Effect of chemical treatments on the degradability of cotton straw by rumen microorganisms and by fungal cellulase

Three different chemical treatments—sulfur dioxide, ozone, and sodium hydroxide—were applied on cotton straw, and the effect on cell-wall degradability was assessed by using rumen microorganism and Trichoderma reesei cellulase. Sulfur dioxide (applied at 70°C for 72 h) did not change the lignin content of cotton straw but reduced the concentration of hemicellulose by 48%. Ozone exerted a dual effect, both on lignin (a 40% reduction) and hemicellulose (a 54% decrease). The treatment with NaOH did not solublize cell-wall components. The in vitro organic matter digestibility with rumen fluid of cotton straw was increased significantly by ozone and SO₂ treatments, by 120% and 50%, respectively, but not by NaOH. T. reesei cellulase was applied on the chemically pretreated cotton straw at a low level (6 filter paper U/g straw, organic matter), and the release of reducing sugars was determined. The highest level of reducing sugars (30.6 g/100 g organic matter) was obtained with the O₃-cellulase combination, which solubilized 64% of the cellulose and 88% of the hemicellulose. the SO₂- and the NaOH-pretreated cotton straw were hydrolyzed by T. reesei cellulase to the same extent (21 g reducing sugars/100 g organic matter). The rumen fluid digestibility of the enzymatic ally hydrolyzed straw was not increased further over the effect already obtained with the chemical pretreatments. However, the fermentability of the combined treatments was increased markedly. In the O₃-cellulase-treated cotton straw, 83% of the rumen fluid digestible material consisted of highly fermentable components. Although ozone proved to be the most potent pretreatment for enzymic saccharification in this study, the absolute result was modest. The limited effect of the combined O₃-cellulase treatment was probably associated with the pretreatment limitations, but not with the enzyme level. Based on the differential response of the chemically treated cotton straw to attack by rumen microorganisms on the one hand, and by T. reesei cellulase on the other hand, a hypothesis has been suggested as to the location of lignin and hemicellulose in the cellwall unit of cotton straw.     

Stopped-flow system with ozonizer for the estimation of low biochemical oxygen demand in environmental samples

The stopped-flow system with an ozonizer was developed to estimate low biochemical oxygen demand (BOD) in rivers. Rivers contain many biopersistent organic compounds such as humic acid, lignin, and gum arabic. Free radicals generated by self-decomposition of ozone were used as powerful oxidants to split organic compounds. Ozonysis of the samples was carried out by 42.4 g N⁻¹ m⁻³ ozone for 3 min at pH 7.0. Artificial wastewater (AWW) solutions were employed as standard solutions for the calibrations of the BOD sensor. At a BOD of 1 mg l⁻¹, the sensor response after ozonation was 1.6-fold higher than that before ozonation. The response time of the BOD sensor was only 5 min, being independent of the concentrations, and the lower detection limit was 0.5 mg l⁻¹ BOD. The degradations of lignin and tannic acid by ozonation were 54.1 and 42.3%, respectively. In the biosensor responses by ozonation, lignin, gum arabic, and surfactant increased by double or more compared with previous responses. BOD in rivers was estimated using the stopped-flow system. Environmental samples pretreated with ozone gave high responses to the biosensor that were similar to those of the conventional BOD₅ method. Accordingly, a good correlation between the sensor and the conventional BOD₅ was obtained (r = 0.989). The system has to evolve the highly sensitive BOD determination.     

Combined Biological Treatment/Ozonation of Wastewaters for the Mineralisation of Non‐Biodegradable Naphthalene‐1,5‐Disulphonic Acid

This paper deals with the ozonation and biological treatment for achieving the mineralisation of the non‐biodegradable compound naphthalene‐1,5‐disulphonic acid (NADSA; C₁₀H₈O₆S₂), a basic chemical for dyes and building materials. This combination of ozonation and biological treatment is expected to minimise the ozone consumption compared to ozonation alone. To examine the influence of ozone on the biodegradability of NADSA, a lab‐scale plant was run semi‐continuously and batch tests were conducted for comparison. For total ozonation, 1.8 mol O₃/mol DOC have to be used. For optimum biodegradation, only 0.8 mol O₃/mol DOC are needed. The results show that by using a combination of ozonation and biological treatment the ozone consumption can be reduced by more than 50% compared to ozonation alone. Some of the intermediate products are isolated and suggestions for their chemical structures are made. A proposal for the first steps of ozonation is also presented to illustrate a possible pathway of ozonation. All the major identifiable compounds are non‐biodegradable. From these results, it is quite clear that further intermediate products are formed, which are biodegradable.     

TCF bleaching of wheat straw pulp using ozone and xylanase. Part B: kinetic studies

The reaction kinetics of ozone bleaching of wheat straw pulp has been studied for the first time. The results were compared with eucalyptus pulp in order to know that both raw materials have a similar behaviour. Ozone treatments were carried out in a special reactor at low consistency (0.5% o.d.p.). The main variables were consumption of ozone by the pulp and application of a xylanase treatment (X) prior to the oxygen stage (O). The responses measured were kappa number, viscosity and brightness, to give the kinetic expressions for delignification, cellulose degradation and elimination of chromophore groups, along with calculation of selectivity. Cellulose degradation and elimination of lignin and chromophore groups show first-order kinetics in all cases. The kinetics of the enzyme pre-treatment effect shows similar behaviour in both raw materials, although the constants of delignification and elimination of chromophore are higher in straw pulp.     

Effect of ozonolysis pretreatment on enzymatic digestibility of wheat and rye straw

Wheat and rye straws were pretreated with ozone to increase the enzymatic hydrolysis extent of potentially fermentable sugars. Through a 2(5-1) factorial design, this work studies the influence of five operating parameters (moisture content, particle size, ozone concentration, type of biomass and air/ozone flow rate) on ozonization pretreatment of straw in a fixed bed reactor under room conditions. The acid insoluble lignin content of the biomass was reduced in all experiments involving hemicellulose degradation. Near negligible losses of cellulose were observed. Enzymatic hydrolysis yields of up to 88.6% and 57% were obtained compared to 29% and 16% in non-ozonated wheat and rye straw respectively. Moisture content and type of biomass showed the most significant effects on ozonolysis. Additionally, ozonolysis experiments in basic medium with sodium hydroxide evidenced a reduction in solubilization and/or degradation of lignin and reliable cellulose and hemicellulose degradation.     

Variations of photosynthetic activity and growth of freshwater algae according to ozone contact time in ozone treatment

The activity of freshwater algae in drinking water supply at various ozone contact times was investigated to evaluate ozonation processes used for sterilization of algae. O production rates of algae just after ozonation were severely retarded even by short ozone contact. The algal growth rate was also declined with increase of ozone contact time and completely inhibited by ozonation for 60 min. These results implied that the usual treatment time applied in conventional ozonation processes is not sufficient but at least 60 min of ozonation is required for a complete sterilization of freshwater algae in drinking water source.     

Pretreatment of poplar lignocellulose by gamma-ray or ozone for subsequent fungal biodegradation

Summary Poplar lignocelluloses, ¹⁴C-labelled, on all the cell wall components or only the lignin moieties, were either irradiated with -rays from ⁶⁰Co or treated with ozone. The two pretreatments increase the accessibility of cellulose to commercial cellulase and enhance, to the same extent, lignin and polysaccharide biodegradation by Phanerochaete chrysosporium. As far as delignification is concerned ozone treatment appears, however, to be the most efficient through its effects both on lignin solubilization and lignin biodegradation. Ozone treatment and fungal biodegradation, of poplar sawdust increase its in vitro digestibility when performed independently. Moreover, we have shown that when these treatments are sequentially associated, they make the digestibility of sawdust comparable to that of straw, provided that the pH of the culture medium is controlled. These results open possibilities for the use of such transformed raw materials as animal feed.     

Inactivation of Viruses and Bacteria by Ozone, With and Without Sonication

Selected organisms with public health significance were placed in a reaction chamber for treatment by ozonation, by ozonation and sonication, by sonication, or by sonication during oxygenation. Vesicular stomatitis virus, encephalomyocarditis virus, GDVII virus, Staphylococcus aureus, Pseudomonas fluorescens, Salmonella typhimurium, enteropathogenic Escherichia coli, Vibrio cholerae, and Shigella flexneri were inactivated by treatment with ozone. When microorganisms were suspended in phosphate-buffered saline, they were inactivated rapidly by treatment with ozone. However, microorganisms suspended in secondary effluent from a wastewater treatment plant required longer contact times with ozone for complete inactivation. Simultaneous treatments by ozonation and sonication reduced the contact time for complete inactivation of microorganisms in secondary effluent. Treatment by sonication alone or sonication and oxygenation did not inactivate microorganisms. Therefore, the simultaneous treatment of microorganisms in secondary effluent with ozone and sonication resulted in a synergistic effect.     

Negative effects of tropospheric ozone on the feed value of rice straw are mitigated by an ozone tolerance QTL

High surface ozone concentrations are recognized as an emerging threat to food security in Asia. This study aimed at determining the effects of ozone on the nutritive quality of rice straw, a by‐product of rice grain production and a major feed resource for ruminant livestock. Further, the question was addressed whether negative effects of ozone can be mitigated through molecular breeding. Rice plants from three different genotypes were exposed to four different ozone treatments in fumigation chambers from transplanting to maturity. These genotypes were (i) IR64, one of the most wide spread indica varieties in the world, (ii) Nipponbare, a typical japonica variety, and (iii) SL41, an ozone tolerant breeding line that carried chromosomal inserts at the ozone tolerance QTL OzT9 in the genetic background of Nipponbare. The treatments consisted of (i) charcoal filtered air, (ii) simulated ambient ozone concentration, (iii) 2 × ambient ozone concentration, and (iv) 2.5 × ambient ozone concentration. The effects of ozone on the chemical composition of straw were clearly dependent of the ozone level, and were significant even at ambient ozone concentration. Increases in crude ash, lignin and phenolics concentration adversely affected the digestibility as demonstrated in incubation experiments simulating rumen digestion in vitro. Negative ozone effects included reductions in the rate and extent of gas production due to inhibition of microbial fermentation, reduced formation of short chain fatty acids (SCFA), and a decrease in the true organic matter digestibility. The ozone tolerant genotype SL41 was less responsive to ozone than its more susceptible recurrent parent Nipponbare in terms of lignin and phenolics formation, organic matter digestibility and SCFA production. These data demonstrate that the feed quality of rice straw is affected by ozone even at ambient concentration, and that these negative effects are mitigated by the ozone tolerance QTL OzT9.     

Degradation of polysaccharides and lignin in wood ozonation

Ozonation has been considered as a method for the pretreatment of plant biomass to obtain cellulose and monosaccharides. Ozone consumption by aspen wood with various moisture contents has been investigated. We have considered the gradual transformation of the substrate: wood to ozonated wood to cellulose-containing product (CP) to holocelluloze (HC) and to cellulose. Yields of ozonated wood (OW), the (CP), water-soluble ozonation products, HC, and cellulose have been determined. The lignin content in the CP has been estimated. Both HC and cellulose samples have been studied by IR spectroscopy. The degree of polymerization and molecular mass distribution of cellulose obtained from ozonated wood have been determined. It has been shown that wood destruction by ozone is accompanied by degradation of lignin, hemicelluloses, and cellulose.It has been found that physicochemical properties of cellulose obtained from ozonated wood can be regulated by the variation of the initial moisture content in the substrate. Both molecular ozone and radical species, which are generated in the course of ozone reactions with water present in the substrate structure, participate in wood destruction.     

Degradation of lignin by combined chemical and biological treatment

It has been demonstrated that ozone dosages of 0.06 lb and 0.78 lb per pound of lignin can reduce the optical density (in the visible range) of the latter by 82% and 92%, respectively. The reduction in color is accompanied by a shift in the molecular weight distribution of lignin from a broad peak of between 20,000 and 70,000 to lower molecular weight, species including substantial amounts possessing a molecular weight of 1000 or less. The cost of decolorizing a typical kraft paper bleach effluent is estimated to be under 50¢/1000 gal which compares favorably with competitive decolorizing processes. Lignin ozonation results in the production of a series of decolorized products which can serve as the sole source of carbon for a variety of microorganisms. Feasibility studies indicated that at least 40% of the ozonated material can be transferred into microbial biomass (protein) as well as other products of commercial interest such as fumaric acid and penicillin.     

Hydrolysis of wheat straw hemicellulose with trifluoroacetic acid. Fermentation of xylose with Pachysolen tannophilus

Treatment of wheat straw with 1N trifluoroacetic acid (TFA) for 7 h at reflux temperature yielded 23% xylose based upon initial straw weight. This corresponds to about an 80% xylose yield based on the xylan content of the hemicellulose. The cellulose component of wheat straw was largely unaffected, as evidenced by low glucose yields. Decomposition of xylose by prolonged refluxing (23 h) was minimal in 1N TFA compared to 1N HCl. Treatment of wheat straw with refluxing 1N TFA converts about 10% of the lignin initially present in straw into water-soluble lignin fragments. Fermentation of the xylose-rich wheat straw hydrolyzate to ethanol with Pachysolen tannophilus was comparable to the fermentation of reagent grade xylose, indicating that furfural and toxic lignin by-products were not produced by 1N TFA in sufficient amounts to impair cell growth and ethanol production. Cellulase treatment of the wheat straw residue after TFA hydrolysis resulted in a 70-75% conversion of the cellulose into glucose.     

Ozone treatment of process water from a dry-mill ethanol plant

Fuel ethanol production in corn dry milling plants is a rapidly expanding industrial sector. Whole stillage, the residue from the distillation of the fermented corn, is centrifuged and the concentrate, thin stillage, is found to have a chemical oxygen demand (COD) of approximately 75,000 mg/L. This thin stillage is partly recycled, but much of it needs to be evaporated to concentrate the solubles for addition to the animal feed coproduct from corn dry milling. This research is an exploration into lowering COD from thin stillage using ozonation as a simple single-step unit process to facilitate a larger reusable fraction. The ozonation would usually be a pretreatment before additional flocculation or biological treatment. Also, COD removal by ozonation with and without a catalyst has been studied. Three different application rates of ozone O(3,1)=7 mg/min, O(3,2)=21 mg/min, and O(3,3)=33 mg/min were used for 8h into samples of 2L each of three dilutions 20x, 30x and 40x. COD removal of 85% was observed with an ozone dosage of 4000 mg/L into a 40x-diluted sample. This would correspond to about 0.5 mg COD removed per mg ozone dosed. However, at lower dosages and smaller dilutions, more than 1mg COD removal was achieved per mg ozone dosed. Two different catalysts, Fe(II) and Fe(III), were used and the samples were ozonated for 4 h. Five different dosages of each of the two catalysts were used and better COD removal was observed compared to ozonation alone with all 5 dosages. COD removal rate was increased from 45% to 74% with Fe(III) and was increased to 77% with Fe(II). Up to 10mg/L COD was removed per mg O(3) dosed well below the maximum dosage. Both the catalysts resulted in almost the same improved COD removal rates when compared to ozonation alone.     

木质纤维素的定量测定及降解规律的初步研究

为了准确地测定稻草及其发酵物中纤维素、半纤维素、木质素的含量,通过差重法进行定量测定,并以此评价白腐菌株Pleurotus sapidus对稻草秸秆的降解状况,结果表明：利用差重法测定稻草发酵物中纤维素、半纤维索、木质素的百分含量是可行的,并能很好地评价白腐菌对稻草的降解规律,即降解过程中纤维素、半纤维素、本质素在前20d降解的很快,之后降解减缓,在50d内,纤维素被降解34．02％,半纤维素被降解56．29％,木质素被降解61．65％。     

Ozone inactivation of cell-associated viruses

The inactivation of HEp-2 cell-associated poliovirus (Sabin 1) and coxsackievirus A9 was investigated in three experimental systems, using ozone as a disinfectant. The cell-associated viral samples were adjusted to a turbidity of 5 nephelometric turbidity units. The cell-associated poliovirus and coxsackievirus samples demonstrated survival in a continuous-flow ozonation system at applied ozone dosages of 4.06 and 4.68 mg/liter, respectively, for 30 s. Unassociated viral controls were inactivated by the application of 0.081 mg of ozone per liter for 10 s. Ultrasonic treatment of cell-associated enteric viruses did not increase inactivation of the cell-associated viruses. The batch reactor with a declining ozone residual did not effect total inactivation of either cell-associated enteric virus. These cell-associated viruses were completely inactivated after exposure to ozone in a batch reactor using continuous ozonation. Inactivation of cell-associated poliovirus required a 2-min contact period with an applied ozone dosage of 6.82 mg/liter and a residual ozone concentration of 4.70 mg/liter, whereas the coxsackievirus was completely inactivated after a 5-min exposure to an applied ozone dosage of 4.81 mg/liter with an ozone residual of 2.18 mg/liter. These data indicate that viruses associated with cells or cell fragments are protected from inactivation by ozone concentrations that readily inactivate purified virus. The cell-associated viral samples used in this research contained particles that were 10 to 15 microns in size. Use of a filtration system before ozonation would remove these particles, thereby facilitating inactivation of any remaining viruses associated with cellular fragments.     

Ozone inactivation of cell-associated viruses.

The inactivation of HEp-2 cell-associated poliovirus (Sabin 1) and coxsackievirus A9 was investigated in three experimental systems, using ozone as a disinfectant. The cell-associated viral samples were adjusted to a turbidity of 5 nephelometric turbidity units. The cell-associated poliovirus and coxsackievirus samples demonstrated survival in a continuous-flow ozonation system at applied ozone dosages of 4.06 and 4.68 mg/liter, respectively, for 30 s. Unassociated viral controls were inactivated by the application of 0.081 mg of ozone per liter for 10 s. Ultrasonic treatment of cell-associated enteric viruses did not increase inactivation of the cell-associated viruses. The batch reactor with a declining ozone residual did not effect total inactivation of either cell-associated enteric virus. These cell-associated viruses were completely inactivated after exposure to ozone in a batch reactor using continuous ozonation. Inactivation of cell-associated poliovirus required a 2-min contact period with an applied ozone dosage of 6.82 mg/liter and a residual ozone concentration of 4.70 mg/liter, whereas the coxsackievirus was completely inactivated after a 5-min exposure to an applied ozone dosage of 4.81 mg/liter with an ozone residual of 2.18 mg/liter. These data indicate that viruses associated with cells or cell fragments are protected from inactivation by ozone concentrations that readily inactivate purified virus. The cell-associated viral samples used in this research contained particles that were 10 to 15 microns in size. Use of a filtration system before ozonation would remove these particles, thereby facilitating inactivation of any remaining viruses associated with cellular fragments.     

Laccase-Mediator Pretreatment of Wheat Straw Degrades Lignin and Improves Saccharification

Agricultural by-products such as wheat straw are attractive feedstocks for the production of second-generation bioethanol due to their high abundance. However, the presence of lignin in these lignocellulosic materials hinders the enzymatic hydrolysis of cellulose. The purposes of this work are to study the ability of a laccase-mediator system to remove lignin improving saccharification, as a pretreatment of wheat straw, and to analyze the chemical modifications produced in the remaining lignin moiety. Up to 48 % lignin removal from ground wheat straw was attained by pretreatment with Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) as mediator, followed by alkaline peroxide extraction. The lignin removal directly correlated with increases (～60 %) in glucose yields after enzymatic saccharification. The pretreatment using laccase alone (without mediator) removed up to 18 % of lignin from wheat straw. Substantial lignin removal (37 %) was also produced when the enzyme-mediator pretreatment was not combined with the alkaline peroxide extraction. Two-dimensional nuclear magnetic resonance (2D NMR) analysis of the whole pretreated wheat straw material swollen in dimethylsulfoxide-d ₆ revealed modifications of the lignin polymer, including the lower number of aliphatic side chains involved in main β-O-4′ and β-5′ inter-unit linkages per aromatic lignin unit. Simultaneously, the removal of p-hydroxyphenyl, guaiacyl, and syringyl lignin units and of p-coumaric and ferulic acids, as well as a moderate decrease of tricin units, was observed without a substantial change in the wood polysaccharide signals. Especially noteworthy was the formation of Cα-oxidized lignin units during the enzymatic treatment.     

Functional group analysis during ozonation of sunflower oil methyl esters by FT-IR and NMR

Ozonation of neat sunflower oil (SFO) methyl esters was monitored by FT-IR and 1H and 13C NMR spectroscopy. During the early stage of ozonation, ozone absorption was essentially quantitative. This was accompanied by the formation of 1,2,4-trioxolane. IR and NMR spectra of ozonated samples showed that scission of ozonide to give aldehyde were minimal. 1H NMR analysis revealed that the amount of ozonide relative to aldehyde was more than 90% regardless of the extent of ozonation. Complete ozonation was attained after supplying around 0.20 g O3/ml methyl ester after which ozone absorption suddenly dropped to around 25%. At the latter part of ozonation, ozonide and aldehyde reacted with excess ozone to give carboxylic acid. Reaction products were identified according to Criegee mechanism.     

Water disinfection using the novel approach of ozone and a liquid whistle reactor

A novel approach of ozone treatment assisted by a liquid whistle reactor (LWR), which generates hydrodynamic cavitation, has been explored for water disinfection using a simulated effluent containing Escherichia coli (E. coli), one of the dominant markers in faecal coliforms. A suspension having an E. coli concentration of approximately 10⁸ to 10⁹ CFU mL⁻¹ was introduced into the LWR to examine the effect of hydrodynamic cavitation alone and in combination with ozone. Operating conditions of inlet pressure and ozone doses as well as time of ozonation for individual operation along with the combined operation have been varied with the aim of maximizing the extent of disinfection and arriving at an optimum strategy for treatment. It has been observed that nearly 75% disinfection can be achieved in about 3 h of treatment time using an optimized combination of hydrodynamic cavitation and ozonation. This combination has been found to be a cost-effective technique for achieving maximum disinfection compared to the individual operation of hydrodynamic cavitation (lower extent of disinfection) and ozonation (higher costs of treatment usually due to higher cost of ozone generation).     

pH-Sensitive mutagenic activity of ozone-treated 1,2-dimethylhydrazine in the Salmonella/microsome assay

Treatment with ozone inactivates the mutagenicity of many carcinogens in aqueous solution. The colon carcinogen, 1,2-dimethylhydrazine (DMH) has been reported an exception; ozone treatment convenrts dimethylhydrazine from a non-mutagen into a mutagen. In the Salmonella/microsone assay, the mutagenicity of ozone-treated dimethylhydrazine was dependent on pH. The ozonation product was a strong mutagen in acidic but was not mutagenic in basic solution. The mutagenicity of the acidic ozonation product was inactivated by raising the pH of the solution. Unlike untreated dimethylhydrazine, its ozonation product in basic solution was not converted to a mutagen in this ozone-low pH system.