Solid-state fermentation production of tetracycline by <Emphasis Type="Italic">Streptomyces</Emphasis> strains using some agricultural wastes as substrate

The ability of Streptomyces sp. OXCI, S. rimosus NRRL B2659, S. rimosus NRRL B2234, S. alboflavus NRRL B1273 S. aureofaciens NRRL B2183 and S. vendagensis ATCC 25507 to produce tetracycline using some local agricultural wastes as solid state media, were assessed. The wastes employed include peanut (groundnut) shells, corncob, corn pomace and cassava peels. Bacillus subtilis ATCC 6633 was used to assay antimicrobial activity. All the strains produced tetracycline in a solid-state fermentation process containing peanut (groundnut) as the carbohydrate source. Streptomyces sp. OXC1 had the highest ability for tetracycline production with peanut shells as the substrate in solid fermentation (13.18 mg/g), followed by S. vendagensis ATCC 25507 (11.08 mg/g), S.rimosus NRRL B1679 (8.46 mg/g), S. alboflavus NRRL B1273 (7.59 mg/g), S. rimosus NRRL B2234 (6.37 mg/g), S. aureofaciens NRRL B2183 (4.27 mg/g). Peanut (groundnut) shells were the most effective substrate (4.36 mg/g) followed by corncob (2.64 mg/g), cassava peels (2.16 mg/g) and corn pomace (1.99 mg/g). The composition of the peanut (groundnut) shell medium optimal for tetracycline production were peanut shells 100 g, organic nitrogen (peanut meal) 10 g, (NH ₄)₂ SO₄ 1 g, KH₂ PO ₄ 0.5 g, CaCO₃ > 0.5 g, NaCl 0.5 g, MgSO₄ · 7H₂ O 0.5 g, soluble starch 10 g, peanut oil 0.25 ml with initial moisture content of 65–68%, and initial pH 5.3–6.3. Substrate (1 g dry weight) was inoculated with 1.0 × 10 ⁸ conidia per ml and incubated at 28–31 °C for 5–7 days, producing 13.18 mg/g of total tetracycline. Tetracycline detection started on day 3 and attained its maximum level on day 5.     

Bioconversion of grape must into modulated gluconic acid production by Aspergillus niger ORS-4.410

AIMS: Analysis of regulators for modulated gluconic acid production under surface fermentation (SF) condition using grape must as the cheap carbohydrate source, by mutant Aspergillus niger ORS-4.410. Replacement of conventional fermentation condition by solid-state surface fermentation (SSF) for semi-continuous production of gluconic acid by pseudo-immobilization of A. niger ORS-4.410. METHODS AND RESULTS: Grape must after rectification was utilized for gluconic acid production in batch fermentation in SF and SSF processes using mutant strain of A. niger ORS-4.410. Use of rectified grape must led to the improved levels of gluconic acid production (80-85 g l(-1)) in the fermentation medium containing 0.075% (NH4)2HPO4; 0.1% KH2PO4 and 0.015% MgSO4.7H2O at an initial pH 6.6 (+/-0.1) under surface fermentation. Gluconic acid production was modulated by incorporating the 2% soybean oil, 2% starch and 1% H2O2 in fermentation medium at continuously high aeration rate (2.0 l min(-1)). Interestingly, 95.8% yield of gluconic acid was obtained when A. niger ORS-4.410 was pseudo-immobilized on cellulose fibres (bagasse) under SSF. Four consecutive fermentation cycles were achieved with a conversion rate of 0.752-0.804 g g(-1) of substrate into gluconic acid under SSF. CONCLUSIONS: Use of additives modulated the gluconic acid production under SF condition. Semi-continuous production of gluconic acid was achieved with pseudo-immobilized mycelia of A. niger ORS-4.410 having a promising yield (95.8%) under SSF condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioconversion of grape must into modulated gluconic acid production under SSF conditions can further be employed in fermentation industries by replacing the conventional carbohydrate sources and expensive, energy consuming fermentation processes.     

Effects of air pressure oscillation amplitude on oxygen transfer rate and biomass productivity in a solid-state fermenter

The modified sulfite oxidation method was adapted for estimation of the overall oxygen transfer rate in a pressure oscillating, solid-state fermenter. At 4.5 atm and 30 °C, the oxygen transfer rate reached 717 mmol kg^–1 initial dry matter h^–1 in this system against 37 mmol kg^–1 initial dry matter h^–1 in a static tray fermenter. At 30 °C and 3 atm, Azotobacter vinelandii grew on wheat straw and reached 4.7×10¹⁰ c.f.u. g^–1 substrate dry matter after 36 h, while only 8.2×10⁹ c.f.u. g^–1 substrate dry matter was obtained in a static tray system.     

Selection of side-chain carbons in a high-molecular-weight, hydrophobic peptide using solid-state spectral editing methods

Solid-state spectral editing techniques have been used by others to simplify ¹³C CPMAS spectra of small organic molecules, synthetic organic polymers, and coals. One approach utilizes experiments such as cross-polarization-with-polarization-inversion and cross-polarization-with-depolarization to generate subspectra. This work shows that this particular methodology is also applicable to natural-abundance ¹³C CPMAS NMR studies of high-molecular-weight biopolymers. The editing experiments are demonstrated first with model peptides and then with -elastin, a high-molecular-weight peptidyl preparation obtained from the elastic fibers in mammalian tissue. The latter has a predominance of small, nonpolar residues, which is evident in the crowded aliphatic region of typical ¹³C CPMAS spectra. Spectral editing is particularly useful for simplifying the aliphatic region of the NMR spectrum of this elastin preparation.     

Pilot-scale production of mosquitocidal toxins by Bacillus thuringiensis and Lysinibacillus sphaericus under solid-state fermentation

Mosquitocidal toxins of Bacillus thuringiensis israelensis (Bti) and Lysinibacillus sphaericus 14N1 (Ls14N1) were produced under solid-state fermentation using agro-industrial wastes. Sugar beet pulp–sesame meal (1:1) and wheat germ meal–linen meal (1:1) at 9% were the efficient substrate mixtures for the growth and toxin production of Bti and Ls14N1, respectively. Bti was more active after the addition of beef extract (0.2%) or yeast extract (0.5%) to the medium. On the other hand, the addition of yeast extract (0.2%) or NYSM salts (2%) significantly enhanced the toxicity produced by Ls14N1. The optimum conditions for the maximum toxicity of Bti were at pH 7–8, 20–30% moisture, 4–10% inoculum and 7 days incubation. For Ls14N1, the best conditions were pH 6.5–7.5, 20–30% moisture, 4–10% inoculum and 5 days incubation. It was found that the best thickness of carrier-substrates in the plate (15?cm in diameter) for the maximum mosquitocidal activity was about 0.5?cm for Bti and 0.5–1?cm for Ls14N1. Pilot-scale production in aluminium trays applying the above conditions showed a decrement of toxicity of fermented cultures and some plates were contaminated. These problems were dissolved by reducing the moisture content to 15%, increasing inoculum to 10% and manual agitation of trays every-day.     

Interface Welding via Thermal Pulse Sintering to Enable 4.6 V Solid-State Batteries

NASICON-type Li_1.3Al_0.3Ti_1.7(PO4)₃ (LATP) is one of the most promising solid-state electrolytes (SSEs) to achieve high-energy-density solid-state batteries (SSBs) due to its high ionic conductivity, high-voltage stability, and low cost. However, its practical application is constrained by inadequate interfacial compatibility with cathode materials and significant incompatibility with lithium metal. In this work, a cost-effective interface welding approach is reported, utilizing an innovative thermal pulse sintering (TPS) to fabricate LATP-based solid-state batteries. Initially, the rapid thermal pulses enhance the ionic conductivity of LATP SSE by inducing selective growth of LATP nanowires, effectively occupying interparticle voids. Additionally, this process results in the formation of a dense layer (GCM) comprising graphene oxide, carbon nanotubes, and MXene with a controlled Li⁺ transport pathway, facilitating lithium stripping and plating processes. Moreover, these thermal pulses facilitate the interfacial fusion between LATP and cathode materials, while avoiding undesired phase diffusion. As a result, SSBs with a LiCoO₂ cathode deliver favorable cycle stability at 4.6 V, marking significant progress. This facile interface welding strategy represents a substantial step toward high-energy-density SSB development.     

Triosephosphate Isomerase: N and C Chemical Shift Assignments and Conformational Change upon Ligand Binding by Magic-Angle Spinning Solid-State NMR Spectroscopy

Microcrystalline uniformly ¹³C,¹⁵N-enriched yeast triosephosphate isomerase (TIM) is sequentially assigned by high-resolution solid-state NMR (SSNMR). Assignments are based on intraresidue and interresidue correlations, using dipolar polarization transfer methods, and guided by solution NMR assignments of the same protein. We obtained information on most of the active-site residues involved in chemistry, including some that were not reported in a previous solution NMR study, such as the side-chain carbons of His95. Chemical shift differences comparing the microcrystalline environment to the aqueous environment appear to be mainly due to crystal packing interactions. Site-specific perturbations of the enzyme's chemical shifts upon ligand binding are studied by SSNMR for the first time. These changes monitor proteinwide conformational adjustment upon ligand binding, including many of the sites probed by solution NMR and X-ray studies. Changes in Gln119, Ala163, and Gly210 were observed in our SSNMR studies, but were not reported in solution NMR studies (chicken or yeast). These studies identify a number of new sites with particularly clear markers for ligand binding, paving the way for future studies of triosephosphate isomerase dynamics and mechanism.     

Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase

The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase-S3P-Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native (31)P in S3P and Glp, biosynthetically (13)C-labeled S3P, specifically (13)C and (15)N labeled Glp, and a variety of protein-(15)N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.     

Significance of low-frequency local fluctuation motions in the transmembrane B and C α-helices of bacteriorhodopsin,to facilitate efficient proton uptake from the cytoplasmic surface,as revealed by site-directed solid-state <Superscript>13</Superscript>C NMR

¹³C NMR spectra of [1-¹³C]Val- or -Pro-labeled bacteriorhodopsin (bR) and its single or double mutants, including D85N, were recorded at various pH values to reveal conformation and dynamics changes in the transmembrane -helices, in relation to proton release and uptake between bR and the M-like state caused by modified charged states at Asp85 and the Schiff base (SB). It was found that the D85N mutant acquired local fluctuation motion with a frequency of 10⁴ Hz in the transmembrane B -helix, concomitant with deprotonation of SB in the M-like state at pH 10, as manifested from a suppressed ¹³C NMR signal of the [1-¹³C]-labeled Val49 residue. Nevertheless, local dynamics at Pro50 neighboring with Val49 turned out to be unchanged, irrespective of the charged state of SB as viewed from the ¹³C NMR of [1-¹³C]-labeled Pro50. This means that the transmembrane B -helix is able to acquire the fluctuation motion with a frequency of 10⁴ Hz beyond the kink at Pro50 in the cytoplasmic side. Concomitantly, fluctuation motion at the C helix with frequency in the order of 10⁴ Hz was found to be prominent, due to deprotonation of SB at pH 10, as viewed from the ¹³C NMR signal of Pro91. Accordingly, we have proposed here a novel mechanism as to proton uptake and transport based on a dynamic aspect that a transient environmental change from a hydrophobic to hydrophilic nature at Asp96 and SB is responsible for the reduced pK_a value which makes proton uptake efficient, as a result of acquisition of the fluctuation motion at the cytoplasmic side of the transmembrane B and C -helices in the M-like state. Further, it is demonstrated that the presence of a van der Waals contact of Val49 with Lys216 at the SB is essential to trigger this sort of dynamic change, as revealed from the ¹³C NMR data of the D85N/V49A mutant.     

Solid-state 13C NMR spectroscopic, chemolytic and biological assessment of pretreated municipal solid waste

In Central Europe, composting and anaerobic digestion of municipal solid waste (MSW) is used as pretreatment before landfilling to reduce landfill emissions. MSW samples were analyzed before, during, and after pretreatment to assess the stability of the organic matter. Chemolytic, nuclear magnetic resonance (NMR) spectroscopic, and respiration parameters were correlated to evaluate a substitution of the time-consuming respiration analysis by chemical parameters. ¹³C cross polarization magic angle spinning (CPMAS) NMR spectroscopy showed a preferential biodegradation of O-alkyl carbon (carbohydrates) and a selective accumulation of plastics during all pretreatments, confirming findings from chemolytic analyses. Principal component analysis exhibited a strong association between the respiration rate, the carbohydrate content, and the O-alkyl C content, corroborating that carbohydrates are the most important compounds of MSW with regard to the emission potential. Rank correlation (Spearman) also showed strong relationships between the respiration rate and the content of carbohydrates (r=0.75) and of O-alkyl C (r=0.72). Journal of Industrial Microbiology & Biotechnology (2001) 26, 83–89. Received 20 April 2000/ Accepted in revised form 21 July 2000