Getting the right tail right: Modeling tails of health expenditure distributions.

Health expenditure data almost always include extreme values, implying that the underlying distribution has heavy tails. This may result in infinite variances as well as higher-order moments and bias the commonly used least squares methods. To accommodate extreme values, we propose an estimation method that recovers the right tail of health expenditure distributions. It extends the popular two-part model to develop a novel three-part model. We apply the proposed method to claims data from one of the biggest German private health insurers. Our findings show that the estimated age gradient in health care spending differs substantially from the standard least squares method.

Obstructive sleep apnea severity and prevalent atrial fibrillation in a sleep clinic cohort with versus without excessive daytime sleepiness.

BACKGROUND: Obstructive sleep apnea (OSA) is associated with atrial fibrillation (AF) in cardiac cohorts. Less is known regarding the magnitude of this association in a sleep clinic cohort with vs. without excessive daytime sleepiness (EDS). OBJECTIVES: To explore the association of OSA severity with AF in a sleep clinic cohort stratified by EDS. PATIENTS AND METHODS: All consecutive adults (n = 3814) admitted to the Skaraborg Hospital, Sweden between Jan 2005 and December 2011 were registered in a local database, and the follow-up ended in December 2018. OSA was defined as an apnea-hypopnea index (AHI) ≥5 events/h. Mild OSA was defined as AHI ≥5 & AHI<15 events/h; moderate OSA as AHI ≥15 & AHI<30 events/h; and severe OSA as AHI ≥30 events/h. EDS was defined as an Epworth Sleepiness Scale score ≥11. We conducted cross-sectional analyzes of the prevalent AF across the OSA severity categories and logistic regression analyzes stratified by EDS. RESULTS: In all, 202 patients (5.3%) had AF at baseline, 1.6% in no-OSA, 3.9% in mild OSA, 5.2% in moderate OSA, and 7.6% in severe OSA, respectively (p < 0.001). The stratified analyzes revealed that patients with severe OSA without EDS had an increased risk for prevalent AF (OR 2.54, 95% CI 1.05-6.16; p = 0.039) independent of the confounding factors. CONCLUSIONS: There was an independent dose-response relationship between OSA and prevalent AF among the non-sleepy phenotype in this sleep clinic cohort. Since adherence to OSA treatment is challenging in the absence of EDS, these patients may have increased risk for adverse cardiovascular outcomes.

The performance of EuroSCORE II in CABG patients in relation to sex, age, and surgical risk: a nationwide study in 14,118 patients.

BACKGROUND: To determine the discriminative accuracy and calibration of EuroSCORE II in relation to age, sex, and surgical risk in a large nationwide coronary artery bypass grafting (CABG) cohort. METHODS: All 14,118 patients undergoing isolated CABG in Sweden during 2012-2017 were included. Individual patient data were taken from the SWEDEHEART registry. Patients were divided by age (< 60, 60-69, 70-79, ≥ 80 years), sex, and surgical risk (low: EuroSCORE < 4%, intermediate: 4-8%, high: > 8%). Discriminative accuracy was determined by the area under the receiver operating characteristic curve (AUC) and calibration by the observed/estimated (O/E) mortality ratio at 30 days. RESULTS: AUC and O/E ratio were 0.82 (95% CI 0.79-0.85) and 0.58 (0.50-0.66) overall, 0.82 (0.79-0.86) and 0.57 (0.48-0.66) in men, and 0.79 (0.73-0.85) and 0.60 (0.47-0.75) in women. Regarding age, discriminative accuracy was highest in patients aged 60-69 years (AUC: 0.86 [0.80-0.93]) but was satisfactory in all groups (AUC: 0.74-0.80). O/E ratio varied from 0.26 for patients > 60 years to 0.90 for patients > 80 years. Regarding surgical risk, AUC and O/E ratio were 0.63 (0.44-0.83) and 0.18 (0.09-0.30) in low-risk patients, 0.60 (0.55-0.66) and 0.57 (0.46-0.68) in intermediate-risk patients, and 0.78 (0.73-0.83) and 0.78 (0.64-0.92) in high-risk patients. CONCLUSIONS: EuroSCORE II had good discriminative accuracy independently of sex and age, but markedly overestimated mortality risk, especially in younger patients. Accuracy and calibration were better in high-risk patients than in low-risk and intermediate-risk patients.

Identifying targets for increased biogas production through chemical and organic matter characterization of digestate from full-scale biogas plants: what remains and why?

BACKGROUND: This study examines the destiny of macromolecules in different full-scale biogas processes. From previous studies it is clear that the residual organic matter in outgoing digestates can have significant biogas potential, but the factors dictating the size and composition of this residual fraction and how they correlate with the residual methane potential (RMP) are not fully understood. The aim of this study was to generate additional knowledge of the composition of residual digestate fractions and to understand how they correlate with various operational and chemical parameters. The organic composition of both the substrates and digestates from nine biogas plants operating on food waste, sewage sludge, or agricultural waste was characterized and the residual organic fractions were linked to substrate type, trace metal content, ammonia concentration, operational parameters, RMP, and enzyme activity. RESULTS: Carbohydrates represented the largest fraction of the total VS (32-68%) in most substrates. However, in the digestates protein was instead the most abundant residual macromolecule in almost all plants (3-21 g/kg). The degradation efficiency of proteins generally lower (28-79%) compared to carbohydrates (67-94%) and fats (86-91%). High residual protein content was coupled to recalcitrant protein fractions and microbial biomass, either from the substrate or formed in the degradation process. Co-digesting sewage sludge with fat increased the protein degradation efficiency with 18%, possibly through a priming mechanism where addition of easily degradable substrates also triggers the degradation of more complex fractions. In this study, high residual methane production (> 140 L CH4/kg VS) was firstly coupled to operation at unstable process conditions caused mainly by ammonia inhibition (0.74 mg NH3-N/kg) and/or trace element deficiency and, secondly, to short hydraulic retention time (HRT) (55 days) relative to the slow digestion of agricultural waste and manure. CONCLUSIONS: Operation at unstable conditions was one reason for the high residual macromolecule content and high RMP. The outgoing protein content was relatively high in all digesters and improving the degradation of proteins represents one important way to increase the VS reduction and methane production in biogas plants. Post-treatment or post-digestion of digestates, targeting microbial biomass or recalcitrant protein fractions, is a potential way to achieve increased protein degradation.

Long-term outcome of patients undergoing re-exploration for bleeding following cardiac surgery: a SWEDEHEART study.

OBJECTIVES: Excessive bleeding leading to re-exploration is a severe complication of cardiac surgical procedures, associated with early postoperative morbidity and mortality. Less is known about the long-term outcome of these patients. We evaluated the impact of re-exploration after cardiac surgery on peri- and postoperative morbidity and mortality, as well long-term mortality, in a well-defined nationwide population. METHODS: In this retrospective study, 48 060 consecutive patients undergoing coronary artery bypass grafting (CABG) and/or valve surgery from 2006 to 2015 were analysed. Multivariable logistic regression was used to identify factors associated with re-exploration, morbidity and mortality. Cox regression analysis was implemented to explore the association between re-exploration and long-term mortality. The mean follow-up time was 4.6 years (range 0-10 years) with follow-up time set at 31 December 2015. RESULTS: Overall, 2371 patients (4.9%) underwent re-exploration. Factors associated with re-exploration included advanced age, procedures other than isolated CABG and acute surgery. Re-explored patients had an increased risk of unadjusted mortality at 30, 90 and beyond 90 days (all P < 0.001). Significance was maintained after adjustment at 30 days [odds ratio: 3.94, 95% confidence interval (CI): 3.19-4.85, P < 0.001] and 90 days (odds ratio: 3.79, 95% CI: 3.14-4.55, P < 0.001), but not with long-term mortality (hazard ratio: 1.02, 95% CI: 0.91-1.15, P = 0.712). Furthermore, re-exploration was independently associated with other postoperative complications, e.g. prolonged hospital stay, stroke and renal injury. CONCLUSIONS: Patients who are re-explored for bleeding within 24 h have almost four-fold higher odds of mortality within 3 months post-procedure. However, the increased risk of death following re-exploration is not maintained in the long term.

A comparison of the in vitro effects of three fibrinogen concentrates on clot strength in blood samples from cardiac surgery patients.

BACKGROUND: Fibrinogen concentrate is used clinically to improve hemostasis in bleeding patients. We investigated and compared the efficacy of three commercially available fibrinogen concentrates to improve clot strength in blood samples from cardiac surgery patients. OBJECTIVES: Postoperative blood samples were collected from 23 cardiac surgery patients. Samples were each divided into four vials, each supplemented with 1.125 mg of fibrinogen of one of three fibrinogen concentrates (RiaSTAP® , Fibryga® , FibCLOT® ), or placebo. The fibrinogen dose corresponded to 2.5 g per 70 kg of body weight. Clot strength after supplementation was assessed in duplicate with rotational thromboelastometry (ROTEM® ) using FIBTEM maximum clot firmness, EXTEM clot formation time, and maximum clot firmness assays. RESULTS: In vitro fibrinogen concentrate supplementation of the samples resulted in higher plasma fibrinogen concentrations and improved clot strength with all three concentrates. Supplementation with FibCLOT increased FIBTEM maximum clot firmness (+46% [25th-75th percentile 35-55] compared to placebo) significantly more than did supplementation with Fibryga (+26% [21-35]) and RiaSTAP (+29% [22-47], p < .001). FibCLOT supplementation also shortened EXTEM clot formation time and increased EXTEM maximum clot firmness to a greater extent than did the other concentrates (both p < .001). CONCLUSIONS: At the selected dose, FibCLOT was more effective than Fibryga and RiaSTAP in restoring clot strength in postoperative blood samples from cardiac surgery patients. These results may have implications for the choice of fibrinogen concentrate and dosing.

Disease and fertility: Evidence from the 1918-19 influenza pandemic in Sweden.

What are the consequences of a severe health shock like an influenza pandemic on fertility? Using rich administrative data and a difference-in-differences approach, we evaluate fertility responses to the 1918-19 influenza pandemic in Sweden. We find evidence of a small baby boom following the end of the pandemic, but we show that this effect is second-order compared to a strong long-term negative fertility effect. Within this net fertility decline there are compositional effects: we observe a relative increase in births to married women and to better-off families. Several factors - including disruptions to the marriage market and income effects - contribute to the long-term fertility reduction. The results are consistent with studies that find a positive fertility response following natural disasters, but we show that this effect is short-lived.

Twisted Intramolecular Charge Transfer (TICT) Controlled by Dimerization: An Overlooked Piece of the TICT Puzzle.

Organic dyes have shown high efficiencies in solar cells, which is mainly attributed to the push-pull strategy present in such dyes upon attaching to the semiconductor surfaces. We deeply studied the fundamental photophysical properties of cyanoacrylic dyes, mostly the L1 dye, and found unique emission properties that depend on many factors such as the solvent polarity and the concentration of the dye and could present a complete emission picture about this family of dyes. The L1 dye shows an intramolecular charge transfer (ICT) emission state at low concentrations (approximately nanomolar scale) and shows a twisted intramolecular charge transfer (TICT) emission state in specific solvents upon increasing the concentration to the micromolar scale. Moreover, the associated emission lifetimes of the ICT and TICT states of the L1 dye depend on solvent basicity, highlighting the role of hydrogen bond formation on controlling such states. Density functional theory calculations are performed to gain insight into the photophysical properties of the dye and revealed that H-bonding between the carboxylic groups triggers the dimerization at low concentrations. Using femtosecond transient absorption, we assigned the rate of TICT formation to be in the range (160-650 fs)-1, depending on the size of the studied cyanoacrylic dye. Therefore, we add herein a new dimension for controlling the formation of the TICT state, in addition to the solvent polarity and acceptor strength parameters. These findings are not limited to the studied dyes, and we expect that numerous organic carboxylic acids dyes show similar properties.

The risk of dementia after coronary artery bypass grafting in relation to age and sex.

INTRODUCTION: We examined the long-term risk of dementia after coronary artery bypass grafting (CABG) in relation to age and sex. METHODS: All CABG patients in Sweden 1992-2015 (n = 111,335), and matched controls (n = 222,396) were included in a population-based study. Adjusted hazard ratios (aHR) for all-cause dementia, vascular dementia, and Alzheimer's disease were calculated. RESULTS: There was no difference in the risk for all-cause dementia between CABG patients and control subjects (aHR 0.98 [95% confidence interval 0.95 to 1.02]). CABG patients <65 years and 65 to 74 years had higher risk (aHR 1.29 [1.17-1.42] and 1.08 [1.02-1.13], respectively), and patients ≥75 years had lower risk (aHR 0.76 [0.71-0.81]). The highest risk was observed in women <65 years (aHR 1.64 [1.31-2.05]). DISCUSSION: Overall, the long-term risk for all-cause dementia does not differ between CABG patients and the general population. Younger patients have a higher risk, while older patients have a lower risk, compared to controls.

Does Prolonged Education Causally Affect Dementia Risk When Adult Socioeconomic Status Is Not Altered? A Swedish Natural Experiment in 1.3 Million Individuals.

Intervening on modifiable risk factors to prevent dementia is of key importance, since progress-modifying treatments are not currently available. Education is inversely associated with dementia risk, but causality and mechanistic pathways remain unclear. We aimed to examine the causality of this relationship in Sweden using, as a natural experiment, data on a compulsory schooling reform that extended primary education by 1 year for 70% of the population between 1936 and 1949. The reform introduced substantial exogenous variation in education that was unrelated to pupils' characteristics. We followed 18 birth cohorts (n = 1,341,842) from 1985 to 2016 (up to ages 79-96 years) for a dementia diagnosis in the National Inpatient and Cause of Death registers and fitted Cox survival models with stratified baseline hazards at the school-district level, chronological age as the time scale, and cohort indicators. Analyses indicated very small or negligible causal effects of education on dementia risk (main hazard ratio = 1.01, 95% confidence interval: 0.98, 1.04). Multiple sensitivity checks considering only compliers, the pre-/post- design, differences in health-care-seeking behavior, and the impact of exposure misclassification left the results essentially unaltered. The reform had limited effects on further adult socioeconomic outcomes, such as income. Our findings suggest that without mediation through adult socioeconomic position, education cannot be uncritically considered a modifiable risk factor for dementia.

Socioeconomic Factors, Secondary Prevention Medication, and Long-Term Survival After Coronary Artery Bypass Grafting: A Population-Based Cohort Study From the SWEDEHEART Registry.

Background Low income and short education have been found to be independently associated with inferior survival after coronary artery bypass grafting (CABG), whereas the use of secondary prevention medications is associated with improved survival. We investigated whether underusage of secondary prevention medications contributes to the inferior long-term survival in CABG patients with a low income and short education. Methods and Results Patients who underwent CABG in Sweden between 2006 to 2015 and survived at least 6 months after discharge (n=28 448) were included in a population-based cohort study. Individual patient data from 5 national registries, including the SWEDEHEART (Swedish Web System for Enhancement and Development of Evidence-Based Care in Heart Disease Evaluated According to Recommended Therapies) registry, covering dispensing of secondary prevention medications (statins, platelet inhibitors, ß-blockers, and RAAS inhibitors), socioeconomic factors, patient characteristics, comorbidity, and long-term mortaity were merged. All-cause mortality risk was estimated using multivariable Cox regression models adjusted for patient characteristics, baseline comorbidities, time-updated secondary prevention medications, and socioeconomic status. Long-term mortality was higher in patients with a low income and short education. Statins and platelet inhibitors were dispensed less often to patients with a low income, both at baseline and after 8 years. The decline in dispensing over time was steeper for low-income patients. Short education was not associated with reduced dispensing of any secondary prevention medication. Use of statins (adjusted hazard ratio=0.57 [95% CI, 0.53-0.61]), RAAS inhibitors (adjusted hazard ratio=0.78 [0.73-0.84]), and platelet inhibitors (adjusted hazard ratio=0.74 [0.68-0.80]) were associated with reduced long-term mortality irrespective of socioeconomic status. Conclusions Secondary prevention medications are dispensed less often after CABG to patients with low income. Underusage of secondary prevention medications after CABG is associated with increased mortality risk independently of income and extent of education.

Fibrinogen Concentrate to Cardiac Surgery Patients with Ongoing Bleeding does not Increase the Risk of Thromboembolic Complications or Death.

BACKGROUND: We investigated whether fibrinogen concentrate administration to bleeding patients is associated with an increased risk of thromboembolic complications and death. METHODS: All consecutive patients who underwent first-time cardiac surgery at Sahlgrenska University Hospital from 2009 to 2014 were included. Patients, who had received fibrinogen concentrate, were compared with those who had not received fibrinogen concentrate. The primary endpoint was a composite of thromboembolic complications and death within 1 year after surgery. Secondary endpoints included the composite and mortality within 30 days and mortality within 1 year after surgery. Multivariable logistic regression and Cox regression models were used to compare the groups. Propensity score (PS)-matched models were used for sensitivity analyses. RESULTS: A total of 5,408 patients were included in the present study, of which 564 (10.4%) received fibrinogen concentrate. The composite endpoint occurred in 3.5% of patients at 30 days and 10.5% at 1 year. There was no significant difference between the groups in the composite endpoint at 1 year (adjusted hazard ratio [HR]: 1.11, 95% confidence interval [CI]: 0.84-1.46, p = 0.45) or in the secondary endpoints, that is, mortality at 1 year (adjusted HR: 1.38, 95% CI: 0.93-2.04, p = 0.11), composite at 30 days (adjusted odds ratio [OR]: 1.07, 95% CI: 0.64-1.81, p = 0.79) and mortality at 30 days (adjusted OR: 1.00, 95% CI: 0.51-1.96, p = 0.50). The results of the sensitivity analyses were consistent with those of main analyses. CONCLUSION: Perioperative administration of fibrinogen concentrate to bleeding cardiac surgery patients is not associated with an increased risk of thromboembolic complications or death.

Secondary prevention medications after coronary artery bypass grafting and long-term survival: a population-based longitudinal study from the SWEDEHEART registry.

AIMS: To evaluate the long-term use of secondary prevention medications [statins, ß-blockers, renin-angiotensin-aldosterone system (RAAS) inhibitors, and platelet inhibitors] after coronary artery bypass grafting (CABG) and the association between medication use and mortality. METHODS AND RESULTS: All patients who underwent isolated CABG in Sweden from 2006 to 2015 and survived at least 6 months after discharge were included (n = 28 812). Individual patient data from SWEDEHEART and other mandatory nationwide registries were merged. Multivariable Cox regression models using time-updated data on dispensed prescriptions were used to assess associations between medication use and long-term mortality. Statins were dispensed to 93.9% of the patients 6 months after discharge and to 77.3% 8 years later. Corresponding figures for ß-blockers were 91.0% and 76.4%, for RAAS inhibitors 72.9% and 65.9%, and for platelet inhibitors 93.0% and 79.8%. All medications were dispensed less often to patients ≥75 years. Treatment with statins [hazard ratio (HR) 0.56, 95% confidence interval (95% CI) 0.52-0.60], RAAS inhibitors (HR 0.78, 95% CI 0.73-0.84), and platelet inhibitors (HR 0.74, 95% CI 0.69-0.81) were individually associated with lower mortality risk after adjustment for age, gender, comorbidities, and use of other secondary preventive drugs (all P < 0.001). There was no association between ß-blockers and mortality risk (HR 0.97, 95% CI 0.90-1.06; P = 0.54). CONCLUSION: The use of secondary prevention medications after CABG was high early after surgery but decreased significantly over time. The results of this observational study, with inherent risk of selection bias, suggest that treatment with statins, RAAS inhibitors, and platelet inhibitors is essential after CABG whereas the routine use of ß-blockers may be questioned.

Towards implementing hierarchical porous zeolitic imidazolate frameworks in dye-sensitized solar cells.

A one-pot method for encapsulation of dye, which can be applied for dye-sensitized solar cells (DSSCs), and synthesis of hierarchical porous zeolitic imidazolate frameworks (ZIF-8), is reported. The size of the encapsulated dye tunes the mesoporosity and surface area of ZIF-8. The mesopore size, Langmuir surface area and pore volume are 15 nm, 960-1500 m2 · g-1 and 0.36-0.61 cm3 · g-1, respectively. After encapsulation into ZIF-8, the dyes show longer emission lifetimes (greater than 4-8-fold) as compared to the corresponding non-encapsulated dyes, due to suppression of aggregation, and torsional motions.

Social Factors, Sex, and Mortality Risk After Coronary Artery Bypass Grafting: A Population-Based Cohort Study.

Background Little is known of the impact of social factors on mortality after coronary artery bypass grafting ( CABG ). We explored sex- and age-specific associations between mortality risk after CABG and marital status, income, and education. Methods and Results This population-based register study included 110 742 CABG patients (21.3% women) from the SWEDEHEART registry (Swedish Web-system for Enhancement and Development of Evidence-based Care in Heart Disease Evaluated According to Recommended Therapies) operated 1992 to 2015. Cox regression models were used to study the relation between social factors and all-cause mortality. Never having been married compared with being married/cohabiting was associated with a higher risk in women than in men (hazard ratio 1.32, 95% CI 1.20-1.44) versus 1.17 (1.13-1.22), P=0.030 between sex. The lowest income quintile, compared with the highest, was associated with higher risk in men than in women (hazard ratio 1.44 [1.38-1.51] versus 1.25 [1.14-1.38], P=0.0036). Lowest education level was associated with higher risk without sex difference (hazard ratio 1.15 [1.11-1.19] versus 1.25 [1.16-1.35], P=0.75). For unmarried women aged 60 years at surgery with low income and low education, mortality 10 years after surgery was 18%, compared with 11% in married women with high income and higher education level. The median life expectancy was 4.8 years shorter. Corresponding figures for 60-year-old men were 21% versus 12% mortality risk at 10 years and 5.0 years shorter life expectancy. Conclusions There are strong associations between social factors and mortality risk after CABG in both men and women. These results emphasize the importance of developing and implementing secondary prevention strategies for CABG patients with disadvantages in social factors.

Relative impacts of fishing and eutrophication on coastal fish assessed by comparing a no-take area with an environmental gradient.

Understanding the relative impacts of pressures on coastal ecosystems is central for implementing relevant measures to reach environmental management objectives. Here, survey data on the species and size composition of coastal fish are evaluated in relation to fishing and eutrophication, by comparing a long-standing no-take area to an environmental gradient in the Baltic Sea. The no-take area represents an intermediate eutrophication level, but the species composition resembles that seen at low eutrophication in areas with fishing. The catch biomass of piscivores is 2-3 times higher in the no-take area than in the other areas, while the biomass of Cyprinids, generally benefitted by eutrophication, corresponds to that of areas with low eutrophication. The results support that fishing may generate eutrophication-like effects, and, conversely, that no-take areas may contribute to improving environmental status in impacted areas by enhancing piscivores, which in turn may contribute to further improvement in the food web.

Regional fire scenarios in Spain: Linking landscape dynamics and fire regime for wildfire risk management.

Fire scenarios are multiscale land-type planning units for a fire regime with different applications at national, regional, and local scales. The main aim of this research is to implement a methodology integrating landscape dynamics and wildfire risk from a context-specific approach, to identify current fire scenarios at a regional scale in the Spanish Central Mountain Range. These homogeneous areas are linked to different stages of a territorial dynamics model called wildfire generations and related to land use diversity and land management trends. A place-based methodology has been developed to characterize fire scenarios using Geographic Information Systems and statistical analysis, resulting in analytical and diagnostic mapping that can be used as a decision support tool for spatial planning and wildfire risk management. Its implementation has led to the delimitation of 91 discrete geographic units in the Central Mountain Range, classified according to wildfire generations and land use-land cover criteria. In conclusion, the fire scenarios concept is a potential approach to manage uncertainty by moving from the operational level of fire suppression to the strategic level of integrated fire management.

End-Of-Life Medical Spending In Last Twelve Months Of Life Is Lower Than Previously Reported.

Although end-of-life medical spending is often viewed as a major component of aggregate medical expenditure, accurate measures of this type of medical spending are scarce. We used detailed health care data for the period 2009-11 from Denmark, England, France, Germany, Japan, the Netherlands, Taiwan, the United States, and the Canadian province of Quebec to measure the composition and magnitude of medical spending in the three years before death. In all nine countries, medical spending at the end of life was high relative to spending at other ages. Spending during the last twelve months of life made up a modest share of aggregate spending, ranging from 8.5 percent in the United States to 11.2 percent in Taiwan, but spending in the last three calendar years of life reached 24.5 percent in Taiwan. This suggests that high aggregate medical spending is due not to last-ditch efforts to save lives but to spending on people with chronic conditions, which are associated with shorter life expectancies.

Metaproteomics-guided selection of targeted enzymes for bioprospecting of mixed microbial communities.

BACKGROUND: Hitherto, the main goal of metaproteomic analyses has been to characterize the functional role of particular microorganisms in the microbial ecology of various microbial communities. Recently, it has been suggested that metaproteomics could be used for bioprospecting microbial communities to query for the most active enzymes to improve the selection process of industrially relevant enzymes. In the present study, to reduce the complexity of metaproteomic samples for targeted bioprospecting of novel enzymes, a microbial community capable of producing cellulases was maintained on a chemically defined medium in an enzyme suppressed metabolic steady state. From this state, it was possible to specifically and distinctively induce the desired cellulolytic activity. The extracellular fraction of the protein complement of the induced sample could thereby be purified and compared to a non-induced sample of the same community by differential gel electrophoresis to discriminate between constitutively expressed proteins and proteins upregulated in response to the inducing substance. RESULTS: Using the applied approach, downstream analysis by mass spectrometry could be limited to only proteins recognized as upregulated in the cellulase-induced sample. Of 39 selected proteins, the majority were found to be linked to the need to degrade, take up, and metabolize cellulose. In addition, 28 (72%) of the proteins were non-cytosolic and 17 (44%) were annotated as carbohydrate-active enzymes. The results demonstrated both the applicability of the proposed approach for identifying extracellular proteins and guiding the selection of proteins toward those specifically upregulated and targeted by the enzyme inducing substance. Further, because identification of interesting proteins was based on the regulation of enzyme expression in response to a need to hydrolyze and utilize a specific substance, other unexpected enzyme activities were able to be identified. CONCLUSIONS: The described approach created the conditions necessary to be able to select relevant extracellular enzymes that were extracted from the enzyme-induced microbial community. However, for the purpose of bioprospecting for enzymes to clone, produce, and characterize for practical applications, it was concluded that identification against public databases was not sufficient to identify the correct gene or protein sequence for cloning of the identified novel enzymes.

Enhanced biomethane production rate and yield from lignocellulosic ensiled forage ley by in situ anaerobic digestion treatment with endogenous cellulolytic enzymes.

BACKGROUND: Enzymatic treatment of lignocellulosic material for increased biogas production has so far focused on pretreatment methods. However, often combinations of enzymes and different physicochemical treatments are necessary to achieve a desired effect. This need for additional energy and chemicals compromises the rationale of using enzymes for low energy treatment to promote biogas production. Therefore, simpler and less energy intensive in situ anaerobic digester treatment with enzymes is desirable. However, investigations in which exogenous enzymes are added to treat the material in situ have shown mixed success, possibly because the enzymes used originated from organisms not evolutionarily adapted to the environment of anaerobic digesters. In this study, to examine the effect of enzymes endogenous to methanogenic microbial communities, cellulolytic enzymes were instead overproduced and collected from a dedicated methanogenic microbial community. By this approach, a solution with very high endogenous microbial cellulolytic activity was produced and tested for the effect on biogas production from lignocellulose by in situ anaerobic digester treatment. RESULTS: Addition of enzymes, endogenous to the environment of a mixed methanogenic microbial community, to the anaerobic digestion of ensiled forage ley resulted in significantly increased rate and yield of biomethane production. The enzyme solution had an instant effect on more readily available cellulosic material. More importantly, the induced enzyme solution also affected the biogas production rate from less accessible cellulosic material in a second slower phase of lignocellulose digestion. Notably, this effect was maintained throughout the experiment to completely digested lignocellulosic substrate. CONCLUSIONS: The induced enzyme solution collected from a microbial methanogenic community contained enzymes that were apparently active and stable in the environment of anaerobic digestion. The enzymatic activity had a profound effect on the biogas production rate and yield, comparable with the results of many pretreatment methods. Thus, application of such enzymes could enable efficient low energy in situ anaerobic digester treatment for increased biomethane production from lignocellulosic material.