Nazim Cicek

Biomass pretreatment: Fundamentals toward application

Development of sustainable energy systems based on renewable biomass feedstocks is now a global effort. Lignocellulosic biomass contains polymers of cellulose, hemicellulose, and lignin, bound together in a complex structure. Liquid biofuels, such as ethanol, can be made from biomass via fermentation of sugars derived from the cellulose and hemicellulose within lignocellulosic materials, but the biomass must be subjected to pretreatment processes to liberate the sugars needed for fermentation. Production of value-added co-products along-side biofuels through integrated biorefinery processes creates the need for selectivity during pretreatment. This paper presents a survey of biomass pretreatment technologies with emphasis on concepts, mechanism of action and practicability. The advantages and disadvantages, and the potential for industrial applications of different pretreatment technologies are the highlights of this paper.

Third Generation Biofuels via Direct Cellulose Fermentation

Consolidated bioprocessing (CBP) is a system in which cellulase production, substrate hydrolysis, and fermentation are accomplished in a single process step by cellulolytic microorganisms. CBP offers the potential for lower biofuel production costs due to simpler feedstock processing, lower energy inputs, and higher conversion efficiencies than separate hydrolysis and fermentation processes, and is an economically attractive near-term goal for “third generation” biofuel production. In this review article, production of third generation biofuels from cellulosic feedstocks will be addressed in respect to the metabolism of cellulolytic bacteria and the development of strategies to increase biofuel yields through metabolic engineering.

Importance of the operating pH in maintaining the stability of anoxic ammonium oxidation (anammox) activity in moving bed biofilm reactors

Two bench-scale parallel moving bed biofilm reactors (MBBR) were operated to assess pH-associated anammox activity changes during long term treatment of anaerobically digested sludge centrate pre-treated in a suspended growth partial nitrification reactor. The pH was maintained at 6.5 in reactor R1, while it was allowed to vary naturally between 7.5 and 8.1 in reactor R2. At high nitrogen loads reactor R2 had a 61% lower volumetric specific nitrogen removal rate than reactor R1. The low pH and the associated low free ammonia (FA) concentrations were found to be critical to stable anammox activity in the MBBR. Nitrite enhanced the nitrogen removal rate in the conditions of low pH, all the way up to the investigated level of 50mg NO(2)-N/L. At low FA levels nitrite concentrations up to 250 mg NO(2)-N/L did not cause inactivation of anammox consortia over a 2-days exposure time.

Impact of shear force on the biofilm structure and performance of a membrane biofilm reactor for tertiary hydrogen-driven denitrification of municipal wastewater

Hydrogen-driven denitrification using a hollow-fiber membrane biofilm reactor (MBfR) was evaluated for operation in tertiary wastewater treatment. Specific objectives were to evaluate the impact of different levels of shearing stress caused by mixing and nitrogen sparging on the biofilm structure and denitrification rates. Applying high shear force proved to be effective in improving denitrification rates by reducing the thickness of the biofilm. With intensive mixing a biofilm thickness of approximately 800 microm was maintained, while additional nitrogen sparging could further reduce the biofilm thickness to approximately 300 microm. The highest denitrification rates of 0.93 gN/m(2)d were obtained when biofilm thickness was lower than 500 microm. Lower extracellular polymeric substances (EPS) accumulation and carbohydrates to protein ratio observed in thinner biofilms allowed for higher nitrate removal in the system. No significant sloughing of biomass or change in total and soluble COD in the final effluent was observed under steady-state conditions.

Growth phase-dependant enzyme profile of pyruvate catabolism and end-product formation in Clostridium thermocellum ATCC 27405.

End-product synthesis and enzyme activities involved in pyruvate catabolism, H(2) synthesis, and ethanol production in mid-log (OD(600) approximately 0.25), early stationary (OD(600) approximately 0.5), and stationary phase (OD(600) approximately 0.7) cell extracts were determined in Clostridium thermocellum ATCC 27405 grown in batch cultures on cellobiose. Carbon dioxide, hydrogen, ethanol, acetate and formate were major end-products and their production paralleled growth and cellobiose consumption. Lactate dehydrogenase, pyruvate:formate lyase, pyruvate:ferredoxin oxidoreductase, methyl viologen-dependant hydrogenase, ferredoxin-dependant hydrogenase, NADH-dependant hydrogenase, NADPH-dependant hydrogenase, NADH-dependant acetaldehyde dehydrogenase, NADH-dependant alcohol dehydogenase, and NADPH-dependant alcohol dehydrogenase activities were detected in all extracts, while pyruate dehydrogenase and formate dehydrogenase activities were not detected. All hydrogenase activities decreased (2-12-fold) as growth progressed from early exponential to stationary phase. Alcohol dehydrogenase activities fluctuated only marginally (<45%), while lactate dehydrogenase, pyruvate:formate lyase, and pyruvate:ferredoxin oxidoreductase remained constant in all cell extracts. We have proposed a pathway involved in pyruvate catabolism and end-product formation based on enzyme activity profiles in conjunction with bioinformatics analysis.

Impact of free ammonia on anammox rates (anoxic ammonium oxidation) in a moving bed biofilm reactor

Using a bench scale moving bed bioreactor (MBBR), the effect of free ammonia (FA, NH(3), the un-ionized form of ammonium NH(4)(+)) concentration on anoxic ammonium oxidation (anammox) was evaluated based on the volumetric nitrogen removal rate (NRR). Although, a detailed microbial analysis was not conducted, the major NRR observed was assumed to be by anammox, based on the nitrogen conversion ratios of nitrite to ammonium and nitrate to ammonium. Since the concentration of free ammonia as a proportion of the total ammonia concentration is pH-dependent, the impact of changing the operating pH from 6.9 to 8.2, was investigated under constant nitrogen loading conditions during continuous reactor operation. Furthermore, the effect of sudden nitrogen load changes was investigated under constant pH conditions. Batch tests were conducted to determine the immediate response of the anammox consortium to shifts in pH and FA concentrations. It was found that FA was inhibiting NRR at concentrations exceeding 2 mg N L(-1). In the pH range 7-8, the decrease in anammox activity was independent of pH and related only to the concentration of FA. Nitrite concentrations of up to 120 mg N L(-1) did not negatively affect NRR for up to 3.5 h. It was concluded that a stable NRR in a moving bed biofilm reactor depended on maintaining FA concentrations below 2 mg N L(-1) when the pH was maintained between 7 and 8.

Lipid production in the under-characterized oleaginous yeasts, Rhodosporidium babjevae and Rhodosporidium diobovatum, from biodiesel-derived waste glycerol

The growth, lipid production, and carbon use efficiency of two oleaginous yeasts, Rhodosporidium babjevae and Rhodosporidium diobovatum, were compared under nitrogen-limiting conditions with glycerol as the carbon source. Final biomass concentrations of R. babjevae and R. diobovatum were 9.4±0.80g/L and 12.0±0.82g/L, respectively, after 120h of growth. Final lipid accumulation in for R. babjevae and R. diobovatum were 34.9±3.0% cell dry weight (cdw) and 63.7±4.5% cdw, respectively. Biomass production increased to 9.9±0.2g/L for R. babjevae and 14.1±0.2g/L for R. diobovatum in medium containing biodiesel-derived waste glycerol, but lipid accumulation decreased in both species. In a 7L bioreactor with biodiesel-derived glycerol as carbon source, R. diobovatum produced 13.6±0.4g/L biomass and accumulated lipids to 50.7±2.2% cdw, matching flask experiments.

Linking genome content to biofuel production yields: a meta-analysis of major catabolic pathways among select H2 and ethanol-producing bacteria

BackgroundFermentative bacteria offer the potential to convert lignocellulosic waste-streams into biofuels such as hydrogen (H2) and ethanol. Current fermentative H2 and ethanol yields, however, are below theoretical maxima, vary greatly among organisms, and depend on the extent of metabolic pathways utilized. For fermentative H2 and/or ethanol production to become practical, biofuel yields must be increased. We performed a comparative meta-analysis of (i) reported end-product yields, and (ii) genes encoding pyruvate metabolism and end-product synthesis pathways to identify suitable biomarkers for screening a microorganism’s potential of H2 and/or ethanol production, and to identify targets for metabolic engineering to improve biofuel yields. Our interest in H2 and/or ethanol optimization restricted our meta-analysis to organisms with sequenced genomes and limited branched end-product pathways. These included members of the Firmicutes, Euryarchaeota, and Thermotogae.ResultsBioinformatic analysis revealed that the absence of genes encoding acetaldehyde dehydrogenase and bifunctional acetaldehyde/alcohol dehydrogenase (AdhE) in Caldicellulosiruptor, Thermococcus, Pyrococcus, and Thermotoga species coincide with high H2 yields and low ethanol production. Organisms containing genes (or activities) for both ethanol and H2 synthesis pathways (i.e. Caldanaerobacter subterraneus subsp. tengcongensis, Ethanoligenens harbinense, and Clostridium species) had relatively uniform mixed product patterns. The absence of hydrogenases in Geobacillus and Bacillus species did not confer high ethanol production, but rather high lactate production. Only Thermoanaerobacter pseudethanolicus produced relatively high ethanol and low H2 yields. This may be attributed to the presence of genes encoding proteins that promote NADH production. Lactate dehydrogenase and pyruvate:formate lyase are not conducive for ethanol and/or H2 production. While the type(s) of encoded hydrogenases appear to have little impact on H2 production in organisms that do not encode ethanol producing pathways, they do influence reduced end-product yields in those that do.ConclusionsHere we show that composition of genes encoding pathways involved in pyruvate catabolism and end-product synthesis pathways can be used to approximate potential end-product distribution patterns. We have identified a number of genetic biomarkers for streamlining ethanol and H2 producing capabilities. By linking genome content, reaction thermodynamics, and end-product yields, we offer potential targets for optimization of either ethanol or H2 yields through metabolic engineering.

Pyruvate catabolism and hydrogen synthesis pathway genes of Clostridium thermocellum ATCC 27405

Clostridium thermocellum is a gram-positive, acetogenic, thermophilic, anaerobic bacterium that degrades cellulose and carries out mixed product fermentation, catabolising cellulose to acetate, lactate, and ethanol under various growth conditions, with the concomitant release of H2 and CO2. Very little is known about the factors that determine metabolic fluxes influencing H2 synthesis in anaerobic, cellulolytic bacteria like C. thermocellum. We have begun to investigate the relationships between genome content, gene expression, and end-product synthesis in C. thermocellum cultured under different conditions. Using bioinformatics tools and the complete C. thermocellum 27405 genome sequence, we identified genes encoding key enzymes in pyruvate catabolism and H2-synthesis pathways, and have confirmed transcription of these genes throughout growth on α-cellulose by reverse transcriptase polymerase chain reaction. Bioinformatic analyses revealed two putative lactate dehydrogenases, one pyruvate formate lyase, four pyruvate:formate lyase activating enzymes, and at least three putative pyruvate:ferredoxin oxidoreductase (POR) or POR-like enzymes. Our data suggests that hydrogen may be generated through the action of either a Ferredoxin (Fd)-dependent NiFe hydrogenase, often referred to as “Energy-converting Hydrogenases”, or via NAD(P)Hdependent Fe-only hydrogenases which would permit H2 production from NADH generated during the glyceraldehyde-3-phosphate dehydrogenase reaction. Furthermore, our findings show the presence of a gene cluster putatively encoding a membrane integral NADH:Fd oxidoreductase, suggesting a possible mechanism in which electrons could be transferred between NADH and ferredoxin. The elucidation of pyruvate catabolism pathways and mechanisms of H2 synthesis is the first step in developing strategies to increase hydrogen yields from biomass. Our studies have outlined the likely pathways leading to hydrogen synthesis in C. thermocellum strain 27405, but the actual functional roles of these gene products during pyruvate catabolism and in H 2 synthesis remain to be elucidated, and will need to be confirmed using both expression analysis and protein characterization.

Treatment of Organic Micropollutants in Water and Wastewater by UV-Based Processes: A Literature Review

This paper provides a review of the up-to-date research on the treatment of micropollutants by UV-based processes. More than 140 scientific publications in the past 10 years were reviewed and analyzed, with emphasis on the peer-reviewed papers in the last three years (2009–2011). Previous studies showed that UV-based advanced oxidation processes (AOPs) could be efficient for the removal of a number of micropollutants in drinking water and wastewater although direct UV photolysis at disinfection dosages was proven not effective in removing most micropollutants. Among those UV-based AOPs, UV-oxidation with hydrogen peroxide (H2O2) has attracted great attention and shown numerous advantages as an advanced technology for micropollutants control. The public concern about the impact of micropollutants on the safety of drinking water and the increasing needs of safe water reuse will likely facilitate the application of UV-based processes for micropollutants control in drinking water treatment and water reuse.