A technique for lyopreservation of Clostridium ljungdahlii in a biocomposite matrix for CO absorption.

A system capable of biocatalytic conversion of distributed sources of single carbon gases such as carbon monoxide into hydrocarbons can be highly beneficial for developing commercially viable biotechnology applications in alternative energy. Several anaerobic bacterial strains can be used for such conversion. The anaerobic carbon monoxide-fixing bacteria Clostridium ljungdahlii OTA1 is a model CO assimilating microorganism that currently requires cryogenic temperature for storage of the viable strains. If these organisms can be stabilized and concentrated in thin films in advanced porous materials, it will enable development of high gas fraction, biocomposite absorbers with elevated carbon monoxide (CO) mass transfer rate, that require minimal power input and liquid, and demonstrate elevated substrate consumption rate compared to conventional suspended cell bioreactors. We report development of a technique for dry-stabilization of C. ljungdahlii OTA1 on a paper biocomposite. Bacterial samples coated onto paper were desiccated in the presence of trehalose using convective drying and stored at 4°C. Optimal dryness was ~1g H2O per gram of dry weight (gDW). CO uptake directly following biocomposite rehydration steadily increases over time indicating immediate cellular metabolic recovery. A high-resolution Raman microspectroscopic hyperspectral imaging technique was employed to spatially quantify the residual moisture content. We have demonstrated for the first time that convectively dried and stored C. ljungdahlii strains were stabilized in a desiccated state for over 38 days without a loss in CO absorbing reactivity. The Raman hyperspectral imaging technique described here is a non-invasive characterization tool to support development of dry-stabilization techniques for microorganisms on inexpensive porous support materials. The present study successfully extends and implements the principles of dry-stabilization for preservation of strictly anaerobic bacteria as an alternative to lyophilization or spray drying that could enable centralized biocomposite biocatalyst fabrication and decentralized bioprocessing of CO to liquid fuels or chemicals.

Characterization of Clostridium ljungdahlii OTA1: a non-autotrophic hyper ethanol-producing strain.

A Clostridium ljungdahlii lab-isolated spontaneous-mutant strain, OTA1, has been shown to produce twice as much ethanol as the C. ljungdahlii ATCC 55383 strain when cultured in a mixotrophic medium containing fructose and syngas. Whole-genome sequencing identified four unique single nucleotide polymorphisms (SNPs) in the C. ljungdahlii OTA1 genome. Among these, two SNPs were found in the gene coding for AcsA and HemL, enzymes involved in acetyl-CoA formation from CO/CO2. Homology models of the respective mutated enzymes revealed alterations in the size and hydrogen bonding of the amino acids in their active sites. Failed attempts to grow OTA1 autotrophically suggested that one or both of these mutated genes prevented acetyl-CoA synthesis from CO/CO2, demonstrating that its activity was required for autotrophic growth by C. ljungdahlii. An inoperable Wood-Ljungdahl pathway resulted in higher CO2 and ethanol yields and lower biomass and acetate yields compared to WT for multiple growth conditions including heterotrophic and mixotrophic conditions. The two other SNPs identified in the C. ljungdahlii OTA1 genome were in genes coding for transcriptional regulators (CLJU_c09320 and CLJU_c18110) and were found to be responsible for deregulated expression of co-localized arginine catabolism and 2-deoxy-D-ribose catabolism genes. Growth medium supplementation experiments suggested that increased arginine metabolism and 2-deoxy-D-ribose were likely to have minor effects on biomass and fermentation product yields. In addition, in silico flux balance analysis simulating mixotrophic and heterotrophic conditions showed no change in flux to ethanol when flux through HemL was changed whereas limited flux through AcsA increased the ethanol flux for both simulations. In characterizing the effects of the SNPs identified in the C. ljungdahlii OTA1 genome, a non-autotrophic hyper ethanol-producing strain of C. ljungdahlii was identified that has utility for further physiology and strain performance studies and as a biocatalyst for industrial applications.

A high gas fraction, reduced power, syngas bioprocessing method demonstrated with a Clostridium ljungdahlii OTA1 paper biocomposite.

We propose a novel approach to continuous bioprocessing of gases. A miniaturized, coated-paper strip, high gas fraction, biocomposite absorber has been developed using slowly shaken horizontal anaerobic tubes. Concentrated Clostridium ljungdahlii OTA1 was used as a model system. These gas absorbers demonstrate elevated CO mass transfer with low power input, reduced liquid requirements, elevated substrate consumption, and increased product secretion compared to shaken suspended cells. Concentrated OTA1 cell paste was coated by extrusion onto chromatography paper. The immobilized system shows high, constant reactivity immediately upon rehydration. Cell adhesion was by adsorption to the cellulose fibers; visualized by SEM. The C. ljungdahlii OTA1 coated paper mounted above the liquid level absorbs CO and H2 from a model syngas secreting acetate with minimal ethanol. At 100 rpm shaking speed (7.7 Wm(-3) ) the optimal cell loading is 6.5 gDCW m(-2) to maintain high CO absorbing reactivity without the cells coming off of the paper into the liquid phase. Reducing the medium volume from 10 mL to 4 mL (15% of tube volume) did not decrease CO reactivity. The reduced liquid volume increased secreted product concentration by 80%. The specific CO consumption by paper biocomposites was higher at all shaking frequencies <100 rpm than suspended cells under identical incubation conditions. At 25 rpm the biocomposite outperforms suspended cells for CO absorption by 2.5-fold, with an estimated power reduction of 97% over the power input at 100 rpm. The estimated minimum kL a for miniaturized biocomposite gas-absorbers is ∼100 h(-1) , 10 to 10(4) less power input than other syngas fermentation systems reported in the literature at similar kL a. Specific consumption rates in a biocomposite were ∼14 mmol gDCW-1 h(-1) . This work intensified CO absorption and reactivity by 14-fold to 94 mmol CO m(-2) h(-1) over previous C. ljungdahlii OTA1 work by our group. Specific acetate production rates were 23 mM h(-1) or 46 mmol m(-2) h(-1) . The specific rates and apparent kL a scaled linearly with biocomposite coating area. Biotechnol. Bioeng. 2016;113: 1913-1923. © 2016 Wiley Periodicals, Inc.

Wegener's granulomatosis: clinical manifestations, differential diagnosis, and management of ocular and systemic disease.

Wegener's granulomatosis (WG) is a systemic inflammatory disease whose histopathologic features often include necrosis, granuloma formation, and vasculitis of small-to-medium-sized vessels. WG involves many interrelated pathogenic pathways that are genetic, cell-mediated, neutrophil-mediated, humoral, and environmental. WG most commonly involves the upper respiratory tract, lungs, and kidneys, but has been reported to affect almost any organ. Ophthalmologic involvement is an important cause of morbidity in WG patients, occurring in approximately one-half of patients. The presence of unexplained orbital inflammatory disease, scleritis, peripheral ulcerative keratitis, cicatricial conjunctivitis, nasolacrimal duct stenosis, retinal vascular occlusion, or infrequently uveitis should raise the question of possible WG. A thorough clinical examination, laboratory testing, radiologic imaging, and histologic examination are essential to diagnosing WG and excluding potential mimics. Previously a uniformly fatal disease, treatment with cytotoxic and immunosuppressive agents has greatly improved survival. Treatment-related morbidity is a serious limitation of conventional therapies, leading to numerous ongoing studies of alternative agents.