Selection of a yeast strain with optimal utilization of straw hydrolyzates

The dry nonseparated straw hydrolyzates prepared by mild acid hydrolysis of milled straw contains 25--30% of reducing substances, mostly saccharides with prevalence of xylose. A strain utilizing the neutralized nonseparated hydrolyzates without any growth substances added was obtained by selection and long-term adaptation of an array of yeast strains. The strain, identified as Candida tropicalis 2838, exhibited high cell-growth rate and considerable yield of protein-rich biomass.     

Acid hydrolysis of sunflower seed husks for production of single cell protein

Summary Sunflower seed husks were chosen as a typical lignocellulosic waste product of low value. This model substrate was hydrolyzed with sulphuric acid at 120°C. The hydrolysis was carried out in two steps: hydrolysis of the pentosan fraction and subsequent hydrolysis of the cellulose fraction. The pentosan fraction was nearly quantitatively hydrolyzed. For the cellulose hydrolysis the yield was 79% of the theoretical yield. The hydrolyzates were neutralized to pH 5 with solid calcium hydroxide and used for preparation of growth media forCandida yeasts andPaecilomyces variotii. For the pentosan hydrolyzates the yields of yeast biomass were 35–36 g per 100 g available reducing sugars (supplied to the medium). In cellulose hydrolyzates the corresponding yields were 45–48 g withCandida utilis andC. tropicalis and about 30 g withC. pseudotropicalis. P. variotii was noticeably superior to the yeasts. In pentosan hydrolyzates it produced 63 g dry mycelium from 100 g reducing sugars supplied; in cellulose hydrolyzates, 94 g. This suggests that it must be an effective utilizer of a wide range of compounds, for example, organic acids in the medium.     

Comparative hydrolysis and fermentation of sugarcane and agave bagasse

Sugarcane and agave bagasse samples were hydrolyzed with either mineral acids (HCl), commercial glucanases or a combined treatment consisting of alkaline delignification followed by enzymatic hydrolysis. Acid hydrolysis of sugar cane bagasse yielded a higher level of reducing sugars (37.21% for depithed bagasse and 35.37% for pith bagasse), when compared to metzal or metzontete (agave pinecone and leaves, 5.02% and 9.91%, respectively). An optimized enzyme formulation was used to process sugar cane bagasse, which contained Celluclast, Novozyme and Viscozyme L. From alkaline–enzymatic hydrolysis of sugarcane bagasse samples, a reduced level of reducing sugar yield was obtained (11–20%) compared to agave bagasse (12–58%). Selected hydrolyzates were fermented with a non-recombinant strain of Saccharomyces cerevisiae. Maximum alcohol yield by fermentation (32.6%) was obtained from the hydrolyzate of sugarcane depithed bagasse. Hydrolyzed agave waste residues provide an increased glucose decreased xylose product useful for biotechnological conversion.     

Incorporation of cadmium into proteins in a cadmium tolerant fungi

Aspergillus carbonarius and a strain of Penicillium, a cadmium tolerant fungi, are able to metabolize cadmium chloride up to 2% (w/v). Their amino acids analysis on cadmium free and cadmium chloride containing media indicated certain disorders in their metabolic activities. Cystathionine was only detected in both fungi in the presence of cadmium chloride. However, cadmium was incorporated into several types of low and high molecular weight proteins. The amino acids hydrolyzates of a cadmium containing protein are characterized by the presence of high levels of sulfur amino acids; cysteine and methionine. Ethylasparagine was detected in the hydrolyzate of that cadmium containing protein as well.     

Description of <Emphasis Type="Italic">Streptomyces neopeptinius</Emphasis> sp. nov., an actinobacterium with broad spectrum antifungal activities

A streptomycete strain producing broad-spectrum antifungal substances was taxonomically characterized. The strain, designated KNF 2047(T) (= SH-09(T) = KCTC 10586BP(T)), was found to form extensively branching aerial and substrate mycelia, and produce spiny-ornamented spores with loose spiral chains. The whole cell hydrolyzates contained major amount of LL-diaminopimelic acid. The major fatty acids of the phospholipids were saturated and branched fatty acids containing 14~17 carbons, and the major isoprenoid quinones were hexa-and octa-hydrogenated menaquinones with 9 isoprene units. The phylogenetic analysis using the 16S rRNA gene indicated that the strain belongs to the genus Streptomyces but forms an independent phyletic line. These results clearly demonstrate that strain KNF2047(T) forms a new center of taxonomic variation within Streptomyces, for which the name Streptomyces neopeptinius sp. nov. is proposed.     

Pomegranate peels waste hydrolyzate optimization by Response Surface Methodology for Bioethanol production

Unwanted agricultural waste is largely comprised of lignocellulosic substrate which could be transformed into sugars. The production of bioethanol from garbage manifested an agreeable proposal towards waste management as well as energy causation. The goal of this work is to optimize parameters for generation of bioethanol through fermentation by different yeast strains while Saccharomyces cerevisiae used as standard strain. The low cost fermentable sugars from pomegranate peels waste (PPW) were obtained by hydrolysis with HNO₃ (1 to 5%). The optimum levels of hydrolysis time and temperature were elucidated via RSM (CCD) ranging from 30 to 60 min and 50 to 100 °C respectively. The result shows that optimum values (g/L) for reducing sugars was 61.45 ± 0.01 while for total carbohydrates was 236 ± 0.01. These values were found when PPW was hydrolyzed with 3% HNO₃, at 75 °C for one hour. The hydrolyzates obtained from the dilute HNO₃ pretreated PPW yielded a maximum of 0.43 ± 0.04, 0.41 ± 0.03 g ethanol per g of reducing sugars by both Metchnikowia sp. Y31 and M. cibodasensis Y34 at day 7 of ethanologenic experiment. The current study exhibited that by fermentation of dilute HNO₃ hydrolyzates of PPW could develop copious amount of ethanol by optimized conditions.     

Potential of biotechnological conversion of lignocellulose hydrolyzates by Pseudomonas putida KT2440 as a model organism for a bio‐based economy

Lignocellulose‐derived hydrolyzates typically display a high degree of variation depending on applied biomass source material as well as process conditions. Consequently, this typically results in variable composition such as different sugar concentrations as well as degree and the presence of inhibitors formed during hydrolysis. These key obstacles commonly limit its efficient use as a carbon source for biotechnological conversion. The gram‐negative soil bacterium Pseudomonas putida KT2440 is a promising candidate for a future lignocellulose‐based biotechnology process due to its robustness and versatile metabolism. Recently, P. putida KT2440_xylAB which was able to metabolize the hemicellulose (HC) sugars, xylose and arabinose, was developed and characterized. Building on this, the intent of the study was to evaluate different lignocellulose hydrolyzates as platform substrates for P. putida KT2440 as a model organism for a bio‐based economy. Firstly, hydrolyzates of different origins were evaluated as potential carbon sources by cultivation experiments and determination of cell growth and sugar consumption. Secondly, the content of major toxic substances in cellulose and HC hydrolyzates was determined and their inhibitory effect on bacterial growth was characterized. Thirdly, fed‐batch bioreactor cultivations with hydrolyzate as the carbon source were characterized and a diauxic‐like growth behavior with regard to different sugars was revealed. In this context, a feeding strategy to overcome the diauxic‐like growth behavior preventing accumulation of sugars is proposed and presented. Results obtained in this study represent a first step and proof‐of‐concept toward establishing lignocellulose hydrolyzates as platform substrates for a bio‐based economy.     

Isolation, purification and characterization of antioxidant peptidic fractions from a bovine liver sarcoplasmic protein thermolysin hydrolyzate

Sarcoplasmic proteins isolated from bovine livers were hydrolyzed using the enzyme thermolysin at 37 ?C for 2 h. The hydrolyzates were filtered through molecular weight cut off membranes (MWCO) and filtrates were obtained. The water activity (a_w) of unhydrolysed sarcoplasmic protein, full hydrolyzates, 10-kDa and 3-kDa filtrates were below the limit necessary for microbial growth. The antioxidant activities of both filtrates and fractions were assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay, the ferric ion reducing antioxidant power (FRAP) assay and the Fe²⁺ chelating ability assay. RP-HPLC was used for purification of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates. The peptidic content of the full hydrolyzates, the 10-kDa and the 3-kDa filtrates were assessed using the Dumas method and peptide contents of each fraction were characterized using electrospray quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry with the resultant spectrum analysed using the software programmes Protein Lynx Global Server 2.4. and TurboSEQUEST. Similarities between the amino acid composition of characterized peptides from each fraction and previously reported antioxidant peptides were found. This study demonstrates that meat by-product such as liver can be utilised as raw material for the generation of bioactive peptides with demonstrated antioxidant activities in vitro using the enzyme thermolysin. It is significant as it presents a potential opportunity for meat processors to use their waste streams for the generation of bioactive peptides for potential functional food use.     

On-line control of fed-batch fermentation of dilute-acid hydrolyzates

Dilute-acid hydrolyzates from lignocellulose are, to a varying degree, inhibitory to yeast. In the present work, dilute-acid hydrolyzates from spruce, birch, and forest residue, as well as synthetic model media, were fermented by Saccharomyces cerevisiae in fed-batch cultures. A control strategy based on on-line measurement of carbon dioxide evolution (CER) was used to control the substrate feed rate in a lab scale bioreactor. The control strategy was based solely on the ratio between the relative increase in CER and the relative increase in feed rate. Severely inhibiting hydrolyzates could be fermented without detoxification and the time required for fermentation of moderately inhibiting hydrolyzates was also reduced. The feed rate approached a limiting value for inhibiting media, with a corresponding pseudo steady-state value for CER. However, a slow decrease of CER with time was found for media containing high amounts of 5-hydroxymethyl furfural (HMF). The success of the control strategy is explained by the conversion of furfural and HMF by the yeast during fed-batch operation. The hydrolyzates contained between 1.4 and 5 g/l of furfural and between 2.4 and 6.5 g/l of HMF. A high conversion of furfural was obtained (between 65-95%) at the end of the feeding phase, but the conversion of HMF was considerably lower (between 12-40%).     

Separation of motile populations of spermatozoa prior to freezing is beneficial for subsequent fertilization in vitro: a study with various mouse strains

Success with in vitro fertilization (IVF) using inbred strains of mice varies considerably and appears to be related to the proportion of motile spermatozoa present in epididymal sperm samples of different strains. In this study, motile spermatozoa were separated from the original samples using a column of Sephadex G25. IVF rates were compared between separated and nonseparated samples of epididymal spermatozoa before and after cryopreservation. Oocytes and spermatozoa were obtained from FVB, DBA/2, C57BL/6J, and BALB/c inbred mice; and from F1 (C57BL/6J;ts DBA/2) hybrid mice, and isogenic gametes were used for IVF. These strains of mice were chosen because of their common use in transgenesis and mutagenesis studies. Dulbecco PBS was used for sperm separation on Sephadex, 18% raffinose, and 3% skim milk for cryopreservation; T6 medium for IVF; and mKSOM(AA) for embryo culture. There was a marked improvement in the rate of fertilization using fresh spermatozoa after motile spermatozoa were separated in C57BL/6J and BALB/c strains (92% vs. 58%, 79% vs. 44%) but no differences were found in fertilization rates between separated and nonseparated spermatozoa in F1, FVB, and DBA/2 strains (99% vs. 83%, 95% vs. 93%, 86% vs. 87%, respectively). After cryopreservation, higher rates of fertilization were obtained with separated motile samples in all strains; the greatest improvements were obtained with spermatozoa from C57BL/6J and BALB/c strains (40% vs. 16% and 51% vs. 14% for separated and nonseparated spermatozoa, respectively). No differences were found between the proportions of 14.5-day fetuses developing from embryos derived from separated and nonseparated spermatozoa with or without cryopreservation (33% to 46%). In conclusion, the fertility of frozen-thawed mouse epididymal spermatozoa improves significantly when highly motile populations of spermatozoa are separated for freezing.     

Analysis of cepA encoding an efflux pump-like protein in Corynebacterium glutamicum

A gene encoding a homolog of purine efflux proteins of Escherichia coli and Bacillus subtilis was identified in the genome of Corynebacterium glutamicum and designated as cepA. The gene encoded a putative protein product, containing 12 transmembrane helixes, which is a typical feature of integral membrane transport proteins. To elucidate the function of the gene, we constructed a cepA deletion mutant (ΔcepA) and a cepA-overexpressing strain and analyzed their physiological characteristics. The cepA gene could be deleted with no critical effect on cell growth. However, the cell yield of a ΔcepA strain was decreased by 10% as compared to that of a strain carrying a cepA-overexpression plasmid (P₁₈₀-cepA). Further analysis identified increased resistance of the P₁₈₀-cepA strain to the purine analogues 6-mercaptopurine and 6-mercaptoguanine, but not to 2-aminopurine and purine nucleoside analogues. Moreover, this strain showed increased resistance to the antibiotics nalidixic acid and ampicillin. Collectively, these data suggest that cepA is a novel multidrug resistance gene and probably functions in the efflux of toxic substances from the inside of cells to the environment, thus allowing cells to reach a higher cell yield.     

Kinetic study of detoxification of dilute-acid hydrolyzates by Ca(OH)2

Detoxification of dilute-acid hydrolyzates by addition of Ca(OH)(2) (overliming) and cultivation of the detoxified hydrolyzates by Saccharomyces cerevisiae were examined. The examined overliming involves increasing the pH of the hydrolyzates to 9, 10, 11 or 12, keeping up to 90 min at different temperatures of 30, 45 and 60 degrees C, followed by readjustment of the pH to 5. Increasing the pH, time and/or temperature resulted in more effective degradation of furans and resulted in better fermentability for both of the tested hydrolyzates, but higher loss of the sugars was observed as well. Overliming of glucose and furfural solution at pH 12 showed a rapid decrease in concentration of these chemicals followed by a slow degradation process. Therefore, a kinetic model was proposed for the detoxification, where the sugars or furans make transient complexes with calcium ions and this complex will then be converted to the degradation product. The ANOVA analysis of the model resulted in an average R(2) of 0.99 for the model fitted to all the experimental data points.     

A method for the rapid,automated filtration of protein hydrolyzates

A method for the automated filtration of protein hydrolyzates prior to amino acid analysis is described. Minor modification of a Technicon Sampler II enables it to function simultaneously as a sampler and a filtrate collector. Samples are drawn from cups in the sampler tray and are forced through a Teflon filter (pore size, 0.2 μm) in a Millipore Swinnex filter holder by a variable-speed Technicon proportioning pump. The filtrates are collected in cups in the sampler tray opposite those containing unfiltered hydrolyzates. Using this technique, 12 hydrolyzates can be filtered in 25 min compared to the approximately 2 h of technician time required for their manual filtration. Aliquots from each of 48 samples representing different proteins and hydrolysis conditions are filtered manually and by the automated technique. Analysis of variance of the resulting recoveries of each amino acid indicate little likelihood of effects due to filtration method.     

Preparation of crystalline starch nanoparticles using cold acid hydrolysis and ultrasonication

Waxy maize starch in an aqueous sulfuric acid solution (3.16 M, 14.7% solids) was hydrolyzed for 2–6 days, either isothermally at 40 °C or 4 °C, or at cycled temperatures of 4 and 40 °C (1 day each). The starch hydrolyzates were recovered as precipitates after centrifuging the dispersion (10,000 rpm, 10 min). The yield of starch hydrolyzates depended on the hydrolysis temperature and time, which varied from 6.8% to 78%. The starch hydrolyzed at 40 °C or 4/40 °C exhibited increased crystallinity determined by X-ray diffraction analysis, but melted in broader temperature range (from 60 °C to 110 °C). However, the starch hydrolyzed at 4 °C displayed the crystallinity and melting endotherm similar to those of native starch. The starch hydrolyzates recovered by centrifugation were re-dispersed in water (15% solids), and the dispersion was treated by an ultrasonic treatment (60% amplitude, 3 min). The ultrasonication effectively fragmented the starch hydrolyzates to nanoparticles. The hydrolyzates obtained after 6 days of hydrolysis were more resistant to the ultrasonication than those after 2 or 4 days, regardless of hydrolysis temperatures. The starch nanoparticles could be prepared with high yield (78%) and crystallinity by 4 °C hydrolysis for 6 days followed by ultrasonication. Scanning electron microscopy revealed that the starch nanoparticles had globular shapes with diameters ranging from 50 to 90 nm.     

Debittering of protein hydrolyzates

Enzymatic hydrolysis of proteins frequently results in bitter taste, which is due to the formation of low molecular weight peptides composed of mainly hydrophobic amino acids. Methods for debittering of protein hydrolyzates include selective separation such as treatment with activated carbon, extraction with alcohol, isoelectric precipitation, chromatography on silica gel, hydrophobic interaction chromatography, and masking of bitter taste. Bio-based methods include further hydrolysis of bitter peptides with enzymes such as aminopeptidase, alkaline/neutral protease and carboxypeptidase, condensation reactions of bitter peptides using protease, and use of Lactobacillus as a debittering starter adjunct. The causes for the production of bitter peptides in various food protein hydrolyzates and the development of methods for the prevention, reduction, and elimination of bitterness as well as masking of bitter taste in enzymatic protein hydrolyzates are presented.     

Identification of ccdA in Paracoccus pantotrophus GB17: disruption of ccdA causes complete deficiency in c-type cytochromes

A transposon Tn5-mob insertional mutant of Paracoccus pantotrophus GB17, strain TP43, was unable to oxidize thiosulfate aerobically or to reduce nitrite anaerobically, and the cellular yields were generally decreased by 11 to 20%. Strain TP43 was unable to form functional c-type cytochromes, as determined by difference spectroscopy and heme staining. However, formation of apocytochromes and their transport to the periplasm were not affected, as seen with SoxD, a c-type cytochrome associated with the periplasmic sulfite dehydrogenase homologue. The Tn5-mob-containing DNA region of strain TP43 was cloned into pSUP205 to produce pE18TP43. With the aid of pE18TP43 the corresponding wild-type gene region of 15 kb was isolated from a heterogenote recombinant to produce pEF15. Sequence analysis of 2.8 kb of the relevant region uncovered three open reading frames, designated ORFA, ccdA, and ORFB, with the latter being oriented divergently. ORFA and ccdA were constitutively cotranscribed as determined by primer extension analysis. In strain TP43 Tn5-mob was inserted into ccdA. The deduced ORFA product showed no similarity to any protein in databases. However, the ccdA gene product exhibited similarities to proteins assigned to different functions in bacteria, such as cytochrome c biogenesis. For these proteins at least six transmembrane helices are predicted with the potential to form a channel with two conserved cysteines. This structural identity suggests that these proteins transfer reducing equivalents from the cytoplasm to the periplasm and that the cysteines bring about this transfer to enable the various specific functions via specific redox mediators such as thioredoxins. CcdA of P. pantotrophus is 42% identical to a protein predicted by ORF2, and its location within the sox gene cluster coding for lithotrophic sulfur oxidation suggested a different function.     

Rapid assay for gamma-carboxyglutamic acid in urine and bone by precolumn derivatization and reversed-phase liquid chromatography

A reversed-phase high-performance liquid chromatographic assay for the analysis of gamma-carboxyglutamic acid (Gla) in urine and bone protein hydrolyzates is described. The method employs precolumn derivatization with o-phthalaldehyde and mercaptoethanol. Gla was quantified by reference to an internal standard (beta-carboxyaspartic acid). The "within-run" coefficient of variation of the assay for Gla in urine was between 2.1 and 3.4%, and that for bone protein hydrolyzates was 3.2%. The "between-run" coefficient of variation ranged from 4.1 to 5.5%. There was good agreement between the measurement of urinary Gla by high-performance liquid chromatography and amino acid analyzer. Free Gla could not be detected in serum.     

Bacillus cereus CC-1的亚碲酸盐还原特性及产物表征

【背景】含Te(IV)的工业废水对于生物体具有潜在的毒性作用,可将Te(IV)还原为Te⁰的微生物过程具有重要的研究价值。【目的】探索亚硒酸盐还原菌Bacillus cereus CC-1对Te(IV)的还原能力、还原酶位点以及还原产物的特性。【方法】利用前期筛选的亚硒酸盐还原菌Bacillus cereus CC-1还原Te(IV),根据48h内还原率大小确定最适Te(IV)浓度及pH;考察不同阴阳离子对Te(IV)还原率的影响与Te(IV)还原酶位点;利用表征分析确定还原产物的组成、结晶性与形貌。【结果】菌株CC-1能够将Te(IV)还原,Te(IV)初始浓度为0.5 mmol/L,体系pH为7.0时还原率最高。体系中外加阴阳离子对Te(IV)的还原有一定影响,其中磷酸根、硫酸根、醋酸根、钼酸盐对Te(IV)的去除无明显影响;低浓度的硝酸根抑制Te(IV)的去除,随着硝酸根浓度增加,其对Te(IV)的去除的抑制作用减弱;铅离子和铋离子对Te(IV)的还原有抑制作用;铜离子能够提高Te(IV)的去除率。在胞外、细胞膜组分以及细胞内均检测到Te(IV)还原酶的活性...     

Nutritional stress proteins in Candida albicans

Starving cells of Candida albicans synthesize at least seven proteins that represent nutritional-stress proteins (NSP). Such NSPs are formed by both germination-competent and germination-deficient strains of C. albicans. Heat-shock proteins (HSP) are not formed by starving cells. Germination-competent cells synthesize specific sets of proteins when incubated in a starvation medium that contains the germ-tube-inducing substances N-acetyl-D-glucosamine or L-proline. Both sets of induced proteins were also synthesized by a germination-deficient strain of C. albicans.     

Influence of the metabolism pathway on lactic acid production from hemicellulosic trimming vine shoots hydrolyzates using Lactobacillus pentosus

Hemicellulosic hydrolyzates from trimming wastes of vine shoots were proposed as a carbon source for lactic acid production by Lactobacillus pentosus CECT-4023T (ATCC-8041). These hydrolyzates are composed mainly of glucose (12.0 g/L), xylose (17.5 g/L) and arabinose (4.3 g/L). Acetic acid, the main subproduct, started to be produced after all of the glucose was completely depleted, showing that the acetic acid coproduction came only from the xylose and arabinose consumption. In the absence of glucose, the L. pentosus pathway shifts from homo to heterofermentative. Thus, L. pentosus can be considered a facultative heterofermentative organism, degrading hexoses (glucose) via the Embden-Meyerhoff-Parnas pathway and pentoses (xylose and arabinose) via the phosphoketolase pathway. Hydrolyzates were vacuum evaporated to increase the initial sugars concentration up to 35.4 g/L of glucose, 52.3 g/L of xylose, and 13.0 g/L of arabinose. Under these conditions the lactic acid concentration reached 46.0 g/L (Q(P) = 0.933 g/L.h, Y(P/S) = 0.78 g/g; Y(P/S) theoretical = 91.7%) and a clear product inhibition was observed. Additional experiments with synthetic sugars, in the absence of inhibitory compounds, indicate that this inhibition must be attributed to the metabolic pathway but not to the inhibitory compounds present in the fermentation broth.