Biochemical Conversions of Lignocellulosic Biomass for Sustainable Fuel-Ethanol Production in the Upper Midwest

Biofuels are an increasingly important component of worldwide energy supply. This research aims to understand the pathways and impacts of biofuels production, and to improve these processes to make them more efficient. In Chapter 2, a life cycle assessment (LCA) is presented for cellulosic ethanol production from five potential feedstocks of regional importance to the upper Midwest - hybrid poplar, hybrid willow, switchgrass, diverse prairie grasses, and logging residues - according to the requirements of Renewable Fuel Standard (RFS). Direct land use change emissions are included for the conversion of abandoned agricultural land to feedstock production, and computer models of the conversion process are used in order to determine the effect of varying biomass composition on overall life cycle impacts. All scenarios analyzed here result in greater than 60% reduction in greenhouse gas emissions relative to petroleum gasoline. Land use change effects were found to contribute significantly to the overall emissions for the first 20 years after plantation establishment. Chapter 3 is an investigation of the effects of biomass mixtures on overall sugar recovery from the combined processes of dilute acid pretreatment and enzymatic hydrolysis. Biomass mixtures studied were aspen, a hardwood species well suited to biochemical processing; balsam, a high-lignin softwood species, and switchgrass, an herbaceous energy crop with high ash content. A matrix of three different dilute acid pretreatment severities and three different enzyme loading levels was used to characterize interactions between pretreatment and enzymatic hydrolysis. Maximum glucose yield for any species was 70% oftheoretical for switchgrass, and maximum xylose yield was 99.7% of theoretical for aspen. Supplemental β-glucosidase increased glucose yield from enzymatic hydrolysis by an average of 15%, and total sugar recoveries for mixtures could be predicted to within 4% by linear interpolation of the pure species results. Chapter 4 is an evaluation of the potential for producing Trichoderma reesei cellulose hydrolases in the Kluyveromyces lactis yeast expression system. The exoglucanases Cel6A and Cel7A, and the endoglucanase Cel7B were inserted separately into the K. lactis and the enzymes were analyzed for activity on various substrates. Recombinant Cel7B was found to be active on carboxymethyl cellulose and Avicel powdered cellulose substrates. Recombinant Cel6A was also found to be active on Avicel. Recombinant Cel7A was produced, but no enzymatic activity was detected on any substrate. Chapter 5 presents a new method for enzyme improvement studies using enzyme co-expression and yeast growth rate measurements as a potential high-throughput expression and screening system in K. lactis yeast. Two different K. lactis strains were evaluated for their usefulness in growth screening studies, one wild-type strain and one strain which has had the main galactose metabolic pathway disabled. Sequential transformation and co-expression of the exoglucanase Cel6A and endoglucanase Cel7B was performed, and improved hydrolysis rates on Avicel were detectable in the cell culture supernatant. Future work should focus on hydrolysis of natural substrates, developing the growth screening method, and utilizing the K. lactis expression system for directed evolution of enzymes.

Brain-borne IL-1 adjusts glucoregulation and provides fuel support to astrocytes and neurons in an autocrine/paracrine manner.

It is still controversial which mediators regulate energy provision to activated neural cells, as insulin does in peripheral tissues. Interleukin-1β (IL-1β) may mediate this effect as it can affect glucoregulation, it is overexpressed in the 'healthy' brain during increased neuronal activity, and it supports high-energy demanding processes such as long-term potentiation, memory and learning. Furthermore, the absence of sustained neuroendocrine and behavioral counterregulation suggests that brain glucose-sensing neurons do not perceive IL-1β-induced hypoglycemia. Here, we show that IL-1β adjusts glucoregulation by inducing its own production in the brain, and that IL-1β-induced hypoglycemia is myeloid differentiation primary response 88 protein (MyD88)-dependent and only partially counteracted by Kir6.2-mediated sensing signaling. Furthermore, we found that, opposite to insulin, IL-1β stimulates brain metabolism. This effect is absent in MyD88-deficient mice, which have neurobehavioral alterations associated to disorders in glucose homeostasis, as during several psychiatric diseases. IL-1β effects on brain metabolism are most likely maintained by IL-1β auto-induction and may reflect a compensatory increase in fuel supply to neural cells. We explore this possibility by directly blocking IL-1 receptors in neural cells. The results showed that, in an activity-dependent and paracrine/autocrine manner, endogenous IL-1 produced by neurons and astrocytes facilitates glucose uptake by these cells. This effect is exacerbated following glutamatergic stimulation and can be passively transferred between cell types. We conclude that the capacity of IL-1β to provide fuel to neural cells underlies its physiological effects on glucoregulation, synaptic plasticity, learning and memory. However, deregulation of IL-1β production could contribute to the alterations in brain glucose metabolism that are detected in several neurologic and psychiatric diseases.Molecular Psychiatry advance online publication, 8 December 2015; doi:10.1038/mp.2015.174.

4HPR-X radiation mechanisms of interactions in non-small cell lung cancer (NSCLC) cells in vitro

Lung cancer is the leading cause of cancer death. However, poor survival using conventional therapies fuel the search for more rational interventions. The objective of this study was to design and implement a 4HPR-radiation interaction model in NSCLC, employing a traditional clinical modality (radiation), a relatively new, therapeutically unexplored agent (4HPR) and rationally combining them based on molecular mechanistic findings pertaining to their interactions. To test the hypothesis that 4HPR sensitizes cells to radiation-induced cell death via G2+M accumulation, we designed a working model consisting of H522 adenocarcinoma cells (p53, K-ras mutated) derived from an NSCLC patient; 4HPR at concentrations up to 10 μM; and X radiation up to 6 Gy generated by a patient-dedicated Phillips RT-250 X ray unit at 250 KV, 15 mA, 1.85 Gy/min. We found that 4HPR produced time- and dose-dependent morphological changes, growth inhibition, and DNA damage-inducing enhancement of reactive oxygen species. A transient G2+M accumulation of cells maximal at 24 h of continuous 4HPR exposure was used for irradiation time scheduling. Our data demonstrated enhanced cell death (both apoptotic and necrotic) in irradiated cells pre-treated with 4HPR versus those with either stressor alone. 4HPR's effect of increased NSCLC cells' radioresponse was confirmed by clonogenic assay. To explore these practical findings from a molecular mechanistic perspective, we further investigated and showed that levels of cyclin B1 and p34cdc2 kinase—both components of the mitosis promoting factor (MPF) regulating the G2/M transition—did not change following 4HPR treatment. Likewise, cdc25C phosphatase was not altered. However, enhanced p34cdc2 phosphorylation on its Thr14Tyr15 residues—indicative of its inactivation and increased expression of MPF negative regulators chk1 and wee1 kinases—were supportive of explaining 4HPR-treated cells' accumulation. Hence, p34cdc2 phosphorylation, chk1, and wee1 warrant further evaluation as potential molecular targets for 4HPR-X radiation combination. In summary, we (1) demonstrated that 4HPR not only induces cell death by itself, but also increases NSCLC cells' subsequent radioresponse, indicative of potential clinical applicability, and (2) for the first time, shed light on deciphering 4HPR-X radiation molecular mechanisms of interaction, including the finding of 4HPR's role as a p34cdc2 inactivator via Thr14Tyr15 phosphorylation. ^

Pliocene-Plistocene deposition of carbonate and organic carbon in ODP Hole 159-959C

During Ocean Drilling Program (ODP) Leg 159, four sites (Sites 959-962) were drilled along a depth transect on the Côte d'Ivoire/Ghana Transform Margin. In this study, the Pliocene-Pleistocene history of carbonate and organic carbon accumulation at Hole 959C is reconstructed for the eastern equatorial Atlantic off the Ivory Coast/Ghana based on bulk carbonate, sand fraction, organic carbon, and other organic geochemical records (d13Corg, marine organic matter percentages derived from organic petrology, hydrogen index, C/N). Pliocene-Pleistocene sedimentation off the Ivory Coast/Ghana was strongly affected by low mean sedimentation rates, which are attributed to persistently enhanced bottom-water velocities related to the steep topography of the transform margin. Sand fraction and bulk carbonate records reveal typical glacial/interglacial cycles, preserved, however, with low time resolution. Intermediate carbonate accumulation rates observed throughout the Pliocene-Pleistocene suggest intense winnowing and sediment redistribution superimposed by terrigenous dilution. 'Atlantic-type' sand and carbonate cycles, consistent with records from pelagic areas of the eastern equatorial Atlantic, are encountered at Hole 959C prior to about 0.9 Ma. Total organic carbon (TOC) records are frequently inversely correlated to carbonate contents, indicating mainly productivity-driven carbonate dissolution related to changes in paleoproductivity. During Stages 22-24, 20, 16, 12, 8, and 4, sand and carbonate records reveal a 'Pacific-type' pattern, showing elevated contents during glacials commonly in conjunction with enhanced TOC records. Formation of 'Pacific-type' patterns off the Ivory Coast/Ghana is attributed to drastically increased bottom-water intensities along the transform margin in accordance with results reported from the Walvis Ridge area. Short-term glacial/interglacial changes in paleoproductivity off the Ivory Coast/Ghana are to some extend recognizable during glacials prior to 1.7 Ma and interglacial Stages 21, 19, 13, 9, and 1. Enhanced coastal upwelling during interglacials is attributed to local paleoclimatic and oceanographic conditions off the Ivory Coast/Ghana. Quantitative estimates of marine organic carbon based on organic petrologic and d13Corg records reveal an offset in concentration ranging from 15% to 60%. Highest variabilities of both records are recorded since ~0.9 Ma. Discrepancies between the isotopic and microscopic records are attributed to an admixture of C4 plant debris approaching the eastern equatorial Atlantic via atmospheric dust. Terrestrial organic material likely originated from the grass-savannah-covered Sahel zone in central Africa. Estimated C4 plant concentrations and accumulation rates range from 10% to 37% and from almost zero to 0.006 g/cm**2/k.y., respectively. The strongest eolian supply to the northern Gulf of Guinea is indicated between 1.9 and 1.68 Ma and during glacial isotopic Stages 22-24, 20, 14, and 12. The presence of grass-type plant debris is further supported by organic petrologic studies, which reveal well-preserved cell tissues of vascular plants or tube-shaped, elongated terrestrial macerals showing different levels of oxidation.

Effects of pCO2 and iron on the elemental composition and cell geometry of the marine diatom Pseudo-nitzschia pseudodelicatissima//(Bacillariophyceae)

Partial pressure of CO2 (pCO2) and iron availability in seawater show corresponding changes due to biological and anthropogenic activities. The simultaneous change in these factors precludes an understanding of their independent effects on the ecophysiology of phytoplankton. In addition, there is a lack of data regarding the interactive effects of these factors on phytoplankton cellular stoichiometry, which is a key driving factor for the biogeochemical cycling of oceanic nutrients. Here, we investigated the effects of pCO2 and iron availability on the elemental composition (C, N, P, and Si) of the diatom Pseudo-nitzschia pseudodelicatissima (Hasle) Hasle by dilute batch cultures under 4 pCO2 (~200, ~380, ~600, and ~800 µatm) and five dissolved inorganic iron (Fe'; ~5, ~10, ~20, ~50, and ~100 pmol /L) conditions. Our experimental procedure successfully overcame the problems associated with simultaneous changes in pCO2 and Fe' by independently manipulating carbonate chemistry and iron speciation, which allowed us to evaluate the individual effects of pCO2 and iron availability. We found that the C:N ratio decreased significantly only with an increase in Fe', whereas the C:P ratio increased significantly only with an increase in pCO2. Both Si:C and Si:N ratios decreased with increasing pCO2 and Fe'. Our results indicate that changes in pCO2 and iron availability could influence the biogeochemical cycling of nutrients in future oceans with high- CO2 levels, and, similarly, during the time course of phytoplankton blooms. Moreover, pCO2 and iron availability may also have affected oceanic nutrient biogeochemistry in the past, as these conditions have changed markedly over the Earth's history.

Seawater carbonate chemistry and toxicity of Pseudo-nitzschia fraudulenta in a laboratory experiment

Anthropogenic CO2 is progressively acidifying the ocean, but the responses of harmful algal bloom species that produce toxins that can bioaccumulate remain virtually unknown. The neurotoxin domoic acid is produced by the globally-distributed diatom genus Pseudo-nitzschia. This toxin is responsible for amnesic shellfish poisoning, which can result in illness or death in humans and regularly causes mass mortalities of marine mammals and birds. Domoic acid production by Pseudo-nitzschia cells is known to be regulated by nutrient availability, but potential interactions with increasing seawater CO2 concentrations are poorly understood. Here we present experiments measuring domoic acid production by acclimatized cultures of Pseudo-nitzschia fraudulenta that demonstrate a strong synergism between projected future CO2 levels (765 ppm) and silicate-limited growth, which greatly increases cellular toxicity relative to growth under modern atmospheric (360 ppm) or pre-industrial (200 ppm) CO2 conditions. Cellular Si:C ratios decrease with increasing CO2, in a trend opposite to that seen for domoic acid production. The coastal California upwelling system where this species was isolated currently exhibits rapidly increasing levels of anthropogenic acidification, as well as widespread episodic silicate limitation of diatom growth. Our results suggest that the current ecosystem and human health impacts of toxic Pseudo-nitzschia blooms could be greatly exacerbated by future ocean acidification and 'carbon fertilization' of the coastal ocean.

Seawater carbonate chemistry and protein content, respiration, symbiodinium densities, survivorship of Pocillopora damicornis larvae in a laboratory experiment

Efforts to evaluate the response of coral larvae to global climate change (GCC) and ocean acidification (OA) typically employ short experiments of fixed length, yet it is unknown how the response is affected by exposure duration. In this study, we exposed larvae from the brooding coral Pocillopora damicornis to contrasts of temperature (24.00 °C [ambient] versus 30.49 °C) and pCO2 (49.4 Pa versus 86.2 Pa) for varying periods (1-5 days) to test the hypothesis that exposure duration had no effect on larval response as assessed by protein content, respiration, Symbiodinium density, and survivorship; exposure times were ecologically relevant compared to representative pelagic larval durations (PLD) for corals. Larvae differed among days for all response variables, and the effects of the treatment were relatively consistent regardless of exposure duration for three of the four response variables. Protein content and Symbiodinium density were unaffected by temperature and pCO2, but respiration increased with temperature (but not pCO2) with the effect intensifying as incubations lengthened. Survival, however, differed significantly among treatments at the end of the study, and by the 5th day, 78% of the larvae were alive and swimming under ambient temperature and ambient pCO2, but only 55-59% were alive in the other treatments. These results demonstrate that the physiological effects of temperature and pCO2 on coral larvae can reliably be detected within days, but effects on survival require > or = 5 days to detect. The detection of time-dependent effects on larval survivorship suggests that the influence of GCC and OA will be stronger for corals having long PLDs.

Seawater carbonate chemistry, community calcification and photosynthesis during experiments with coral reefs at Shiraho reef, Ishigaki Island, southwest Japan, 2009

Seven coral reef communities were defined on Shiraho fringing reef, Ishigaki Island, Japan. Net photosynthesis and calcification rates were measured by in situ incubations at 10 sites that included six of the defined communities, and which occupied most of the area on the reef flat and slope. Net photosynthesis on the reef flat was positive overall, but the reef flat acts as a source for atmospheric CO2, because the measured calcification/photosynthesis ratio of 2.5 is greater than the critical ratio of 1.67. Net photosynthesis on the reef slope was negative. Almost all excess organic production from the reef flat is expected to be effused to the outer reef and consumed by the communities there. Therefore, the total net organic production of the whole reef system is probably almost zero and the whole reef system also acts as a source for atmospheric CO2. Net calcification rates of the reef slope corals were much lower than those of the branching corals. The accumulation rate of the former was approximately 0.5 m kyr?1 and of the latter was ~0.7-5 m kyr?1. Consequently, reef slope corals could not grow fast enough to keep up with or catch up to rising sea levels during the Holocene. On the other hand, the branching corals grow fast enough to keep up with this rising sea level. Therefore, a transition between early Holocene and present-day reef communities is expected. Branching coral communities would have dominated while reef growth kept pace with sea level rise, and the reef was constructed with a branching coral framework. Then, the outside of this framework was covered and built up by reef slope corals and present-day reefs were constructed.

Seawater carbonate chemistry and early development of the sea urchin Lytechinus variegatus in a laboratory experiment using artificial seawater

Land-based aquaculture facilities often utilize additional bicarbonate sources such as commercial sea salts that are designed to boost alkalinity in order to buffer seawater against reductions in pH. Despite these preventative measures, many facilities are likely to face occasional reductions in pH and corresponding reductions in carbonate saturation states due to the accumulation of metabolic waste products. We investigated the impact of reduced carbonate saturation states (Omega Ca, Omega Ar) on embryonic developmental rates, larval developmental rates, and echinoplutei skeletal morphometrics in the common edible sea urchin Lytechinus variegatus under high alkalinity conditions. Commercial artificial seawater was bubbled with a mixture of air and CO2 gas to reduce the carbonate saturation state. Rates of embryonic and larval development were significantly delayed in both the low and extreme low carbonate saturation state groups relative to the control at a given time. Although symmetry of overall skeletal body lengths was not affected, allometric relationships were significantly different between treatment groups. Larvae reared under ambient conditions had significantly greater postoral arm and overall body lengths relative to body lengths than larvae grown under extreme low carbonate saturation state conditions, indicating that extreme changes in the carbonate system affected not only developmental rates but also larval skeletal shape. Reduced rates of embryonic development and delayed and altered larval skeletal growth are likely to negatively impact larval culturing of L. variegatus in land-based, intensive culture situations where calcite and aragonite saturation states are lowered by the accumulation of metabolic waste products.

Seawater carbonate chemistry, POC, PIC, TPC, SPM, N, TEP and growth rate during experiments with coccolithophores Emiliania huxleyi (AC472), Calcidiscus leptoporus (AC370) and Syracosphaera pulchra (AC418) during experiments, 2011

The response of three coccolithophores (Emiliania huxleyi, Calcidiscus leptoporus and Syracosphaera pulchra) to elevated partial pressure (pCO2) of carbon dioxide was investigated in batch cultures. For the first time, we also report on the response of the non calcifying (haploid) life stage of these three species. The growth rate, cell size, inorganic (PIC) and organic carbon (POC) of both life stages were measured at two different pCO2 (400and 760 ppm) and their organic and inorganic carbon production calculated. The two lifestages within the same species generally exhibited a similar response to elevated pCO2, theresponse of the haploid stage being often more pronounced than that of the diploid stage. Thegrowth rate was consistently higher at higher pCO2 but the response of other processes varied among species. The calcification rate of C. leptoporus and of S. pulchra did not change at elevated pCO2 while increased in E. huxleyi. The POC production as well as the cell size of both life stages of S. pulchra and of the haploid stage of E. huxleyi markedly decreased at elevated pCO2. It remained unaltered in the diploid stage of E. huxleyi and C. leptoporus and increased in the haploid stage of the latter. The PIC:POC ratio increased in E. huxleyi and was constant in C. leptoporus and S. pulchra. These results suggest that the non-calcifying stage, is more responsive than the calcifying stage and that the most versatile genera will proliferate in a more acidic ocean rather than all coccolithophores will decline.

Seawater carbonate chemistry and processes during experiments with cyanobacterium Nodularia spumigena, 2009

The surface ocean absorbs large quantities of the CO2 emitted to the atmosphere from human activities. As this CO2 dissolves in seawater, it reacts to form carbonic acid. While this phenomenon, called ocean acidification, has been found to adversely affect many calcifying organisms, some photosynthetic organisms appear to benefit from increasing [CO2]. Among these is the cyanobacterium Trichodesmium, a predominant diazotroph (nitrogen-fixing) in large parts of the oligotrophic oceans, which responded with increased carbon and nitrogen fixation at elevated pCO2. With the mechanism underlying this CO2 stimulation still unknown, the question arises whether this is a common response of diazotrophic cyanobacteria. In this study we therefore investigate the physiological response of Nodularia spumigena, a heterocystous bloom-forming diazotroph of the Baltic Sea, to CO2-induced changes in seawater carbonate chemistry. N. spumigena reacted to seawater acidification/carbonation with reduced cell division rates and nitrogen fixation rates, accompanied by significant changes in carbon and phosphorus quota and elemental composition of the formed biomass. Possible explanations for the contrasting physiological responses of Nodularia compared to Trichodesmium may be found in the different ecological strategies of non-heterocystous (Trichodesmium) and heterocystous (Nodularia) cyanobacteria.

Seawater carbonate chemistry and silica during an experiment with a marine diatom Thalassiosira weissflogii, 2004

Opal accumulation rates in sediments have been used as a proxy for carbon flux, but there is poor understanding of the factors that regulate the Si quota of diatoms. Natural variation in silicon isotopes (delta.lc.gif - 54 Bytes30Si) in diatom frustules recovered from sediment cores are an alternative to opal mass for reconstructing diatom Si use and potential C export over geological timescales. Understanding the physiological factors that may influence the Si quota and the delta.lc.gif - 54 Bytes30Si isotopic signal is vital for interpreting biogenic silica as a paleoproxy. We investigated the influence of pCO2 on the Si quota, fluxes across the cell membrane, and frustule dissolution in the marine diatom Thalassiosira weissflogii and determined the effect that pCO2 has on the isotopic fractionation of Si. We found that our Si flux estimates mass balance and, for the first time, describe the Si budget of a diatom. The Si quota rose in cells grown with low pCO2 (100 ppm) compared with controls (370 ppm), and the increased quota was the result of greater retention of Si (i.e., lower losses of Si through efflux and dissolution). The ratio of efflux : influx decreased twofold as pCO2 decreased from 750 to 100 ppm. The efflux of silicon is shown to significantly bias measurements of silica dissolution rates determined by isotope dilution, but no effect on the Si isotopic enrichment factor (epsilon.lc.gif - 51 Bytes) was observed. The latter effect suggests that silicon isotopic discrimination in diatoms is set by the Si transport step rather than by the polymerization step. This observation supports the use of the v signal of biogenic silica as an indicator of the percentage utilization of silicic acid.

Seawater carbonate chemistry and dark respiration and photosynthetic capacity during experiments with coral Acropora formosa, 2010

Ocean acidification is expected to lower the net accretion of coral reefs yet little is known about its effect on coral photophysiology. This study investigated the effect of increasing CO2 on photosynthetic capacity and photoprotection in Acropora formosa. The photoprotective role of photorespiration within dinoflagellates (genus Symbiodinium) has largely been overlooked due to focus on the presence of a carbon-concentrating mechanism despite the evolutionary persistence of a Form II Rubisco. The photorespiratory fixation of oxygen produces phosphoglycolate that would otherwise inhibit carbon fixation though the Calvin cycle if it were not converted to glycolate by phosphoglycolate phosphatase (PGPase). Glycolate is then either excreted or dealt with by enzymes in the photorespiratory glycolate and/or glycerate pathways adding to the pool of carbon fixed in photosynthesis. We found that CO2 enrichment led to enhanced photoacclimation (increased chlorophyll a per cell) to the subsaturating light levels. Light-enhanced dark respiration per cell and xanthophyll de-epoxidation increased, with resultant decreases in photosynthetic capacity (Pnmax) per chlorophyll. The conservative CO2 emission scenario (A1B; 600-790 ppm) led to a 38% increase in the Pnmax per cell whereas the 'business-as-usual' scenario (A1F1; 1160-1500 ppm) led to a 45% reduction in PGPase expression and no change in Pnmax per cell. These findings support an important functional role for PGPase in dinoflagellates that is potentially compromised under CO2 enrichment.