Cellulase and xylanase production at pilot scale by solid-state fermentation from coffee husk using specialized consortia : the consistency of the process and the microbial communities involved

Solid state fermentation is a promising technology however rising concerns related to scale up and reproducibility in a productive process. Coffee husk and a specialized inoculum were used in a 4.5L and then in 50L reactors to assess the reproducibility of a cellulase and hemicellulase production system. Fermentations were consistent in terms of cellulase production and microbial communities. The higher temperatures achieved when operating at 50L generated a shift on the microbial communities and a reduction of nearly 50% on cellulase production at pilot scale. In spite, an overall enzymatic production of 3.1±0.5FPUg⁻¹DM and 48±4Ug⁻¹DM for FPase and Xyl activities was obtained, respectively, with low deviation coefficients of 16 and 19% for FPase and Xyl production. Gaseous emissions assessment revealed an emission factor of 2.6·10⁻³kg volatile organic compounds per Mg of coffee husk and negligible NH₃, CH₄ and N₂O emissions

Long term enhanced solid-state fermentation : inoculation strategies for amylase production from soy and bread wastes by Thermomyces sp. in a sequential batch operation

The effect of two thermophilic amylase producing strains was evaluated using different mixtures of soy and bread wastes. Thermomyces sp. was found to be better inoculum than Geobacillus sp. for a soy and bread waste mixture (90:10 w/w respectively) producing a maximum enzyme activity of 39.9·103 U g−1 dry substrate. Three strategies (a, b, c) were evaluated for solid-state fermentation (SSF) operation in sequential batches. Fermented solids from each batch were used to inoculate the following batch: (a) solids at the moment of maximum biological activity; (b) final solids (end of the process, maximum amylase production); (c) final solids after enzymatic extraction. The evaluated strategies led to an increase in amylase production of 50, 500 and 98% for each strategy, respectively. This indicates the suitability of fermented solids to act as inoculum and the enhancement of amylase production compared to traditional batches. As one of the main challenges of SSF is the maintenance of a productive process along time, these results confirm SSF as an excellent option to produce amylases from organic wastes

Towards a competitive solid state fermentation : cellulases production from coffee husk by sequential batch operation and role of microbial diversity

The cost of cellulases is the main bottleneck for bioethanol production at commercial scale. Solid-state fermentation (SSF) is a promising technology that can potentially reduce cellulases cost by using wastes as substrates. In this work, a SSF system of 4.5L bioreactors was operated continuously by sequential batch operation using the fermented solids from one batch to inoculate the following batch. Coffee husk was used as lignocellulosic substrate. Compost was used as starter in the first batch to provide a rich microbiota. Two strategies were applied: using 10% fermented solids as inoculum in 48h batches (SB90) and using 50% solids in 24h batches (SB50). A consistent and robust production process was achieved by sequential batch operation. Similar cellulase activities around 10 Filter Paper Units per gram of dry solids were obtained through both strategies. Microbial diversity in the starting materials and in the final fermented solids was characterized by next generation sequencing. Microbial composition of both fermented solids was similar but the relative abundance of families and species was affected by the operation strategy used. Main bacteria in the final solids came from compost (families Sphingobacteriaceae, Paenibacillaceae and Xanthomonadaceae), while main fungi families came from coffee husk (families Phaffomycetaceae, Dipodascaceae and two unidentified families of the class of Tramellomycetes). There was a high presence of non-identified mycobiota in the fermented solids. Main identified species were the bacteria Pseudoxanthonomas taiwanensis (12.3% in SB50 and 6.1% in SB90) and Sphingobacterium composti (6.1% in SB50 and 2.6% in SB90) and the yeasts Cyberlindnera jardinii and Barnettozyma californica (17.8 and 4.1% respectively in SB50 and 34 and 9.1% in SB90), all four previously described as lignocellulose degraders. The development of these operational strategies and further biological characterization of the end product could eventually benefit the process economics by providing a standard and specialized inoculum for a continuous SSF for cellulases production

Microbial strategies for cellulase and xylanase production through solid-state fermentation of digestate from biowaste

Altres ajuts: Generalitat de Catalunya pre-doctoral grant (reference 2017 DI019)Solid-state fermentation (SSF) is a promising technology for producing bioproducts from organic wastes. The objective of this study is to assess the feasibility of using digestate as substrate to produce hydrolytic enzymes, mainly cellulase and xylanase, by exploring three different inoculation strategies: (i) SSF with autochthonous microbiota; (ii) non-sterile SSF inoculated with Trichoderma reesei and (iii) sequential batch operation to select a specialized inoculum, testing two different residence times. Native microbial population did not show a significant cellulase production, suggesting the need for a specialized inoculum. The inoculation of Trichoderma reesei did not improve the enzymatic activity. On the other hand, inconsistent operation was achieved during sequential batch reactor in terms of specific oxygen uptake rate, temperature and enzymatic activity profile. Low cellulase and xylanase activities were attained and the main hypotheses are non-appropriate biomass selection and some degree of hydrolysis by non-targeted proteases produced during fermentation

Composting of food wastes : status and challenges

This review analyses the main challenges of the process of food waste composting and examines the crucial aspects related to the quality of the produced compost. Although recent advances have been made in crucial aspects of the process, such composting microbiology, improvements are needed in process monitoring. Therefore, specific problems related to food waste composting, such as the presence of impurities, are thoroughly analysed in this study. In addition, environmental impacts related to food waste composting, such as emissions of greenhouse gases and odours, are discussed. Finally, the use of food waste compost in soil bioremediation is discussed in detail

Valorisation of digestate from biowaste through solid-state fermentation to obtain value added bioproducts : a first approach

Altres ajuts: Laura Mejias thanks Generalitat de Catalunya for her pre-doctoral grant (reference 2017 DI019)Digestate from biowaste was assessed as a potential source of bioproducts of commercial and industrial interest through solid-state fermentation. The targeted bioproducts were hydrolytic enzymes (cellulases and proteases from autochthonous microbiome), biosurfactants (sophorolipids produced from Starmella bombicola) and biopesticides (produced from Bacillus thuringiensis). Low cellulase production was observed within the range of 0.5-1.5 FPU g⁻¹ DM while protease production showed two discrete peaks of 66 ± 8 and 65 ± 3 U g⁻¹ DM at 3.5 and 48 h, respectively. Low sophorolipids production was also obtained, with a maximum yield of 0.02 g g⁻¹ DM using hygienised digestate supplemented with external sugar and fat sources. Biopesticides produced by B. thuringiensis were successfully at 72 h of operation, reaching a maximum spore production of 8.15 ± 0.04 (10⁷) CFU g⁻¹ DM and 2.85 ± 0.22 (10⁷) CFU g⁻¹ DM using sterile and hygienised digestate, respectively. These biopesticides could contribute to the substitution of chemically produced pesticides, moving towards a sustainable digestate management in a circular economy scheme

From wastes to high value added products : novel aspects of SSF in the production of enzymes

Solid-state fermentation (SSF), a process that occurs in the absence or near absence of water, has been used for the production of various high value added products such as enzymes and other organic components. This paper reviews the recent studies reported on the use of SSF for the production of enzymes: lipases, proteases, cellulases, hemicellulases, ligninases, glucoamylases, pectinases, and inulinases. The microorganisms used for fermentation are mostly fungi, and substrates are waste materials from the agriculture and food industry. This shows the advantages of SSF from an economical and environmental viewpoint. The paper provides an update on several issues, viz. wastes, microorganisms, and issues of scaling up and controlling the process of fermentation in solid state

Gaseous emissions during the solid state fermentation of different wastes for enzyme production at pilot scale

The emissions of volatile organic compounds (VOC), CH₄, N₂O and NH₃ during the solid state fermentation process of some selected wastes to obtain different enzymes have been determined at pilot scale. Orange peel + compost (OP), hair wastes + raw sludge (HW) and winterization residue + raw sludge (WR) have been processed in duplicate in 50 L reactors to provide emission factors and to identify the different VOC families present in exhaust gaseous emissions. Ammonia emission from HW fermentation (3.2 ± 0.5 kg Mg⁻¹ dry matter) and VOC emission during OP processes (18 ± 6 kg Mg⁻¹ dry matter) should be considered in an industrial application of these processes. Terpenes have been the most emitted VOC family during all the processes although the emission of sulphide molecules during HW SSF is notable. The most emitted compound was dimethyl disulfide in HW and WR processes, and limonene in the SSF of OP

Sustainable carbohydrase production using organic wastes through solid-state fermentation: operational strategies and microbial communities assessment

De acuerdo con la línea de investigación de fermentación en estado sólido (FES) que actualmente se desarrolla en el grupo de investigación de compostaje (GICOM) el objetivo principal de esta tesis es desarrollar un proceso sustentable y a una escala representativa para la producción de celulasas utilizando residuos orgánicos. Con el fin de lograr este objetivo se analizaron diferentes residuos orgánicos y su potencial para la producción de celulasas a escala de laboratorio. Además, se utilizó compost como inoculo mixto, con el objetivo de incorporar el matriz sólida microorganismos capaces de degradar material lignocelulósicos. Luego de la selección de los mejores substratos en reactores de 0.5 L, se llevó a cabo el escalamiento del proceso en reactores de 10 L and 50 L. Todos los experimentos fueron desarrollados en condiciones cercanas a adiabáticas utilizando reactores aislados térmicamente. Durante el desarrollo del proceso, se realizó la monitorización de la actividad biológica reflejada como la velocidad especifica de consumo de oxígeno (sVCO) y temperatura. El principal desafío a superar cuando se trabaja con FES y residuos orgánicos son, por un lado, el desarrollo de un proceso en continuo y, por otro lado, que el proceso desarrollado sea reproducible y consistente. Ambas temáticas fueron abordadas durante el desarrollo de la tesis. Un primer acercamiento a un proceso en continuo se desarrolló para la producción de amilasas utilizando fibra de soja y residuos de pan como substrato y Thermomyces sp como inoculo. Se evaluaron tres estrategias de operación en reactores secuenciales, retirando del reactor una parte del sólido fermentado en el momento de: máxima sVCO y máxima actividad de amilasas. En esta última se evaluó la reinoculación con y sin extracción enzimática. En todas las estrategias se obtuvo un incremento por sobre el 200%. Debido al éxito de la operación en continuo para la producción de amilasas, se desarrolló un proceso similar para la producción de celulasas utilizando las condiciones operacionales desarrolladas a escala laboratorio. Inicialmente se consideró la incorporación de compost como inóculo (10%) y cascarilla de café como substrato (90%). Después de conseguir el máximo de actividad de celulasas, el sistema se continuó operando en ciclos, substrayendo una cantidad determinada de material y reemplazándolo por substrato fresco. Se trabajó con tasas de intercambio de 50% y 90%. Finalmente se pudo desarrollar exitosamente un proceso en continuo para la producción de celulasas, logrando producciones sostenidas en el tiempo entre 8-9 unidades de actividad por gramos de materia seca. Adicionalmente, se caracterizó el sólido fermentado obtenido al final de la fermentación con la finalidad de identificar las poblaciones microbianas presentes. En este sólido se identificaron una amplia gama de bacterias y hongos capaces de degradar material lignocelulósico, por lo que se catalogó como un inóculo especializado que podría ser utilizado con la finalidad de desarrollar un proceso reproducible; sin necesidad de la adición continua de nuevo inóculo. Finalmente, se evaluaron a escala piloto (50 L) la reproducibilidad y consistencia del proceso utilizando el inóculo especializado y cascarilla de café como substrato en triplicados. Adicionalmente, se analizaron las emisiones gaseosas generadas durante el proceso con la finalidad de determinar los posibles requerimientos de una unidad de tratamiento de gases. Los resultados obtenidos en estos experimentos mostraron que tanto la dinámica del proceso como la producción enzimática fueron consistentes y robustas. Además, el proceso puede ser catalogado como amigable con el medioambiente, debido a la valorización de residuos orgánicos, así como también debido a los bajos factores de emisión generados por el sistema.According to the solid-state fermentation (SSF) research line of the composting research group GICOM, the main goal of this thesis is to develop a sustainable process for the production of carbohydrases using organic wastes in a representative scale. To achieve this goal, several organic wastes were screened on its potential to produce cellulase at laboratory scale. Also, compost was added as a complex mixture of biomass in order to provide cellulose-degrading microorganisms. After the selection of the two best substrates: orange peels and coffee husk using 0.5 L reactors, the scale up was assessed in 10 and 50 L reactors. All SSF were undertaken in near-adiabatic conditions using thermally isolated reactors with a continuous monitoring of the biological activity reflected as specific oxygen uptake rate (sOUR) and temperature. The main challenges to overcome in SSF using organic wastes is on one hand the possibility to work in a continuous configuration and, on the other hand, the reproducibility of the proposed process. These two aspects were assessed in this thesis. A first approach towards a continuous process was carried out for the production of amylase using bread waste and soy fiber as substrate and adding Thermomyces sp as inoculum. Three different strategies were developed in a sequential batch operation by removing part of the substrate in three diffeerent stages of the process: during maximum sOUR, maximum amylase activity and maximum amylase activity with a posterior enzymatic extraction. An increase of above 200% in amylase activity production was obtained in all the assessed strategies. Due to the success of the amylase production in a continuous configuration, the next step was to use the operational conditions obtained at laboratory scale for cellulase production and develop a sequential batch operation in 4.5L reactors. An initial compost addition as inoculum was considered only at the beginning of the fermentation. After reaching the maximum cellulase production the system started working in cycles, removing one part of the substrate and replacing it for fresh substrate. Two substrate exchange ratios were evaluated: 90% and 50%. Using this configuration, a continuous process was carried out, with a continuous production of cellulase and valorization of organic wastes and eliminating the requirement for fresh inoculum in each cycle. The sequential batch operation resulted in the successful production of cellulase with sustained values around 8-9 filter paper units per grams of dry matter in both configurations. In addition. the microbial communities present at the end of this operation was characterized, identifying several cellulose, lignin and hemicellulose degraders. This fermented solid was described as a specialized inoculum able to colonize the solid matrix of the reactor and provides the opportunity to develop a reproducible process. Finally, the reproducibility and consistency of the process was assessed in triplicates in a 50 L reactor using the specialized inoculum and coffee husk as the substrate. In addition the gaseous emissions were measured in order to fully understand the process and to determine the requirement of a gas treatment unit. Results showed that the process in consistent and robust with minor emissions of pollutants

Cellulase and xylanase production at pilot scale by solid-state fermentation from coffee husk using specialized consortia : the consistency of the process and the microbial communities involved

Solid state fermentation is a promising technology however rising concerns related to scale up and reproducibility in a productive process. Coffee husk and a specialized inoculum were used in a 4.5L and then in 50L reactors to assess the reproducibility of a cellulase and hemicellulase production system. Fermentations were consistent in terms of cellulase production and microbial communities. The higher temperatures achieved when operating at 50L generated a shift on the microbial communities and a reduction of nearly 50% on cellulase production at pilot scale. In spite, an overall enzymatic production of 3.1±0.5FPUg⁻¹DM and 48±4Ug⁻¹DM for FPase and Xyl activities was obtained, respectively, with low deviation coefficients of 16 and 19% for FPase and Xyl production. Gaseous emissions assessment revealed an emission factor of 2.6·10⁻³kg volatile organic compounds per Mg of coffee husk and negligible NH₃, CH₄ and N₂O emissions