprieties.

β-glucosidases are important components of the cellulase enzyme system in which they not only hydrolyse cellobiose to glucose, but also remove the feedback inhibition effects of cellobiose on exoglucanase and endoglucanase thereby increasing the rate of cellulose degradation to fermentable sugars. A total of 166 proteins were identified as β-glucosidases after manual BLASTp search on the Aspergillus comparative database from eight species. Evidence for Horizontal Gene Transfer (HGT) of bacterial origin of some β-glucosidase genes was provided by their lack of introns, absence of some fungal specific amino acid insertions in their sequences and unusual positions in phylogenetic trees showing similarities to bacterial proteins. A rapid plate assay based on Congo red methods was developed to study the optimum parameters such as pH and temperature for growth of strains and activities of the enzymes produced. Bacterial cellulose (BC) was produced by Gluconacetobacter xylinus. For the first time a fully detailed characterization by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD), Differential scanning calorimeter (DSC), Thermogravimetric analysis (TGA) and 13Carbon Solid State Nuclear Magnetic Resonance (SSNMR) of pure BC before and after treatment with a commercially available Aspergillus cellulase enzyme was demonstrated. Two encoding sequences for novel Aspergillus nidulans hydrophobin genes ANID_05290.1 and ANID_07327 that do not fall into either the class I or class II category of hydrophobins were successfully cloned. Two encoding sequences for a novel β-glucosidase gene from an Aspergillus niger strain from Nigeria were amplified and cloned from genomic DNA using PCR. Aspergillus nidulans β-glucosidases (AN2227 and AN1804) expressed in Pichia were purified to homogeneity by using ammonium sulphate precipitation and DEAE-Sephadex A-50 chromatography. Both enzymes had a remarkably broad pH and temperature profile. Further experiments on the development of a technology for lignocellulose degradation based on co-production of β-glucosidase with hydrophobin for biofuel production are suggested.