The dry basis elemental composition of the feedstock, shown in Table 2, is identical to previous NREL and PNNL design reports [20,21]. The composition was originally assumed to come from pulpwood. Recent feedstock logistics work at the Idaho National Laboratory (INL) suggests that the use of blended material may be required to meet a cost target of $80/dry U.S. ton while still meeting these specifications [22]. For the purpose of this report, it is assumed that any blended material provided to meet this feedstock elemental composition will not adversely affect fast pyrolysis conversion efficiencies. Ongoing studies being conducted jointly by INL, NREL, and PNNL will provide experimental evidence of the impact of blended feedstocks on fast pyrolysis and gasification processes. Future TEA will be modified to reflect conversion impacts inferred from such studies.This report is available at no cost from the National Renewable Energy Laboratory (NREL) at www.nrel.gov/publications. not considered in this design in order to focus on the core technology of in situ and ex situ fast pyrolysis vapor upgrading. Aspen Plus ModelAn Aspen Plus Version 7.2 simulation was used as the basis for this report. Since the products in pyrolysis are numerous and varied, only selected model compounds were used to represent the product slate. Additional hydrocarbon species were added to represent hydroprocessing products. Many of the desired molecular species in the desired boiling ranges for light and heavy fractions did not exist in Aspen Plus databanks and physical property parameters needed to be estimated. The biomass feedstock, ash, char, and coke were modeled as non-conventional components. Appendix F provides information about compounds selected to represent the process. The Peng-Robinson with Boston-Mathias modifications (PR-BM) equation of state was used throughout most of the process simulation. The ASME 1967 steam table correlations (STEAM-TA) were used for the steam cycle calculations. Combustor/Regenerator Temperature, °C (°F) 650 (1,202) 720 (1,328) 650 (1,202) Pressure, psia (bar) 117 (8.1) 117 (8.1) 113 (7.8) Excess air (%) 20 20 20 Solids temperature before transfer to reactor, °C (°F) 650 (1,202) 720 (1,328) 341 (645) No. of cyclones per combustor 2 2 2 Area 200 Equipment Cost EstimationsCapital costs for the equipment in this area were estimated by Harris Group. A previously developed spreadsheet tool for gasifier costs was leveraged for this exercise. Cost estimates from this tool were compared with order of magnitude estimates from technology vendors and documented in Appendix I of Worley et al.

Dormancy is a common strategy adopted by bacterial cells as a means of surviving adverse environmental conditions. For Streptomyces bacteria, this involves developing chains of dormant exospores that extend away from the colony surface. Both spore formation and subsequent spore germination are tightly controlled processes, and while significant progress has been made in understanding the underlying regulatory and enzymatic bases for these, there are still significant gaps in our understanding. One class of proteins with a potential role in spore-associated processes are the so-called resuscitation-promoting factors, or Rpfs, which in other actinobacteria are needed to restore active growth to dormant cell populations. The model species Streptomyces coelicolor encodes five Rpf proteins (RpfA to RfpE), and here we show that these proteins have overlapping functions during growth. Collectively, the S. coelicolor Rpfs promote spore germination and are critical for growth under nutrient-limiting conditions. Previous studies have revealed structural similarities between the Rpf domain and lysozyme, and our in vitro biochemical assays revealed various levels of peptidoglycan cleavage capabilities for each of these five Streptomyces enzymes. Peptidoglycan remodeling by enzymes such as these must be stringently governed so as to retain the structural integrity of the cell wall. Our results suggest that one of the Rpfs, RpfB, is subject to a unique mode of enzymatic autoregulation, mediated by a domain of previously unknown function (DUF348) located within the N terminus of the protein; removal of this domain led to significantly enhanced peptidoglycan cleavage. Streptomyces species are Gram-positive bacteria that abound in soil environments. They are renowned for their secondary metabolic capabilities; they produce a multitude of compounds that have found utility in both medicine and agriculture, including a vast array of antibiotics, chemotherapeutics, immunosuppressants, and antiparasitic agents (1). They are also well known for their complex, multicellular developmental cycle (2). The Streptomyces life cycle can be broadly divided into two stages: vegetative growth and reproductive development. Unlike most bacteria, Streptomyces organisms are filamentous, and during the vegetative phase of their life cycle, they grow by hyphal tip extension and branching, ultimately forming an interwoven network of cells known as the vegetative mycelium (3). Under certain as yet poorly defined stress conditions, vegetative growth ceases, and reproductive growth ensues. Here, unbranched filamentous cells, termed aerial hyphae, grow away from the vegetative mycelium and extend into the air. These cells then undergo a synchronous round of chromosome segregation, cell division, and cell wall modification to yield chains of dormant exospores (4). These spores are resistant to a wide variety of environmental insults and can be widely dispersed.A dormant cell state is not unique to the streptomycetes, and indeed many well-studied bacteria (e...

Studying bacterial cell envelope architecture with electron microscopy is challenging due to the poor preservation of microbial ultrastructure with traditional methods. Here, we established and validated a super-resolution cryo-correlative light and electron microscopy (cryo-CLEM) method, and combined it with cryo-focused ion beam (cryo-FIB) milling and scanning electron microscopy (SEM) volume imaging to structurally characterize the bacterium Deinococcus radiodurans . Subsequent cryo-electron tomography (cryo-ET) revealed an unusual diderm cell envelope architecture with a thick layer of peptidoglycan (PG) between the inner and outer membranes, an additional periplasmic layer, and a proteinaceous surface S-layer. Cells grew in tetrads, and division septa were formed by invagination of the inner membrane (IM), followed by a thick layer of PG. Cytoskeletal filaments, FtsA and FtsZ, were observed at the leading edges of constricting septa. Numerous macromolecular complexes were found associated with the cytoplasmic side of the IM. Altogether, our study revealed several unique ultrastructural features of D. radiodurans cells, opening new lines of investigation into the physiology and evolution of the bacterium.

Many species of Gram-positive bacteria use sortase transpeptidases to covalently affix proteins to their cell wall or to assemble pili. Sortase-displayed proteins perform critical and diverse functions for cell survival, including cell adhesion, nutrient acquisition, and morphological development, among others. Based on their amino acid sequences, there are at least six types of sortases (class A to F enzymes); however, class E enzymes have not been extensively studied. Class E sortases are used by soil and freshwater-dwelling Actinobacteria to display proteins that contain a non-canonical LAXTG sorting signal, which differs from 90% of known sorting signals by substitution of alanine for proline. Here we report the first crystal structure of a class E sortase, the 1.93 Å resolution structure of the SrtE1 enzyme from Streptomyces coelicolor. The active site is bound to a tripeptide, providing insight into the mechanism of substrate binding. SrtE1 possesses β3/β4 and β6/β7 active site loops that contact the LAXTG substrate and are structurally distinct from other classes. We propose that SrtE1 and other class E sortases employ a conserved tyrosine residue within their β3/β4 loop to recognize the amide nitrogen of alanine at position P3 of the sorting signal through a hydrogen bond, as seen here. Incapability of hydrogen-bonding with canonical proline-containing sorting signals likely contributes to class E substrate specificity. Furthermore, we demonstrate that surface anchoring of proteins involved in aerial hyphae formation requires an N-terminal segment in SrtE1 that is presumably positioned within the cytoplasm. Combined, our results reveal unique features within class E enzymes that enable them to recognize distinct sorting signals, and could facilitate the development of substrate-based inhibitors of this important enzyme family.

The success of Mycobacterium tuberculosis as a pathogen is well established: tuberculosis is the leading cause of death by a single infectious agent worldwide. The threat of multi- and extensively drug-resistant bacteria has renewed global concerns about this pathogen and understanding its virulence strategies will be essential in the fight against tuberculosis. The current review will focus on phthiocerol dimycocerosates (PDIMs), a long-known and well-studied group of complex lipids found in the M. tuberculosis cell envelope. Numerous studies show a role for PDIMs in several key steps of M. tuberculosis pathogenesis, with recent studies highlighting its involvement in bacterial virulence, in association with the ESX-1 secretion system. Yet, the mechanisms by which PDIMs help M. tuberculosis to control macrophage phagocytosis, inhibit phagosome acidification and modulate host innate immunity, remain to be fully elucidated.