Production of bioethanol from multiple waste streams of rice milling

This work describes the feasibility of using rice milling by-products as feedstock for bioethanol. Starch-rich residues (rice bran, broken, unripe and discolored rice) were individually fermented (20% w/v) through Consolidated Bioprocessing by two industrial engineered yeast secreting fungal amylases. Rice husk (20% w/v), mainly composed by lignocellulose, was pre-treated at 55 degrees C with alkaline peroxide, saccharified through optimized dosages of commercial enzymes (Cellic (R) CTec2) and fermented by the recombinant strains. Finally, a blend of all the rice by-products, formulated as a mixture (20% w/v) according to their proportions at milling plants, were co-processed to ethanol by optimized pre-treatment, saccharification and fermentation by amylolytic strains. Fermenting efficiency for each by-product was high (above 88% of the theoretical) and further confirmed on the blend of residues (nearly 52 g/L ethanol). These results demonstrated for the first time that the co-conversion of multiple waste streams is a promising option for second generation ethanol production

Engineering robust Saccharomyces cerevisiae yeast for consolidated bioprocessing of lignocellulose into bioethanol

Second generation bioethanol, making use of the polysaccharides included in the lignocellulosic biomass, represents a promising alternative approach to overcome the limitations revealed by first generation bioethanol. The main issue hindering the effective industrial scale utilization of biomass is the lack of low-cost technology. In fact, lignocellulose hydrolysis requires expensive pre-treatments and large dosages of commercial enzymes. Moreover, feedstock pre-treatment results in the formation of inhibitors, mainly weak acids (acetic and formic), furans (furfural and 5-hydroxymethyl-2-furaldehyde) and phenolics, which affect the fermentation phase. Consolidated BioProcessing (CBP) of lignocellulosic biomass is gaining increasing attention as a potential strategy to reduce production costs both by integrating different production steps and by lowering the need for supplying of commercial cellulases. As no naturally occurring fermenting microorganism suitable for CBP has been described yet, genetic engineering of highly fermentative microorganisms, particularly yeast, will be required. To further improve the economic feasibility of the process in industrial scenarios, the search of robust yeast with high inhibitors tolerance as platform for genetic engineering would be desired. In this study, a collection of wild type Saccharomyces cerevisiae yeast, previously selected for their robustness and high ethanol yield, was characterized for inhibitors tolerance on synthetic mixtures of the inhibitors typical of lignocellulosic pre-hydrolysates and on real pre-hydrolysates, rich in these toxic compounds. The best performing strain was chosen as a robust candidate for the expression of three fungal β-glucosidases by δ-integration, together with the benchmark strain Ethanol Red®, currently utilized in industrial bioethanol production. Similarly, two wild-type yeast that were previously successfully used as parental to develop CBP strains, were used for the same purpose. Among the cellulases required for cellulose degradation, β-glucosidase was selected as it plays a key role in the process, representing the rate limiting enzyme. A large amount of recombinant clones, secreting β-glucosidases from the fungal species Saccharomycopsis fibuligera and Phanerochaete chrysosporium, were obtained. The engineered clones were firstly screened for high enzyme activity in a quantitative assay, using esculin as substrate. The β-glucosidase activity of the best performing strains was then quantified on pNPG. One recombinant able to produce high amounts of β-glucosidase demonstrated to be mitotically-stable and capable of sustaining the growth in presence of cellobiose as sole carbon source. The enzymatic activity of the recombinant was characterized in vitro in terms of enzyme localization, optimal pH and temperature, and stability. The fermentative abilities were assessed in defined medium containing cellobiose. The obtained recombinant showed comparable performances to the parental strain on glucose, indicating that β-glucosidase secretion does not cause any severe metabolic burden to the host. Further, the engineered strain could display high ethanol yield when fermenting cellobiose, comparable to those of a laboratory yeast strain expressing the same β-glucosidase via multicopy episomal plasmid, despite the remarkable disadvantage of lower gene copy number guaranteed by gene integration. This study reports the successful construction of S. cerevisiae strains capable of tolerating high inhibitors concentrations and expressing fungal β-glucosidases. To our knowledge, this work represents the first attempt to produce a CBP microorganism for lignocellulosic bioethanol via integration of β-glucosidases into tolerant yeast selected for thermotolerance and resistance to the inhibitors typically present in lignocellulosic pre-hydrolysates. The fermentation performances of the engineered strain will next be studied on sugarcane bagasse hydrolysate, with the aim to confirm the inhibitors tolerance traits

Utilisation of wheat bran as a substrate for bioethanol production using recombinant cellulases and amylolytic yeast

Wheat bran, generated from the milling of wheat, represents a promising feedstock for the production of bioethanol. This substrate consists of three main components: starch, hemicellulose and cellulose. The optimal conditions for wheat bran hydrolysis have been determined using a recombinant cellulase cocktail (RCC), which contains two cellobiohydrolases, an endoglucanase and a beta-glucosidase. The 10% (w/v, expressed in terms of dry matter) substrate loading yielded the most glucose, while the 2% loading gave the best hydrolysis efficiency (degree of saccharification) using unmilled wheat bran. The ethanol production of two industrial amylolytic Saccharomyces cerevisiae strains, MEL2[TLG1-SFA1] and M2n [TLG1-SFA1], were compared in a simultaneous saccharification and fermentation (SSF) for 10% wheat bran loading with or without the supplementation of optimised RCC. The recombinant yeasts. cerevisiae MEL2[TLG1-SFA1] and M2n[TLG1-SFA1] completely hydrolysed wheat bran's starch producing similar amounts of ethanol (5.3 +/- 0.14 g/L and 5.0 +/- 0.09 g/L, respectively). Supplementing SSF with RCC resulted in additional ethanol production of about 2.0 g/L. Scanning electron microscopy confirmed the effectiveness of both RCC and engineered amylolytic strains in terms of cellulose and starch depolymerisatio

Metabolomic alterations do not induce metabolic burden in the industrial yeast M2n[pBKD2-Pccbgl1]-C1 engineered by multiple δ-integration of a fungal β-glucosidase gene

CITATION: Favaro L., et al. 2019. Metabolomic alterations do not induce metabolic burden in the industrial yeast M2n[pBKD2-Pccbgl1]-C1 engineered by multiple d-integration of a fungal b-glucosidase gene. Frontiers in Bioengineering and Biotechnology. 7:376. doi:10.3389/fbioe.2019.00376The original publication is available at https://www.frontiersin.org/articles/10.3389/fbioe.2019.00376/fullIn the lignocellulosic yeast development, metabolic burden relates to redirection of resources from regular cellular activities toward the needs created by recombinant protein production. As a result, growth parameters may be greatly affected. Noteworthy, Saccharomyces cerevisiae M2n[pBKD2-Pccbgl1]-C1, previously developed by multiple d-integration of the b-glucosidase BGL3, did not show any detectable metabolic burden. This work aims to test the hypothesis that the metabolic burden and the metabolomic perturbation induced by the d-integration of a yeast strain, could differ significantly. The engineered strain was evaluated in terms of metabolic performances and metabolomic alterations in different conditions typical of the bioethanol industry. Results indicate that the multiple d-integration did not affect the ability of the engineered strain to grow on different carbon sources and to tolerate increasing concentrations of ethanol and inhibitory compounds. Conversely, metabolomic profiles were significantly altered both under growing and stressing conditions, indicating a large extent of metabolic reshuffling involved in the maintenance of the metabolic homeostasis. Considering that four copies of BGL3 gene have been integrated without affecting any parental genes or promoter sequences, deeper studies are needed to unveil the mechanisms implied in these metabolomic changes, thus supporting the optimization of protein production in engineered strains.Padova Universityhttps://www.frontiersin.org/articles/10.3389/fbioe.2019.00376/fullPublisher’s versio

The impact of COVID-19 quarantine on patients with dementia and family caregivers: a nation-wide survey.

Quarantine for COVID-19 is associated with an acute worsening of clinical symptoms in patients with dementia as well as increase of caregivers’ burden. These findings emphasize the importance to implement new strategies to mitigate the effects of quarantine in patients with dementia

Behavioral and psychological effects of coronavirus disease-19 quarantine in patients with dementia

Background: In March 2020, the World Health Organization declared a global pandemic due to the novel coronavirus SARS-CoV-2 and several governments planned a national quarantine in order to control the virus spread. Acute psychological effects of quarantine in frail elderly subjects with special needs, such as patients with dementia, have been poorly investigated. The aim of this study was to assess modifications of neuropsychiatric symptoms during quarantine in patients with dementia and their caregivers. Methods: This is a sub-study of a multicenter nation-wide survey. A structured telephone interview was delivered to family caregivers of patients with diagnosis of Alzheimer disease (AD), dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), and vascular dementia (VD), followed regularly at 87 Italian memory clinics. Variations in behavioral and psychological symptoms (BPSD) were collected after 1 month since quarantine declaration and associations with disease type, severity, gender, and caregiver\u2019s stress burden were analyzed. Results: A total of 4,913 caregivers participated in the survey. Increased BPSD was reported in 59.6% of patients as worsening of preexisting symptoms (51.9%) or as new onset (26%), and requested drug modifications in 27.6% of these cases. Irritability, apathy, agitation, and anxiety were the most frequently reported worsening symptoms and sleep disorder and irritability the most frequent new symptoms. Profile of BPSD varied according to dementia type, disease severity, and patients\u2019 gender. Anxiety and depression were associated with a diagnosis of AD (OR 1.35, CI: 1.12\u20131.62), mild to moderate disease severity and female gender. DLB was significantly associated with a higher risk of worsening hallucinations (OR 5.29, CI 3.66\u20137.64) and sleep disorder (OR 1.69, CI 1.25\u20132.29), FTD with wandering (OR 1.62, CI 1.12\u20132.35), and change of appetite (OR 1.52, CI 1.03\u20132.25). Stress-related symptoms were experienced by two-thirds of caregivers and were associated with increased patients\u2019 neuropsychiatric burden (p<0.0001). Conclusion: Quarantine induces a rapid increase of BPSD in approximately 60% of patients and stress-related symptoms in two-thirds of caregivers. Health services need to plan a post-pandemic strategy in order to address these emerging needs

"Delirium Day": A nationwide point prevalence study of delirium in older hospitalized patients using an easy standardized diagnostic tool

Background: To date, delirium prevalence in adult acute hospital populations has been estimated generally from pooled findings of single-center studies and/or among specific patient populations. Furthermore, the number of participants in these studies has not exceeded a few hundred. To overcome these limitations, we have determined, in a multicenter study, the prevalence of delirium over a single day among a large population of patients admitted to acute and rehabilitation hospital wards in Italy. Methods: This is a point prevalence study (called "Delirium Day") including 1867 older patients (aged 65 years or more) across 108 acute and 12 rehabilitation wards in Italian hospitals. Delirium was assessed on the same day in all patients using the 4AT, a validated and briefly administered tool which does not require training. We also collected data regarding motoric subtypes of delirium, functional and nutritional status, dementia, comorbidity, medications, feeding tubes, peripheral venous and urinary catheters, and physical restraints. Results: The mean sample age was 82.0 \ub1 7.5 years (58 % female). Overall, 429 patients (22.9 %) had delirium. Hypoactive was the commonest subtype (132/344 patients, 38.5 %), followed by mixed, hyperactive, and nonmotoric delirium. The prevalence was highest in Neurology (28.5 %) and Geriatrics (24.7 %), lowest in Rehabilitation (14.0 %), and intermediate in Orthopedic (20.6 %) and Internal Medicine wards (21.4 %). In a multivariable logistic regression, age (odds ratio [OR] 1.03, 95 % confidence interval [CI] 1.01-1.05), Activities of Daily Living dependence (OR 1.19, 95 % CI 1.12-1.27), dementia (OR 3.25, 95 % CI 2.41-4.38), malnutrition (OR 2.01, 95 % CI 1.29-3.14), and use of antipsychotics (OR 2.03, 95 % CI 1.45-2.82), feeding tubes (OR 2.51, 95 % CI 1.11-5.66), peripheral venous catheters (OR 1.41, 95 % CI 1.06-1.87), urinary catheters (OR 1.73, 95 % CI 1.30-2.29), and physical restraints (OR 1.84, 95 % CI 1.40-2.40) were associated with delirium. Admission to Neurology wards was also associated with delirium (OR 2.00, 95 % CI 1.29-3.14), while admission to other settings was not. Conclusions: Delirium occurred in more than one out of five patients in acute and rehabilitation hospital wards. Prevalence was highest in Neurology and lowest in Rehabilitation divisions. The "Delirium Day" project might become a useful method to assess delirium across hospital settings and a benchmarking platform for future surveys