Biotechnological processes for conversion of corn into ethanol

Ethanol has been utilized as a fuel source in the United States since the turn of the century. However, it has repeatedly faced significant commercial viability obstacles relative to petroleum. Renewed interest exists in ethanol as a fuel source today owing to its positive impact on rural America, the environment and United States energy security. Today, most fuel ethanol is produced by either the dry grind or the wet mill process. Current technologies allow for 2.5 gallons (wet mill process) to 2.8 gallons (dry grind process) of ethanol (1 gallon = 3.785 l) per bushel of corn. Valuable co-products, distillers dried grains with solubles (dry grind) and corn gluten meal and feed (wet mill), are also generated in the production of ethanol. While current supplies are generated from both processes, the majority of the growth in the industry is from dry grind plant construction in rural communities across the corn belt. While fuel ethanol production is an energy-efficient process today, additional research is occurring to improve its long-term economic viability. Three of the most significant areas of research are in the production of hybrids with a higher starch content or a higher extractable starch content, in the conversion of the corn kernel fiber fraction to ethanol, and in the identification and development of new and higher-value co-products.     

Ectopic expression of bacterial amylopullulanase enhances bioethanol production from maize grain

Key message

Heterologous expression of amylopullulanase in maize seeds leads to partial starch degradation into fermentable sugars, which enhances direct bioethanol production from maize grain.

Abstract

Utilization of maize in bioethanol industry in the United States reached ±13.3 billion gallons in 2012, most of which was derived from maize grain. Starch hydrolysis for bioethanol industry requires the addition of thermostable alpha amylase and amyloglucosidase (AMG) enzymes to break down the α-1,4 and α-1,6 glucosidic bonds of starch that limits the cost effectiveness of the process on an industrial scale due to its high cost. Transgenic plants expressing a thermostable starch-degrading enzyme can overcome this problem by omitting the addition of exogenous enzymes during the starch hydrolysis process. In this study, we generated transgenic maize plants expressing an amylopullulanase (APU) enzyme from the bacterium Thermoanaerobacter thermohydrosulfuricus. A truncated version of the dual functional APU (TrAPU) that possesses both alpha amylase and pullulanase activities was produced in maize endosperm tissue using a seed-specific promoter of 27-kD gamma zein. A number of analyses were performed at 85 °C, a temperature typically used for starch processing. Firstly, enzymatic assay and thin layer chromatography analysis showed direct starch hydrolysis into glucose. In addition, scanning electron microscopy illustrated porous and broken granules, suggesting starch autohydrolysis. Finally, bioethanol assay demonstrated that a 40.2 ± 2.63 % (14.7 ± 0.90 g ethanol per 100 g seed) maize starch to ethanol conversion was achieved from the TrAPU seeds. Conversion efficiency was improved to reach 90.5 % (33.1 ± 0.66 g ethanol per 100 g seed) when commercial amyloglucosidase was added after direct hydrolysis of TrAPU maize seeds. Our results provide evidence that enzymes for starch hydrolysis can be produced in maize seeds to enhance bioethanol production.     

Hydrocarbon-and rubber-producing crops

Agricultural production of rubber and other hydrocarbons in the United States may be compatible with increased food and fiber production if entire plants are harvested and processed for fiber, protein, and carbohydrate as well. Thus, procedures and criteria have been established for the preliminary evaluation of plant species as potential multi-use hydrocarbon-producing crops. Previously, 106 species representing 44 families and 81 genera were evaluated. Now an additional 100 species representing 13 additional families and 60 additional genera have been evaluated, and some of these species also offer promise as future crops. Several Labiatae are high in natural rubber (NR) content;Pycnanthemum incanum (Mountain Mint) andTeucrium canadense (American Germander) were evaluated as promising species. Three Compositae,Cacalia atriplicifolia (Pale Indian-Plant),Solidago graminifolia (Grass-leafed Golden-rod), andSolidago rigida (Stiff Goldenrod) were also evaluated as promising species for NR.Campanula americana (Tall Bellflower, Campanulaceae) has potential as a source of both oil and NR.Euphorbia dentata (Euphorbiaceae ) does not produce NR but is very high in protein and oil contents.Sassafras albidum (Sassafras, Lauraceae ) is of interest for its rapid growth rate in combination with a high oil content. A few other species offer some promise.     

Microscopic analysis of corn fiber using starch- and cellulose-specific molecular probes

Ethanol is the primary liquid transportation fuel produced from renewable feedstocks in the United States today. The majority of corn grain, the primary feedstock for ethanol production, has been historically processed in wet mills yielding products such as gluten feed, gluten meal, starch, and germ. Starch extracted from the grain is used to produce ethanol in saccharification and fermentation steps; however the extraction of starch is not 100% efficient. To better understand starch extraction during the wet milling process, we have developed fluorescent probes that can be used to visually localize starch and cellulose in samples using confocal microscopy. These probes are based on the binding specificities of two types of carbohydrate binding modules (CBMs), which are small substrate-specific protein domains derived from carbohydrate degrading enzymes. CBMs were fused, using molecular cloning techniques, to a green fluorescent protein (GFP) or to the red fluorescent protein DsRed (RFP). Using these engineered probes, we found that the binding of the starch-specific probe correlates with starch content in corn fiber samples. We also demonstrate that there is starch internally localized in the endosperm that may contribute to the high starch content in corn fiber. We also surprisingly found that the cellulose-specific probe did not bind to most corn fiber samples, but only to corn fiber that had been hydrolyzed using a thermochemical process that removes the residual starch and much of the hemicellulose. Our findings should be of interest to those working to increase the efficiency of the corn grain to ethanol process.     

Identification of oleaginous yeast strains able to accumulate high intracellular lipids when cultivated in alkaline pretreated corn stover

Microbial oil is a potential alternative to food/plant-derived biodiesel fuel. Our previous screening studies identified a wide range of oleaginous yeast species, using a defined laboratory medium known to stimulate lipid accumulation. In this study, the ability of these yeasts to grow and accumulate lipids was further investigated in synthetic hydrolysate (SynH) and authentic ammonia fiber expansion (AFEX?)-pretreated corn stover hydrolysate (ACSH). Most yeast strains tested were able to accumulate lipids in SynH, but only a few were able to grow and accumulate lipids in ACSH medium. Cryptococcus humicola UCDFST 10-1004 was able to accumulate as high as 15.5 g/L lipids, out of a total of 36 g/L cellular biomass when grown in ACSH, with a cellular lipid content of 40 % of cell dry weight. This lipid production is among the highest reported values for oleaginous yeasts grown in authentic hydrolysate. Preculturing in SynH media with xylose as sole carbon source enabled yeasts to assimilate both glucose and xylose more efficiently in the subsequent hydrolysate medium. This study demonstrates that ACSH is a suitable medium for certain oleaginous yeasts to convert lignocellullosic sugars to triacylglycerols for production of biodiesel and other valuable oleochemicals.     

Coumarin in vanilla extracts: Its detection and significance

Coumarin was a component of some commercial vanilla preparations until banned in the United States by the Food and Drug Administration in 1954 because of evidence of hepatotoxicity in animals. However, in the present investigation coumarin was detected by capillary column gas chromatography/mass spectrometry in Mexican vanilla extracts commercially available or introduced in the United States by tourists or travelers. The significance of the presence of coumarin in Mexican vanilla extracts is discussed in light of recent evidence suggesting a lack of significant coumarin toxicity in humans.     

Direct ethanol production from cassava pulp using a surface-engineered yeast strain co-displaying two amylases, two cellulases, and β-glucosidase

In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying α-amylase (α-AM), glucoamylase, endoglucanase, cellobiohydrase, and β-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley β-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes.     

Growth regulation,plastid differentiation and the development of a photosynthetic system in cultured carrot root explants as influenced by exogenous sucrose and various phytohormones

Chromoplasts, which exist in the cells of freshly isolated carrot root explants, seemed to be transformed in thylakoid containing plastids, and chlorophyll formation was initiated if the explants were cultured in a liquid medium containing inositol and IAA as a hormonal supplement. This process was intensified when kinetin was also added, but no dependence on a sucrose supply could be found. A sucrose supply of 2% in conjunction with the combination of all three hormones, however, was needed to achieve maximal thylakoid formation including stacking in individual chloroplasts and for the very extensive chloroplast multiplication in explants growing with high cell division activity. It should be noted that the number of plastids per cell is strongly increased by the sucrose supplement which leads also to starch accumulation. However, no transformation into chloroplasts occurred without the hormonal stimulus.     

Fermentation of Barley by Using Saccharomyces cerevisiae: Examination of Barley as a Feedstock for Bioethanol Production and Value-Added Products

The objective of this study was to examine the ethanol yield potential of three barley varieties (Xena, Bold, and Fibar) in comparison to two benchmarks, corn and wheat. Very high gravity (VHG; 30% solids) fermentations using both conventional and Stargen 001 enzymes for starch hydrolysis were carried out as simultaneous saccharification and fermentation. The grains and their corresponding dried distiller''s grain with solubles (DDGS) were also analyzed for nutritional and value-added characteristics. A VHG traditional fermentation approach utilizing jet-cooking fermentation revealed that both dehulled Bold and Xena barley produced ethanol concentrations higher than that produced by wheat (12.3, 12.2, and 11.9%, respectively) but lower than that produced by corn (13.8%). VHG-modified Stargen-based fermentation of dehulled Bold barley demonstrated comparable performance (14.3% ethanol) relative to that of corn (14.5%) and wheat (13.3%). Several important components were found to survive fermentation and were concentrated in DDGS. The highest yield of phenolics was detected in the DDGS (modified Stargen 001, 20% solids) of Xena (14.6 mg of gallic acid/g) and Bold (15.0 mg of gallic acid/g) when the hull was not removed before fermentation. The highest concentration of sterols in DDGS from barley was found in Xena (3.9 mg/g) when the hull was included. The DDGS recovered from corn had the highest concentration of fatty acids (72.6 and 77.5 mg/g). The DDGS recovered from VHG jet-cooking fermentations of Fibar, dehulled Bold, and corn demonstrated similar levels of tocopherols and tocotrienols. Corn DDGS was highest in crude fat but was lowest in crude protein and in vitro energy digestibility. Wheat DDGS was highest in crude protein content, similar to previous studies. The barley DDGS was the highest in in vitro energy digestibility.The growing need for energy independence and proposed renewable fuels has led recently to a major expansion of fuel ethanol production. In North America, this activity primarily uses corn as a feedstock. The need to find other cost-effective and efficient grains for ethanol production has increased in significance. Cereal grains are high in starch and are currently being utilized for ethanol production (26, 41). To ensure long-term viability of the industry, fermentation strategies that focus on holistic utilization of the feedstock that maximize value addition will increase in importance. The focus of industry is slowly moving from biorefineries that anticipate subsidy and government policy to integrated biorefineries that produce multiple products. Multiple product streams and integrated by-product management are thought to ensure better financial stability and opportunities for diversified income streams.Barley is a potential candidate for industrial ethanol production (10) since its ethanol yield is comparable to that of wheat but below that of American corn, which is currently the preferred industrial feedstock. Barley contains on average 63 to 65% starch, 8 to 13% protein, 2 to 3% fat, 1 to 1.5% soluble gums, 8 to 10% hemicellulose, ca. 2.9% lignin, and 2 to 2.5% ash (15, 27). Barley also contains a hull that could be fermented using cellulolytic enzymes, providing opportunities for integrated biorefineries that utilize more feedstocks than corn. Potential coproducts of ethanol production from barley include protein, fiber, fatty acids, tocopherols, and tocotrienols (40). The nutritional value of barley, based on amino acid content, is greater than that for corn and is not significantly affected by the fermentation process (40). A range of nutraceutical and functional food products, as well as amylase, amylase inhibitors, β-amylase, and oxalate oxidase, are found in barley grains and may have potential for extraction and commercial applications (6, 22, 33). Hull-less barley lines, high in both protein (particularly lysine) and starch, and low in fiber, have recently been developed (11, 14, 32). Since starch recovery and thus ethanol yields are lower for barley than corn, coproduct recovery becomes even more essential for profitability (43).Enzymes used for the pretreatment of grains prior to fermentation have traditionally been α-amylases and glucoamylases. The α-amylase decreases the viscosity of the mash (25) and performs the liquefaction of the pretreatment process. The liquefaction step is typically done at high temperatures of 100 to 120°C (38) with direct steam injection (jet-cooking). The α-amylase action serves to break starch at α-(1,4)-glucosidic bonds, producing smaller dextrin chains. During the saccharification step of the pretreatment, the dextrins produced by α-amylase are then acted on by glucoamylase. This conventional method has a considerable economic drawback, because the mash must undergo a cooking step prior to fermentation. Many industrial ethanol producers use jet-cooking to raise the mash temperature to 100 to 120°C. Because of this temperature requirement, the conventional process uses a large amount of energy to produce ethanol.Recently, a new line of cold starch hydrolyzing enzymes was developed. An example of these enzymes is Stargen 001, which is referred to as a raw starch hydrolyzing enzyme because starch is hydrolyzed to fermentable sugars while the temperature remains at or below a temperature of 48°C (38). Stargen 001 replaces the liquefaction and saccharification steps performed by conventional digestion enzymes (i.e., α-amylase and glucoamylase) and releases free glucose and other fermentable sugars for use by yeast cells. Stargen 001 is a cocktail of modified α-amylase and glucoamylase enzymes that work together to convert starch into dextrins, followed by the hydrolysis of dextrins to fermentable sugars (37, 38). With the absence of a cooking stage in the cold hydrolysis method, the potential exists that the dried distiller''s grain plus solubles (DDGS) produced by fermentation would have less damage so that the proteins contained in the DDGS could be of more value (18).The objectives of the present study were to examine the ethanol yield potentials of three barley varieties (Xena, Bold, and Fibar) and two benchmark grains (Pioneer Hi-Bred corn and CPS wheat) using conventional (jet-cooking) and cold starch hydrolysis with Stargen 001. In addition, dehulling was tested for the potential to increase ethanol yields, because hull does not contain fermentable starch; both hulled and dehulled mashes were studied where possible. The grains and their corresponding DDGS were analyzed for nutritional value and the presence of potential value-added products such as fatty acids, tocopherols, tocotrienols, sterols, and polyphenols.     

Chromosome numbers in some cacti Of western North America—II

Documented chromosome numbers and meiotic behavior are recorded for an additional 30 taxa representing 25 species of Cactaceae of southwestern United States and northern Mexico. Diploid and polyploid taxa including two triploids were observed, all of which indicate the same base number,x = 11. Trisomism and inversions are reported for the first time in cacti.     

Introduction of the soybean to Illinois

Events leading to the planting of the first soybeans in Illinois and the subsequent distribution of the seed throughout the United States are documented. In December 1850 theAuckland left Hong Kong for San Francisco. The ship came across a Japanese junk floundering on the sea. The Japanese crew was removed from the junk and placed on board theAuckland, which continued on to San Francisco. In March 1851 Dr. Benjamin Franklin Edwards obtained soybean seeds from the Japanese and brought the seeds to Alton, Illinois. Mr. John H. Lea of Alton planted the soybeans in his garden in summer 1851. These events took place 3 yr prior to the famous introduction of soybeans from Japan into the United States by the Perry Expedition.     

Starch self-processing in transgenic sweet potato roots expressing a hyperthermophilic α-amylase

Sweet potato is a major crop in the southeastern United States, which requires few inputs and grows well on marginal land. It accumulates large quantities of starch in the storage roots and has been shown to give comparable or superior ethanol yields to corn per cultivated acre in the southeast. Starch conversion to fermentable sugars (i.e., for ethanol production) is carried out at high temperatures and requires the action of thermostable and thermoactive amylolytic enzymes. These enzymes are added to the starch mixture impacting overall process economics. To address this shortcoming, the gene encoding a hyperthermophilic α-amylase from Thermotoga maritima was cloned and expressed in transgenic sweet potato, generated by Agrobacterium tumefaciens-mediated transformation, to create a plant with the ability to self-process starch. No significant enzyme activity could be detected below 40°C, but starch in the transgenic sweet potato storage roots was readily hydrolyzed at 80°C. The transgene did not affect normal storage root formation. The results presented here demonstrate that engineering plants with hyperthermophilic glycoside hydrolases can facilitate cost effective starch conversion to fermentable sugars. Furthermore, the use of sweet potato as an alternative near-term energy crop should be considered.     

Genetic diversity and population structure of sickleweed (Falcaria vulgaris; Apiaceae) in the upper Midwest USA

Sickleweed (Falcaria vulgaris), an introduced species native to Europe and Asia, grows as an aggressive weed in some areas of the upper Midwest in the United States. We are reporting genetic diversity and population structure of sickleweed populations using microsatellite markers and nuclear and chloroplast DNA sequences. Populations showed high genetic differentiation but did not show significant geographic structure, suggesting random establishment of different genotypes at different sites was likely due to human mediated multiple introductions. Three genetic clusters revealed by microsatellite data and presence of six chlorotypes supported our hypothesis of multiple introductions. Chloroplast DNA sequence data revealed six chlorotypes nested into two main lineages suggesting at least two introductions of sickleweed in the upper Midwest. Some individuals exhibited more than two alleles at several microsatellite loci suggesting occurrence of polyploidy, which could be a post-introduction development to mitigate the inbreeding effects. High genetic variation in the introduced range attributable to multiple introductions and polyploidy may be inducing the evolution of invasiveness in sickleweed. Results of this study provide valuable insights into the evolution of sickleweed and baseline data for designing proper management practices for controlling sickleweed in the United States.     

cDNA library construction from meristematic tissue of finger millet panicle

A protocol to obtain full-length cDNA using a SuperScript® Full-Length cDNA Library Construction Kit II (Invitrogen, United States) was developed, and a high quality cDNA library of meristem tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was constructed. The titer of the constructed cDNA library was 3.01 × 10⁵ CFU/mL, the average length of the insert was approximately 1070 base pairs, and the average efficiency of insertion of cDNA fragments was 99.5%. The sequencing of randomly selected clones created cDNA library was carried out. The cDNA sequences of clones were identified by BLAST search. The cDNA library analysis and selective sequencing indicate good functionality and full size of cDNA inserts of the clones. The constructed cDNA library from meristematic tissue of finger millet panicle is a good and reliable source for isolation and identification of key genes of metabolism and development of meristem as well for creation of new genetic markers for genetic research and molecular selection.     

Economic botany: Past and future

Economic botany, like botany, had its origin with the herbalists. Its development was encouraged by various European rulers during the Age of Exploration and Discovery in the 16th century. Recent developments in the United States include A. F. Hill’s textbook, Economic Botany, in 1937 the founding of the journal Economic Botany by E. H. Fulling in 1947 and the formation of the Society for Economic Botany in 1959 Some of the breakthroughs in the development of agriculture, beginning with its origin some 10,000 yr ago, are given. It is concluded that we now need another significant breakthrough to feed the world’s population adequately in the year 2000 Some look to genetic engineering to provide such a breakthrough.     

Ginseng: America’s botanical drug connection to the orient

Ginseng has long been one of this country’s major botanical drugs in foreign trade. Once harvested only in the forests of the Eastern frontiers, it became a domesticated crop in the late 1800s and now is raised largely in northcentral Wisconsin. Growers there produce an estimated 90% of the cultivated ginseng in the United States. Most American ginseng has been consumed in the Orient, as is reflected in export records dating back to 1821. Over 95% of the nearly 21,000 metric tons (T) shipped in the period 1821–1983 went to the Far East. Hong Kong has served as the center for re-exporting ginseng to China and Southeast Asia. Ginseng has been used in Asia for many purposes, mostly as a curative agent. It has also gained increasing acceptance elsewhere in the world for its alleged value.     

Phytochemical alteration and new occurring compounds in hairy root cultures of Mitracarpus hirtus L. induced by phenylurea cytokinin (CPPU)

Hairy root cultures of Mitracarpus hirtus L. were obtained after transforming leaf-disc explants with wild strain Agrobacterium rhizogenes A13. The root cultures of M. hirtus showed high efficiency of shoot formation in both transformed and non-transformed cultures when illuminated with light. However, transformed hairy root proliferation was approximately 3.8–5 times higher than the control in both solidified and liquid plant growth regulator free media. Putatively transformed roots were identified by the presence of the rol gene via PCR. Integration of the rol gene into the plant genome was confirmed via Southern blot analysis after 5 months with no detection in non-transformed roots. In addition, the effect of 2-chloro-4-pyridyl-N-phenylurea (CPPU), a synthetic cytokinin, when applied as an elicitor for hairy root cultures of M. hirtus was investigated. The 24-day-old hairy root cultures of high root proliferation line R107-3, were incubated for 48 h in media supplemented with 0 or 5 mg l^?1 CPPU. The methanolic extracts of root tissues were subsequently analyzed for biochemical constituents using Gas Chromatography Mass Spectrometry (GC-MS). The alteration of plant secondary metabolites produced after CPPU treatment was analyzed. Compared to the control (with quality higher than 80 %), six unique compounds were found, five original compounds absent, 11 with increased, and five with decreased contents. Increased contents of two metabolites, chrysophanol and 2-methoxy-4-vinylphenol, showed pharmaceutical potential. CPPU was also found to elicit the alkaloid compound, Eseroline, 7-bromo-, methylcarbamate (ester), which could not be detected in the non-treated sample. The findings of this study demonstrate the establishment of transgenic hairy root of M. hirtus and the application of CPPU as an elicitor to induce variations in plant secondary metabolite that shows its potential to apply for bio-reactor system.     

The Effect of Nutritional Factors and Plant Growth Regulators on Micropropagation and Production of Phenolic Acids and Saponins from Plantlets and Adventitious Root Cultures of Eryngium maritimum L.

Eryngium maritimum L. is a valuable medicinal species, but since it is protected plant, collection from natural populations is forbidden. Therefore, establishing an efficient system for micropropagation of this species is desirable. To determine the optimal nutritional factors needed for shoot multiplication, root development and secondary metabolites accumulation, different media and plant growth regulators were tested. The highest plant regeneration efficiency (over 96 %), with 4.4 shoots per explant was induced on Murashige and Skoog (MS) medium supplemented with 1.0 mg L^?1 benzyladenine (BA) and 0.1 mg L^?1 indole-3-acetic acid (IAA). The in vitro-regenerated shoots were rooted (83.3–100 %) and transferred to an experimental plot with 62 % efficiency. Flow cytometric analysis revealed no variation in nuclear DNA content in field- and in vitro-delivered plant material. Ultra high performance liquid chromatography (UHPLC) indicated that multiple shoots and roots from in vitro-regenerated plantlets and adventitious root cultures maintained the production of rosmarinic (RA) and chlorogenic (CGA) acids and triterpenoid saponins found in the rosette leaves and roots of E. maritimum intact plants. UHPLC revealed a 12-fold increase of RA and CGA and 3.2-fold higher accumulation of triterpenoid saponins in roots of in vitro-derived plantlets in comparison to roots from field-grown plants. Adventitious root cultures allowed continuous growth of excised root in liquid media with or without exogenous auxins. The roots grown in liquid medium supplemented with 0.1 mg L^?1 IAA showed higher (227-fold) phenolic acids accumulation than those without auxin. Obtained results confirmed that micropropagation is a useful strategy in the protection of endangered species and a renewable source of a high quality plant material for secondary metabolites production.     

New Eoblattida from the Permian of Russia and the United States and the origin of earwigs (Insecta: Eoblattida,Forficulida)

New taxa of the order Eoblattida are described from the Lower Permian of the United States and Russia. Pryg absurdus gen. et sp. nov. of the family Prygidae fam. nov. is described from the Leonardian of Elmo (Kansas, United States). Parapryg alogus gen. et sp. nov. of the same family is described from the Vyatkian of Nedubrovo (Vologda Region, Russia). Kamamica promota gen. et sp. nov. of the family Tillyardembiidae is described from the Ufimian of Tyul’kino (Perm Region, Russia). The similarity of the new family with Tillyardembiidae and with basal earwigs, especially Bardacoleidae, is discussed. A hypothesis is proposed about the monophyletic origin of all three groups from spanioderids or other eoblattids close to them.     

Acclimation of denitrifying activated sludge to a single vs. complex external carbon source during a start-up of sequencing batch reactors treating ammonium-rich anaerobic sludge digester liquors

In this study, denitrification of ammonium-reach anaerobic sludge digester liquor was investigated during start-up periods of two laboratory-scale “fill-and-draw” reactors. One reactor was fed with a single carbon source (ethanol), whereas the other reactor was fed with a complex carbon source (fusel oil). During two acclimation experiments, the structure of microbial community involved in denitrification was analyzed using 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis fingerprints and fluorescent in situ hybridization. The characteristics of the mixed liquor were additionally supported by regular measurements of nitrate uptake rates. The addition of fusel oil and ethanol resulted in a significant enhancement of the denitrification rate and efficiency combined with the increasing volumetric addition of sludge digester liquor up to 15 % of the reactor volume. The microbiological analyses revealed that the addition of sludge digester liquor as well as both external carbon sources (fusel oil and ethanol) did not affect the structure of microbial communities in a severe way. In both reactors, Curvibacter sp. and Azoarcus sp. were found as the most abundant representatives of denitrifiers.