Effect of varying feedstock–pretreatment chemistry combinations on the formation and accumulation of potentially inhibitory degradation products in biomass hydrolysates

A variety of potentially inhibitory degradation products are produced during pretreatment of lignocellulosic biomass. Qualitative and quantitative interrogation of pretreatment hydrolysates is paramount to identifying potential correlations between pretreatment chemistries and microbial inhibition in downstream bioconversion processes. In the present study, corn stover, poplar, and pine feedstocks were pretreated under eight different chemical conditions, which are representative of leading pretreatment processes. Pretreatment processes included: 0.7% H₂SO₄, 0.07% H₂SO₄, liquid hot water, neutral buffer solution, aqueous ammonia, lime, lime with oxygen pressurization, and wet oxidation. Forty lignocellulosic degradation products resulting from pretreatment were analyzed using high performance liquid chromatography in combination with UV spectroscopy or tandem mass spectrometry detection (HPLC‐PDA‐MS/MS) and ion chromatography (IC). Of these compounds, several have been reported to be inhibitory, including furfural, hydroxymethyl furfural, ferulic acid, 3,4‐dihydroxybenzaldehyde, syringic acid among others. Formation and accumulation of monitored compounds in hydrolysates is demonstrated to be a function of both the feedstock and pretreatment conditions utilized. Biotechnol. Bioeng. 2010;107: 430–440. © 2010 Wiley Periodicals, Inc.     

Using low temperature to balance enzymatic saccharification and furan formation during SPORL pretreatment of Douglas-fir

Comparing analytical results for Sulfite Pretreatment to Overcome the Recalcitrance of Lignocelluloses (SPORL) of Douglas-fir (Pseudotsuga menziesii) at two different temperatures shows that the apparent activation energy of sugar degradation is higher than that of hemicellulose hydrolysis, approximately 161 kJ/mole versus 100 kJ/mole. Thus, one can balance the production of degradation products against hemicellulose hydrolysis and therefore the enzymatic saccharification efficiency of the resultant substrate by changing pretreatment temperature and duration. Specifically, pretreatment at 165 °C for 75 min significantly reduced furan formation compared with the pretreatment at 180 °C for 30 min while maintaining the same pretreatment severity and therefore the same substrate enzymatic digestibility (SED). Obtaining high SED with Douglas-fir is also limited by lignin content. Fortunately, the bisulfite in SPORL provides delignification activity. By combining kinetic models for hemicelluloses hydrolysis, sugar degradation, and delignification, the performance of pretreatment can be optimized with respect to temperature, duration, acid, and bisulfite loading. The kinetic approach taken in this study is effective to design viable low temperature pretreatment processes for effective bioconversion of lignocelluloses.     

工程大肠杆菌合成游离脂肪酸的研究进展

游离脂肪酸作为一种重要的平台化合物,其衍生产品被广泛应用到能源、化学工业中。作为更加可持续、绿色的生产策略,利用工程微生物合成游离脂肪酸是以石油基和动植物为原料生产脂肪酸类产品的重要补充。大肠杆菌作为经典的模式微生物,通过对其进行代谢工程改造,脂肪酸的积累已经从痕量提高到了约9g/L,展示了其作为脂肪酸合成菌株的巨大应用潜力。随着合成生物学技术的涌现,“感应-调控器”、体外重构、β氧化逆循环、异源合成途径的整合等思路的引入极大地加快了工程大肠杆菌脂肪酸合成的进化速率,并赋予大肠杆菌合成多种脂肪酸产品的能力。对近年来通过代谢工程和合成生物学手段改造大肠杆菌合成游离脂肪酸的研究进展进行综述,对其发展前景进行展望。     

Biodegradation of 2-Chlorobenzoic Acid by Enterobacter cloacae: Growth Kinetics and Effect of Growth Conditions

Enterobacter cloacae was originally isolated from soil irrigated with wastewater on the basis of its ability to grow with linear alkylbenzene sulfonate (LAS) as the sole source for carbon and energy. The isolated bacterium was grown in batch cultures using a 2-chlorobenzoic acid (2-CBA)-containing minimal salt medium (MSM). 2-CBA was found to be the sole source for carbon and energy. 2-CBA inhibited the growth rate with a maximum concentration of 10 mM, after which no growth occurred. The Haldane model was used to predict the specific growth rate concentration data. 2-CBA degradation by starved E. cloaca cells was faster than that of nonstarved cells. The maximum growth rates on 2-CBA (2 mM) for starved and nonstarved cells reached only 0.34 and 0.28 h^?1, respectively. Glucose, lactose, sucrose, maltose, succinic acid, and mannitol as additional carbon sources at a fixed concentration (0.2%) caused the degradation rate of 2-CBA to proceed faster at ranges between 1.08- and 1.5-fold higher than that of the control. In contrast, using only fructose and sorbitol as the carbon sources showed catabolic repression of the degradation activity of 2-CBA by E. cloaca cells, although their cell mass was improved. All nitrogen sources supplied caused an increase in cell mass, whereas only lysine, alanine, glutamine, casein, and yeast extract caused a decrease in the degradation rate of 2-CBA, with a range between 12% and 28%. The activity of C120 could be detected in a crude extract of E. cloacae cells, indicating that the chloroaromatic ring fission occurs through the ortho pathways, not through the meta pathways. The data showed that different initial cell (inocula) densities did not affect the induction time for 2-CBA degradation. However, doubling the initial cell densities reduced the time required for reaching the complete degradation. 2-CBA degradation was optimally achieved at a 37°C incubation temperature and a pH of 7.5.     

Thermal decomposition kinetics of wheat straw treated by Phanerochaete chrysosporium

Combining biological pretreatment with thermal processing may offer an alternative strategy for efficient conversion of lignocellulosic biomass into fuels and chemicals. The thermal decomposition kinetics of biologically pretreated wheat straw by Phanerochaete chrysosporium was investigated in this study using thermogravimetry (TG) - deconvoluted thermogravimetry (DTG) techniques and the Friedman method. This study revealed that biological pretreatment reduced the thermal degradation temperature of the biomass significantly. Relying on the thermal behavior of the biologically pretreated wheat straw, we proposed two biomass degradation phases during the biological degradation of wheat straw. The first phase of biodegradation (within 10 days of biological pretreatment) improved the efficiency of pyrolysis by reducing the temperature demand. In the second phase (after 10 days), although the efficiency of pyrolysis displayed the similar trend as the first phase, it showed a significant increase in activation energy demand. This process is greatly influenced by the residual lignin and cellulose ratios in the biomass. These experimental results will be useful in developing a biological pretreatment based thermochemical conversion process for lignocellulosic biomass.     

Microwave, ultrasonic and chemo-mechanical pretreatments for enhancing methane potential of pulp mill wastewater treatment sludge

Microwave (2450 MHz, 1250 W), ultrasonic (20 kHz, 400 W) and chemo-mechanical (MicroSludge® with 900 mg/L NaOH followed by 83,000 kPa) pretreatments were applied to pulp mill waste sludge to enhance methane production and reduce digester sludge retention time. The effects of four variables (microwave temperature in a range of 50-175 °C) and sonication time (15-90 min), sludge type (primary or secondary) and digester temperature (mesophilic and thermophilic) were investigated. Microwave pretreatment proved to be the most effective, increasing specific methane yields of WAS samples by 90% compared to controls after 21 days of mesophilic digestion. Sonication solubilized the sludge samples better, but resulted in soluble non-biodegradable compounds. Based on the laboratory scale data, MicroSludge® was found the least energy intensive pretreatment followed by sonication for 15 min alternative with net energy profits of 1366 and 386 kWh/tonne of total solids (TS), respectively. Pretreatment benefits were smaller for thermophilic digesters.     

连续温度变化对β-1,3-葡聚糖酶酶解酵母β-葡聚糖的影响

为了探索反应温度对产物组分的影响,利用自制连续变化的温度梯度实验装置,研究了22 ℃~60 ℃ (±0.1 ℃) 区间内温度对一内切β-1,3-葡聚糖酶酶解酵母β-葡聚糖的影响,获得了酶解过程多点温度特性数据。分析表明：该酶酶解酵母β-葡聚糖的活化能为84.17 kJ/mol;以产物积累表示的最适酶解温度随时间延长呈指数下降;酶解产物组分受温度的影响,低温较高温获得的寡糖链长,高温区大于46 ℃可以获得以昆布二糖、昆布三糖为主的组分,而低温区小于30 ℃可以获得昆布五糖及更大分子量的产物。研究结果可为寡糖     

THE BIOTRANSFORMATION,BIODEGRADATION, AND BIOREMEDIATION OF ORGANIC COMPOUNDS BY MICROALGAE1

Rapid growth in the biotechnological industry and production has put tremendous pressure on the biological methods that may be used according to the guidelines of green chemistry. However, despite continuing dramatic increases in published research on organic biotransformation by microorganisms, more research exists with microalgae. Our efforts in transforming chemicals such as organic compounds for the production of functionalized products help to lessen the environmental effects of organic synthesis. These biotransformations convert organic contaminants to obtain carbon or energy for growth or as cosubstrates. This review aims to focus on the potential of microalgae in transformation, conversion, remediation, accumulation, degradation, and synthesis of various organic compounds. However, these technologies have the ability to provide the most efficient and environmentally safe approach for inexpensive biotransforming of a variety of organic contaminants, which are most industrial residues. In addition, the recent advances in microalgal bioactivity were discussed.     

Molecular analysis of polyphosphate accumulation in bacteria

The dynamic behavior of inorganic polyphosphate (polyP), its accumulation and disappearance, is the most striking aspect of polyP metabolism in bacteria. Imbalance between polyP synthesis and degradation results in fluctuations of polyP by 100- to 1000-fold. We here review recent results with respect to this polyP metabolism in bacteria. PolyP accumulation in response to amino acid starvation, accompanied by increased levels of stringent factors, has been observed in Escherichia coli. Inhibition by stringent factors of polyphosphatase interrupts the dynamic balance between the synthesis and degradation of polyP, accounting for polyP accumulation. Polyphosphate kinase is required for activation of intracellular protein degradation, which is required for adaptation at the onset of amino acid starvation. The adaptation to amino acid starvation is mediated by the network of stringent response and polyP metabolism. PolyP accumulation independent of stringent response has also been observed. Novobiocin, an inhibitor for DNA gyrase, stimulated accumulation of polyP but not that of stringent factors. However, a temperature-sensitive DNA gyrase mutant did not exhibit polyP accumulation at the non-permissive temperature. Antagonistic relationship of polyP to nucleic acid synthesis, explored by Harold, appears to be more complicated. We discuss relationship of Pi regulation to polyP accumulation in E. coli and Klebsiella aerogenes. A function of polyP as an in vivo phosphagen affecting polyP accumulation is also discussed.     

Results from demineralized bone creep tests suggest that collagen is responsible for the creep behavior of bone

Cortical and trabecular bone have similar creep behaviors that have been described by power-law relationships, with increases in temperature resulting in faster creep damage accumulation according to the usual Arrhenius (damage rate approximately exp (-Temp.-1)) relationship. In an attempt to determine the phase (collagen or hydroxyapatite) responsible for these similar creep behaviors, we investigated the creep behavior of demineralized cortical bone, recognizing that the organic (i.e., demineralized) matrix of both cortical and trabecular bone is composed primarily of type I collagen. We prepared waisted specimens of bovine cortical bone and demineralized them according to an established protocol. Creep tests were conducted on 18 specimens at various normalized stresses sigma/E0 and temperatures using a noninvasive optical technique to measure strain. Denaturation tests were also conducted to investigate the effect of temperature on the structure of demineralized bone. The creep behavior was characterized by the three classical stages of decreasing, constant, and increasing creep rates at all applied normalized stresses and temperatures. Strong (r2 > 0.79) and significant (p < 0.01) power-law relationships were found between the damage accumulation parameters (steady-state creep rate d epsilon/dt and time-to-failure tf) and the applied normalized stress sigma/E0. The creep behavior was also a function of temperature, following an Arrhenius creep relationship with an activation energy Q = 113 kJ/mole, within the range of activation energies for cortical (44 kJ/mole) and trabecular (136 kJ/mole) bone. The denaturation behavior was characterized by axial shrinkage at temperatures greater than approximately 56 degrees C. Lastly an analysis of covariance (ANCOVA) of our demineralized cortical bone regressions with those found in the literature for cortical and trabecular bone indicates than all three tissues creep with the same power-law exponents. These similar creep activation energies and exponents suggest that collagen is the phase responsible for creep in bone.     

Elucidating acetate tolerance in E. coli using a genome-wide approach

Engineering organisms for improved performance using lignocellulose feedstocks is an important step towards a sustainable fuel and chemical industry. Cellulosic feedstocks contain carbon and energy in the form of cellulosic and hemicellulosic sugars that are not metabolized by most industrial microorganisms. Pretreatment processes that hydrolyze these polysaccharides often also result in the accumulation of growth inhibitory compounds, such as acetate and furfural among others. Here, we have applied a recently reported strategy for engineering tolerance towards the goal of increasing Escherichia coli growth in the presence of elevated acetate concentrations (Lynch et al., 2007). We performed growth selections upon an E. coli genome library developed using a moderate selection pressure to identify genomic regions implicated in acetate toxicity and tolerance. These studies identified a range of high-fitness genes that are normally involved in membrane and extracellular processes, are key regulated steps in pathways, and are involved in pathways that yield specific amino acids and nucleotides. Supplementation of the products and metabolically related metabolites of these pathways significantly increased growth rate (a 130% increase in specific growth) at inhibitory acetate concentrations. Our results suggest that acetate tolerance will not involve engineering of a single pathway; rather we observe a range of potential mechanisms for overcoming acetate based inhibition.     

Post-translational non-enzymatic modification of proteins I. Chromatography of marker adducts with special emphasis to glycation reactions

Analytical methods for marker compounds formed during post-translational modifications of proteins are reviewed. Only adducts arising either in vivo or under in vitro conditions simulating the in vivo situations are discussed. All of these compounds stem from either the reaction of free amino groups (i.e., lysine, arginine or N-terminal amino acid). In most cases the reactive counterpart is an aldehydic moiety containing endogenous compound; however, other functional groups containing metabolites are considered as well. The main demand put upon such marker compounds is that they are stable in acid or enzymatic hydrolysis or, alternatively, can be stabilized by simple sample pretreatment (e.g., by reduction). Practically all categories of separation procedures can be applied provided that the chemical characteristics of a particular marker are adequately respected; frequently combination of two different separation procedures based on different principles must be used. Because of the low level of such marker compounds under in vivo conditions, an appropriate sample enrichment step must be involved. Emphasis is put upon the analysis of Amadori products, pentosidine (and pentosidine related compounds), pyrraline, furosine, N-carboxymethylamino acids, amino acid hydantoins and stabilized dicarbonyl intermediates     

Time-dependent mediators of HPA axis activation following live Escherichia coli

The hypothalamus-pituitary-adrenal (HPA) axis is activated during an immune challenge to liberate energy and modulate immune responses via feedback and regulatory mechanisms. Inflammatory cytokines and prostaglandins are known contributors to HPA activation; however, most previous studies only looked at specific time points following LPS administration. Since whole bacteria have different immune stimulatory properties compared with LPS, the aim of the present studies was to determine whether different immune products contribute to HPA activation at different times following live Escherichia coli challenge. Sprague-Dawley rats were injected intraperitoneally with E. coli (2.5 × 10(7) CFU) and a time course of circulating corticosterone, ACTH, inflammatory cytokines, and PGE(2) was developed. Plasma corticosterone peaked 0.5 h after E. coli and steadily returned to baseline by 4 h. Plasma PGE(2) correlated with the early rise in plasma corticosterone, whereas inflammatory cytokines were not detected until 2 h. Pretreatment with indomethacin, a nonselective cyclooxygenase inhibitor, completely blocked the early rise in plasma corticosterone, but not at 2 h, whereas pretreatment with IL-6 antibodies had no effect on the early rise in corticosterone but attenuated corticosterone at 2 h. Interestingly, indomethacin pretreatment did not completely block the early rise in corticosterone following a higher concentration of E. coli (2.5 × 10(8) CFU). Further studies revealed that only animals receiving indomethacin prior to E. coli displayed elevated plasma and liver cytokines at early time points (0.5 and 1 h), suggesting prostaglandins suppress early inflammatory cytokine production. Overall, these data indicate prostaglandins largely mediate the early rise in plasma corticosterone, while inflammatory cytokines contribute to maintaining levels of corticosterone at later time points.     

Kinetics of the diamine oxidase reaction

1. The oxidation of p-dimethylaminomethylbenzylamine was followed spectrophotometrically by measuring the change in E(250) caused by the p-dimethylaminomethylbenzaldehyde produced under a wide variety of experimental conditions. 2. The effect of variations in concentrations of both substrates (amine and oxygen) and all products (aminoaldehyde, hydrogen peroxide and ammonia) on this reaction was studied and the results used to develop a formal mechanism. 3. The nature of the rate-limiting step was elucidated by studying the effects of alterations in ionic strength, dielectric constant and deuterium substitution on the velocity of the forward reaction. 4. Thermodynamic activation energy parameters were obtained at several pH values from the effects of temperature on the reaction.     

Use of recombinant retroviruses to study the regulation of integrated adenovirus early promoters.

Adenovirus E1A gene products are capable of modulating the expression of a variety of integrated genes. To study the mechanisms by which this regulation occurs, recombinant retroviruses have been utilized to establish cell lines containing an integrated copy of either the adenovirus E2 or E3 promoter adjacent to the bacterial guanine phosphoribosyl transferase (GPT) gene. These cell lines have been characterized with respect to both basal and E1A-induced levels of GPT gene expression. Cell lines with low levels of GPT gene expression showed increased expression in the presence of E1A, whereas cell lines with high basal levels of GPT gene expression had decreased GPT RNA levels in the presence of E1A. Further characterization of these cell lines revealed E1A modulation of the accumulation of RNA initiating at a retrovirus promoter adjacent to the E2 or E3 promoter. The use of the GPT gene as a marker of E2 or E3 promoter activity has allowed the isolation of cell lines which have spontaneously increased their levels of GPT RNA. A preliminary characterization of four of these cell lines has indicated that GPT gene expression is increased as a result of cis activation of the E2 promoter.     

GC-MS detection of chiral markers in cocoa beans of different quality and geographic origin

Fermented cocoa beans (Theobroma cacao L., Sterculiaceae) from different countries of origin (Ecuador, Ghana, Trinidad) and cocoa beans roasted under defined conditions (industrial roasting; 150-220 degrees C for 20 min, dry roasting in conventional oven) were analyzed for their contents of certain chiral hydroxy acids, catechins, and amino acids. Cocoa beans are fermented, dried, and industrially transformed by roasting for the production of chocolate, cocoa powders, and other cocoa-related products. Fermentation and roasting conditions influence the contents of chiral compounds such as hydroxy acids, amino acids, and polyphenols, depending on technological procedures as well as some technical parameters. The aim of this work was to check if the content and nature of the named chiral compounds present both in fermented and roasted cocoa beans could be related to the traditional parameters used to classify the variety of seeds and the degree of fermentation. The extent of racemization of amino acids in fermented cocoa beans was low while it slowly increased during roasting, depending on the temperature applied. L-lactic acid was always higher than the D-form while citric acid was generally the most abundant hydroxy acid detected in beans. A correlation was found between polyphenol content and degree of fermentation, while epimerization of (-)-epicatechin to (+)-catechin was observed during roasting. On the whole, results showed that several chiral compounds could be considered as good quality markers for cocoa seeds and cocoa-related products of different quality and geographic origin.     

From time temperature integrator kinetics to time temperature integrator tolerance levels: heat-treated milk

Six milk compounds were studied as potential intrinsic time temperature integrators (TTIs) for the assessment of heat-treated milk. These include the enzymes alkaline phosphatase and lactoperoxidase, the whey protein beta-lactoglobulin and the chemical compounds hydroxymethylfurfural, lactulose and furosine. In previous research the inactivation/denaturation/formation kinetics of these compounds were analyzed under isothermal and nonisothermal conditions and evaluated for variability of the milk composition. The present paper focuses on the implementation of the TTIs. TTIs are validated with respect to microbiological indices and quality attributes, and a quantitative relationship between the denaturation, inactivation or formation of the TTIs and technological processes is established by construction of general time temperature tolerance (TTT) diagrams. In these diagrams temperature time combinations are presented, which lead to the same formation, inactivation or denaturation of TTIs, or result in the same level of microbiological destruction or quality degradation of the product. TTT-diagrams are very informative since they allow visualization of the impact of a thermal process on milk and evaluation of criteria for evaluating milk authenticity (conformity of the product with the terminology applied). Moreover, the optimum combination of temperature and time of heating may be readily deduced from these diagrams.     

Quantitative trait loci for glucosinolate accumulation in Brassica rapa leaves

Glucosinolates and their breakdown products have been recognized for their effects on plant defense, human health, flavor and taste of cruciferous vegetables. Despite this importance, little is known about the regulation of the biosynthesis and degradation in Brassica rapa. Here, the identification of quantitative trait loci (QTL) for glucosinolate accumulation in B. rapa leaves in two novel segregating double haploid (DH) populations is reported: DH38, derived from a cross between yellow sarson R500 and pak choi variety HK Naibaicai; and DH30, from a cross between yellow sarson R500 and Kairyou Hakata, a Japanese vegetable turnip variety. An integrated map of 1068 cM with 10 linkage groups, assigned to the international agreed nomenclature, is developed based on the two individual DH maps with the common parent using amplified fragment length polymorphism (AFLP) and single sequence repeat (SSR) markers. Eight different glucosinolate compounds were detected in parents and F(1)s of the DH populations and found to segregate quantitatively in the DH populations. QTL analysis identified 16 loci controlling aliphatic glucosinolate accumulation, three loci controlling total indolic glucosinolate concentration and three loci regulating aromatic glucosinolate concentrations. Both comparative genomic analyses based on Arabidopsis-Brassica rapa synteny and mapping of candidate orthologous genes in B. rapa allowed the selection of genes involved in the glucosinolate biosynthesis pathway that may account for the identified QTL.     

Actions of a versatile fluorene-degrading bacterial isolate on polycyclic aromatic compounds.

Pseudomonas cepacia F297 grew with fluorene as a sole source of carbon and energy; its growth yield corresponded to an assimilation of about 40% of fluorene carbon. The accumulation of a ring meta-cleavage product during growth and the identification of 1-indanone in growth media and washed-cell suspensions suggest that strain F297 metabolizes fluorene by mechanisms analogous to those of naphthalene degradation. In addition to fluorene, strain F297 utilized for growth a wide variety of polycyclic aromatic compounds (PACs), including naphthalene, 2,3-dimethylnaphthalene, phenanthrene, anthracene, and dibenzothiophene. Fluorene-induced cells of the strain also transformed 2,6-dimethylnaphthalene, biphenyl, dibenzofuran, acenaphthene, and acenaphthylene. The identification of products formed from those substrates (by gas chromatography-mass spectrometry) in washed-cell suspensions indicates that P. cepacia F297 carries out the following reactions: (i) aromatic ring oxidation and cleavage, apparently using the pyruvate released for growth, (ii) methyl group oxidations, (iii) methylenic oxidations, and (iv) S oxidations of aromatic sulfur heterocycles. Strain F297 grew with a creosote-PAC mixture, producing an almost complete removal of all aromatic compounds containing 2 to 3 rings in 14 days, as demonstrated by gas chromatography analysis of the remaining PACs recovered from cultures. The identification of key chemicals confirmed that not only are certain compounds depleted but also the anticipated reaction products are found.