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The pulp and paper industry is a major source of lignocellulose-containing streams. The components of lignocellulose material are lignin, hemicellulose, and cellulose that may be hydrolyzed into their smaller components and used as feedstocks for valorization efforts. Much of this material is contained in underutilized streams and waste products, such as black liquor, pulp and paper sludge, and wastewater. Bacterial fermentation strategies have suitable potential to upgrade lignocellulosic biomass contained in these streams to value-added chemicals. Bacterial conversion allows for a sustainable and economically feasible approach to valorizing these streams, which can bolster and expand applications of the pulp and paper industry. This review discusses the composition of pulp and paper streams, bacterial isolates from process streams that can be used for lignocellulose biotransformations, and technological approaches for improving valorization efforts. KEY POINTS: • Reviews the conversion of pulp and paper industry waste by bacterial isolates. • Metabolic pathways for the breakdown of lignocellulose components. • Methods for isolating bacteria, determining value-added products, and increasing product yields.

14 páginas, 7 figuras, Pitch control is an important aspect in pulp and paper manufacture, and the first example where microbial biotechnology provided successful solutions in this industrial sector. Triglycerides cause deposits in softwood mechanical pulping, and both microbial and enzymatic products have been commercialized to be applied on wood and pulp, respectively. The former are based on colorless strains of sapstain fungi. The latter are improved lipases, including thermostable variants from directed evolution. These enzymes are among the additives of choice in pulping of high-resin-content softwoods. However, lipases are not useful when pitch originates from other lipids, such as steroids and terpenes, and the sapstain inocula are also only partially effective. In the search for stronger biocatalysts to degrade recalcitrant lipids, the potential of white-rot fungi and their enzymes has been demonstrated. When inocula of these fungi are used, wood treatment must be controlled to avoid cellulose degradation. However, the efficiency and selectivity of the laccase-mediator system permits its integration as an additional bleaching stage. A double benefit can be obtained from these treatments since pitch is controlled at the same time that residual lignin is removed facilitating the implementation of totally chlorine free pulp bleaching., This work was supported by the Spanish projects AGL2008-00709, BIO2005-03569 and BIO2008-01533, and the BIORENEW project of the European Union (contract NMP2-CT-2006-026456).

1Instituto de Recursos Naturales y Agrobiología, CSIC, PO Box 1052, E-41080, Seville, Spain; and 2Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain, Lipophilic extractives in wood and other lignocellulosic materials exert a negative impact in pulp and paper manufacturing causing the so-called pitch problems. In this work, the appropriateness of an enzymatic treatment using the laccase-mediator system for pitch biocontrol is evaluated. With this purpose, three pulp types representative for different raw materials and pulping processes - eucalypt kraft pulping, spruce thermomechanical pulping and flax soda-anthraquinone pulping - were treated with a high-redox potential laccase from the basidiomycete Pycnoporus cinnabarinus in the presence of 1-hydroxybenzotriazole as a redox mediator. The gas chromatography and gas chromatography/mass spectrometry analyses of the lipophilic extractives from the enzymatically-treated pulps revealed that the laccase-mediator treatment completely (or greatly) removed most of the pitch-causing lipophilic compounds present in the different pulps including: i) free and conjugated sitosterol in eucalypt paper pulp; ii) resin acids, sterol esters and triglycerides in spruce pulp; and iii) sterols and fatty alcohols in the flax pulp. Different amounts of free and conjugated 7-oxosterols were found as intermediate products in the oxidation of pulp sterols. Therefore, the laccase-mediator treatment is reported as an efficient method for removing pitch-causing lipophilic compounds from paper pulps obtained from hardwood, softwood and nonwoody plants., This study has been funded by the Spanish projects AGL2002-393 and BIO2002-1166, the EU projects QLK5-99-1357 and QLK3-99-590, and by ENCE-CSIC contracts.

Next to xylanases, laccases from fungi and alkali-tolerant bacteria are the most important biocatalysts that can be employed for eco-friendly biobleaching of hard and soft wood pulps in the paper industry. Laccases offer a potential alternative to conventional, environmental-polluting chlorine and chlorine-based bleaching and has no reductive effect on the final yield of pulp as compared to hemicellulases (xylanases and mannanases). In the last decade, reports on biobleaching with laccases are based on laboratory observations only. There are several critical challenges before this enzyme can be implemented for pulp bleaching at the industrial scale. This review discusses significant factors like redox potential, laccase mediator system (LMS)—synthetic or natural, pH, temperature, stability of enzyme, unwanted grafting reactions of laccase, and cost-intensive production at large scale which constitute a great hitch for the successful implementation of laccases at industrial level.

Cellulose (in the form of printed paper) has always been the prime medium for displaying information in our society and is far better than the various existing display technologies. This is because of its high reflectivity, contrast, low cost and flexibility. There is a major initiative to push for a dynamic display technology that emulates paper (popularly known as "electronic paper"). We have successfully demonstrated the proof of the concept of developing a dynamic display on cellulose. To the best of our knowledge, this is the first significant effort to achieve an electronic display using bacterial cellulose. First, bacterial cellulose is synthesized in a culture of Acetobacter xylinum in standard glucose-rich medium. The bacterial cellulose membrane thus formed (not pulp) is dimensionally stable, has a paper-like appearance and has a unique microfibrillar nanostructure. The technique then involves first making the cellulose an electrically conducting (or semi-conducting) sheet by depositing ions around the microfibrils to provide conducting pathways and then immobilizing electrochromic dyes within the microstructure. The whole system is then cased between transparent electrodes, and upon application of switching potentials (2-5 V) a reversible color change can be demonstrated down to a standard pixel-sized area (ca. 100 microm2). Using a standard back-plane or in-plane drive circuit, a high-resolution dynamic display device using cellulose as substrate can be constructed. The major advantages of such a device are its high paper-like reflectivity, flexibility, contrast and biodegradability. The device has the potential to be extended to various applications, such as e-book tablets, e-newspapers, dynamic wall papers, rewritable maps and learning tools.

International audience; Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l–1. Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (K m) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using laccases expressed in P. cinnabarinus or A. niger with 1-hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation).

Enzyme-aided bleaching of softwood and hardwood kraft pulps by glycosyl hydrolase family-10 and -11 xylanases and a family-26 mannanase was investigated. The ability to release reducing sugar from pulp xylan and to enhance bleachability is not a characteristic shared by all xylanases. Of the six enzymes tested, two xylanases belonging to family 11 were most effective at increasing bleachability and improving final paper brightness. None of the enzymes had a deleterious effect on pulp fibre integrity. The efficiency of individual xylanases as bleach enhancers was not dependent on the source microorganism, and could not be predicted solely on the basis of the quantity or nature of products released from pulp xylan. Cooperative interactions between xylanase/xylanase and xylanase/mannanase combinations, during the pretreatment of softwood and hardwood pulps, were investigated. Synergistic effects on reducing-sugar release and kappa number reduction were elicited by a combination of two family-10 xylanases. Pretreatment of kraft pulp with mannanase A from Pseudomonas fluorescens subsp. cellulosa and any one of a number of xylanases resulted in increased release of reducing sugar and a larger reduction in kappa number than obtained with the xylanases alone, confirming the beneficial effects of family-26 mannanases on enzyme-aided bleaching of paper pulp.

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase A (XylA) from Neocallimastix patriciarum in combination with mannanase and alpha-galactosidase. Mannanase A (ManA) from Pseudomonas fluorescens subsp. cellulosa and ManA from Clostridium thermocellum, both family 26 glycosyl hydrolases, are structurally diverse and exhibit different pH and temperature optima. Although neither mannanase was effective in pretreating softwood pulp alone, both enzymes were able to enhance the production of reducing sugar and the reduction of single-stage bleached kappa number when used with the xylanase. Sequential incubations with XylA and P. fluorescens ManA produced the largest final kappa number reduction in comparison to control pretreated pulp. The release of galactose from softwood pulp by alpha-galactosidase A (AgaA) from P. fluorescens was enhanced by the presence of ManA from the same microorganism, and a single pretreatment with these enzymes, in combination with XylA. gave the most effective kappa number reduction using a single incubation. Results indicated that mixtures of hemicellulase activities can be chosen to enhance pulp bleachability.

The world economy is currently moving towards more sustainable approaches. Lignocellulosic biomass has been widely used as a substitute for fossil sources since it is considered a low-cost bio-renewable resource due to its abundance and continuous production. Compost habitats presenting high content of lignocellulosic biomass are considered a promising source of robust lignocellulose-degrading enzymes. Recently, several novel biocatalysts from different environments have been identified using metagenomic techniques. A key point of the metagenomics studies is the extraction and purification of nucleic acids. Nevertheless, the isolation of high molecular weight DNA from soil-like samples, such as compost, with the required quality for metagenomic approaches remains technically challenging, mainly due to the complex composition of the samples and the presence of contaminants like humic substances. In this work, a rapid and cost-effective protocol for metagenomic DNA extraction from compost samples composed of lignocellulosic residues and containing high content of humic substances was developed. The metagenomic DNA was considered as representative of the global environment and presented high quality (>\thinspace99\\% of humic acids effectively removed) and sufficient quantity (10.5--13.8 \textmug g1 of compost) for downstream applications, namely functional metagenomic studies. The protocol takes about 4 h of bench work, and it can be performed using standard molecular biology equipment and reagents available in the laboratory., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the Project LIG-NOZYMES—Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-01–0145-FEDER-029773)., info:eu-repo/semantics/publishedVersion

Biocatalysts provide a major advantage to bio-based economy over chemical catalysts by catalyzing various useful transformations in an environment friendly manner along with other major benefits of selectivity, specificity, and low energy consumption. Since last decade, cellulase is the 3rd highest used enzyme in industry in various processes. Xylanase is also one amongst the widely used enzymes, and many industrial applications require synergistic action of both of these enzymes. These applications predominantly include bioethanol production, deinking of waste paper, animal feed processing, food processing, paper and pulp production, removal of fine fibers from textile material (biostoning), and pharmaceuticals. These enzymes are produced by microorganisms (fungi and bacteria), and hence, the microorganisms producing both cellulases and xylanases are in high demand by these industries. This review focuses on the synergistic applications of cellulase and xylanase enzymes across various industrial sectors. It also discusses the potential applications and the need of the microbial systems (fungi and bacteria) secreting both of these enzymes and the future prospects of their development into an integral part of various industrial processes.

The world economy depends heavily on crude oil. With a conventional oil recovery process, only one-third of crude oil is extracted. Various technologies have been developed to maximize the recovery of oil resources from natural reservoirs. Polymer technology has been used in many oil fields around the world. The biopolymer pullulan, produced by some Aureobasidium species, has been used in many industrial applications, but no research has been conducted regarding its use in the microbial enhancement of oil recovery (MEOR). Here, we investigate the potential of pullulan produced by newly isolated species Aureobasidium mangrovei SARA-138H for enhancement of oil recovery. Our results indicate that under optimized conditions, that is, sucrose as the carbon source in the medium, a pH of 9, incubation at 25 °C, and 250 rpm agitation, the fungus was able to produce 10 g/L of pullulan. The maximum viscosity achieved under these conditions was 318 cP after 15 days of incubation. Pullulan solution (10 g/L) showed the ability to recover 36.7% of heavy crude oil after 34.2% of secondary oil recovery. However, diluted pullulan in brine at the ratio (1:1) resulted in the recovery of 20.23% of oil from the residual oil in the core after 22.6% of secondary oil recovery. A 20-day injectivity test revealed that pullulan passed smoothly through the core, causing no blockage. It was concluded that pullulan from A. mangrovei SARA-138H was able to increase oil recovery to a degree comparable to that achieved with many polymers used in oil fields around the world. KEY POINTS: • First report of biopolymer "pullulan" from A. mangrovie. • Optimum conditions for pullulan production were obtained. • Pullulan recovered 36.7% of heavy oil from residual oil in place, with good injectivity.

Pectate lyase treatment can be an alternative strategy of the chemical processing, which causes severe environmental pollution, and has been broadly studied and applied for diverse industrial applications including textile industry, beverage industry, pulp processing, pectic wastewater pretreatment, and oil extraction. This review gave a brief description of the origins, enzymatic characterizations, structure, and applications of pectate lyases (Pels). Most of the reported pectate lyases are originated from microorganisms with a small number of them from plants and animals. Due to the diverse environments that these microorganisms exist, Pels present diversified features, especially for the range of optimal pH and temperature. The diversified biochemical properties of Pels define their applications in different industries, and the applications of alkaline Pels on cotton bioscouring and ramie degumming in textile industry were focused in this review. This review also discussed the perspectives of the development and applications of Pels. KEY POINTS: • The first review on pectate lyase focusing on biotechnological applications. • Origins, features, structures, applications of pectate lyases reviewed. • Applications of alkaline Pels in textile industry demonstrated. • Perspectives on future development and applications of Pels discussed.

Aerobic granular sludge (AGS) is a microbial biofilm self-aggregation, which is effective for nutrient and pollutant removal, through the development of dense microbial layers bound together with extracellular polymeric substances (EPSs). However, long start-up times and granule disintegration are still challenges ahead. An array of external additives, including ion chelating agents, sludge-based enhancers, and magnetic influence have been tested to overcome these barriers. The application of such additives may promote enhanced EPS production, neutralization of charges on the bacterial surface, acts as a core-induced agent, or as a bridge to connect EPSs and cell surfaces. Although additives may improve the granule formation without reducing treatment efficiencies, there are still environmental concerns due to the fate and toxicity of discharged excess sludge. This mini-review identifies an array of external additives and their mechanisms to improve granulation properties, and proposes discussion about the technical and economic viability of these additives. KEY POINTS: • Additives reduce granulation time and repair granule disintegration. • Biopolymer-based additives fulfill technical and environmental requirements. • Sludge-based additives are cheap and in line with the resource recovery concept. • The need for environmental-friendly additives for aerobic granular sludge process. • External additives affect granular biomass size distribution.

With today's environmental concerns and the diminishing supply of the world's petroleum-based chemicals and materials, much focus has been directed toward alternative sources. Woody biomass presents a promising option due to its sheer abundance, renewability, and biodegradability. Lignin, a highly irregular polyphenolic compound, is one of the major chemical constituents of woody biomass and is the second most abundant biopolymer on Earth, surpassed only by cellulose. The pulp and paper and cellulosic ethanol industries produce lignin on the scale of millions of tons each year as a by-product. Traditionally, lignin has been viewed as a waste material and burned as an inefficient fuel. However, in recent decades, research has focused on more economical ways to convert lignin into value-added commodities, such as biofuels, biomaterials, and biochemicals, thus developing and strengthening the concept of fully integrated biorefineries. Owing to the phenolic structure of lignin, it is possible to enzymatically graft molecules onto its surface using laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) to create exciting novel biomaterials. These environmentally friendly enzymes use oxygen as their only co-substrate and produce water as their sole by-product, and have thus found great industrial application. This mini-review highlights recent advances in the field of laccase-facilitated functionalization of lignin as well as promising future directions for lignin-based polymers.

Renewable energy can boost the growing population's need and rapid budgetary development. To reduce fossil fuel consumption is the initial purpose of renewable and sustainable energy, producing valuable bio-based products. The fermenters, using for pretreatment of swine manure, and involvement of swine carcasses are reported to enhance organic loading rate followed by improved biogas yield on household digesters. The compositions such as animal residues, pathogenic microbes, pharmaceutical residues and nutrient compositions including undigested feed are still confused. Therefore, it is mandatory to optimize and stabilize anaerobic practice and digestate filtration purification for consequential fertilizer consumption. The effective bio-methane recovery from energy-rich compounds is challenging due to slow degradation procedures. The pretreatment procedure could enhance lipid depolymerization and improve anaerobic fermentation. This article deeply focuses on biodegradation of swine manure. The components of this manure were evaluated and established several approaches to improve biogas production. Furthermore, recycling of co-digestates was discussed in detail as fertilizer consumption including hygienic aspects of manure and pretreatment strategies of biomass residues. KEY POINTS: • Co-digestion of manure and carcasses enhance bio-methane production. • Removel of ammonia from biogas digester may improve bio-methane gas. • A strong antimicrobial influence has been reported on biogas production.

Industrially produced bleached recycled pulp (R) comprising essentially hardwood fibres was subjected to enzymatic treatment with endo-xylanase from Thermomyces lanuginosus with or without ultra-high hydrostatic pressure (UHP) pre-treatment at 300–600 MPa for 10 min. The kinetics and the extent of enzymatic hydrolysis after UHP pre-treatment under different conditions have been evaluated by released reducing sugars and the analysis of neutral sugars in pulps, respectively. The changes in surface chemical composition of pulps were assessed by UV-vis diffuse reflectance spectroscopy. UHP-pre-treated R under optimal conditions (400 MPa), with or without posterior enzymatic treatment, was used for the production of handsheets and evaluation of its mechanical properties. It was suggested that enzymatic modification improves significantly the papermaking properties of recycled pulp. These improvements were related with selective removal of xylan bound to impurities and to aggregated cellulose fibrils on the fibre surface, thus favouring the ensuing swelling and inter-fibre bonding in paper. UHP pre-treatment and posterior enzymatic treatment revealed a synergetic effect on the mechanical properties of recycled pulp. This fact was assigned to enhanced accessibility of fibres towards xylanase and by forced hydration and favourable rearrangement of cellulosic fibrils in fibres after UHP pre-treatment. The increase of basic strength properties after UHP-promoted xylanase treatment was up to 30 % being the most pronounced for the tensile strength and the burst resistance.

In this paper, the use of secondary-treated wastewater as the culture medium for the production of Muriellopsis sp. microalgal biomass is analyzed. Using this wastewater, a maximum biomass productivity of 0.5 g l(-1) day(-1) was measured, it being only 38 % lower than that achieved using the standard culture medium. Due to the low nitrogen content of secondary-treated wastewater, cultures produced in a medium containing a high percentage of it become nitrate-limited, thus the quantum yield reduces by up to 0.38 g E(-1)--this compares to 0.67 g E(-1) when using a standard culture medium. On the other hand, nitrate limitation enhances the accumulation of lipids and carbohydrates, with values measured at 33 and 66 % dry weight, respectively. It was also demonstrated that secondary-treated wastewater does not have any toxic effect on the growth of Muriellopsis sp. in spite of nitrogen being in the form of ammonium rather than in nitrate. Moreover, the secondary-treated wastewater was depurated when used to produce Muriellopsis sp., with the outlet biological oxygen demand and chemical oxygen demand being lower than at the inlet; the nitrate and phosphate concentrations were zero. Therefore, Muriellopsis sp. production using secondary-treated wastewater allows a reduction in the process cost by decreasing freshwater and fertilizer use, as well as by depurating the water, thus greatly enhancing process sustainability.

The development of biodegradable plastic mulch films for use in agriculture has been ongoing for decades. These films consist of mixtures of polymers with various additives. As a result, their physical and chemical properties differ from those of the pure polymers often used for in vitro enzymatic and microbial degradation studies, raising questions about the biodegradation capability of mulch films. Currently, standards exist for the biodegradation of plastics in composting conditions but not in soil. Biodegradation in soil or compost depends on a complex synergy of biological and abiotic degradative processes. This review discusses the physicochemical and structural properties of biodegradable plastic mulches, examines their potential for on-site decomposition in light of site-to-site variance due to environmental and biological conditions, and considers the potential for long-term effects on agroecosystem sustainability and functionality.

Sugar beet pulp is a natural carbon source composed mainly of pectin and cellulose, which is utilized and degraded by the ascomycete Penicillium purpurogenum. The fungus also grows on and degrades acetylated xylan which lacks cellulose and pectin. Both carbon sources have been used in our laboratory to grow the fungus and to purify different enzymes secreted to the medium. The enzymes involved in the complex process of degradation of these carbon sources by the fungus have been explored previously under non-denaturing conditions; multienzyme complexes were separated and some subunits identified by Western blots and mass spectrometry. In this work, proteomic profiles show that the secretome is composed of numerous proteins varying in pI and molecular weight. Some enzymes are common to both growth conditions, while others are specific for each carbon source. The results show that the carbon sources utilized exert strong regulatory control over the proteins secreted. This is the first secretome study from a lignocellulolytic Penicillium.

Continuous production of acetone, n-butanol, and ethanol (ABE) was carried out using immobilized cells of Clostridium acetobutylicum DSM 792 using glucose and sugar mixture as a substrate. Among various lignocellulosic materials screened as a support matrix, coconut fibers and wood pulp fibers were found to be promising in batch experiments. With a motive of promoting wood-based bio-refinery concept, wood pulp was used as a cell holding material. Glucose and sugar mixture (glucose, mannose, galactose, arabinose, and xylose) comparable to lignocellulose hydrolysate was used as a substrate for continuous production of ABE. We report the best solvent productivity among wild-type strains using column reactor. The maximum total solvent concentration of 14.32 g L(-1) was obtained at a dilution rate of 0.22 h(-1) with glucose as a substrate compared to 12.64 g L(-1) at 0.5 h(-1) dilution rate with sugar mixture. The maximum solvent productivity (13.66 g L(-1) h(-1)) was obtained at a dilution rate of 1.9 h(-1) with glucose as a substrate whereas solvent productivity (12.14 g L(-1) h(-1)) was obtained at a dilution rate of 1.5 h(-1) with sugar mixture. The immobilized column reactor with wood pulp can become an efficient technology to be integrated with existing pulp mills to convert them into wood-based bio-refineries.

A recently developed color removal method uses a ligninolytic fungus,Phanerochaete chrysosporium, and a rotating biological contactor to decolorize pulp and paper bleach effluents. Bench-scale studies were undertaken with the objective of developing an aeration reactor to remove color. Culture conditions and biomass characteristics of the process were established. Experiments determined that the aeration process had a maximum color removal rate of 1300 color units per liter per day.

In this work, the effect of Trametes pubescens laccase (TpL) used in combination with a low-molecular-weight ultra-filtered lignin (UFL) to improve mechanical properties of kraft liner pulp and chemi-thermo-mechanical pulp was studied. UFL was isolated by ultra-filtration from the kraft cooking black liquor obtained from softwood pulping. This by-product from the pulp industry contains an oligomeric lignin with almost twice the amount of free phenolic moieties than residual kraft pulp lignin. The reactivity of TpL on UFL and kraft pulp was studied by nuclear magnetic resonance spectroscopy and size exclusion chromatography. Laccase was shown to polymerise UFL and residual kraft pulp lignin in the fibres, seen by the increase in their average molecular weight and in the case of UFL as a decrease in the amount of phenolic hydroxyls. The laccase initiated cross-linking of lignin, mediated by UFL, which gives rise to more than a twofold increase in wet strength of kraft liner pulp handsheets without loosing other critical mechanical properties. Hence, this could be an interesting path to decrease mechano-sorptive creep that has been reported to lessen in extent as wet strength is given to papers. The laccase/2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) mediator system showed a greater increase in wet tensile strength of the resulting pulp sheets than the laccase/UFL system. However, other mechanical properties such as dry tensile strength, compression strength and Scott Bond internal strength were negatively affected by the laccase/ABTS system.

A cellulase-free xylanase production by Thermomyces lanuginosus SSBP using bagasse pulp was examined under submerged (SmC) and solid-state cultivation (SSC). Higher level of xylanase activity (19,320 +/- 37 U g(-1) dried carbon source) was obtained in SSC cultures than in SmC (1,772 +/- 15 U g(-1) dried carbon source) after 120 h with 10% inoculum. The biobleaching efficacy of crude xylanase was tested on bagasse pulp, and the maximum brightness of 46.1 +/- 0.06% was observed with 50 U of crude xylanase per gram of pulp, which was 3.8 points higher than the brightness of untreated samples. Reducing sugars (26 +/- 0.1 mg g(-1)) and UV-absorbing lignin-derived compounds in the pulp filtrates were observed as maximum in 50 U of crude xylanase-treated samples. T. lanuginosus SSBP has potential applications due to its high productivity of xylanase and its efficiency in pulp bleaching.

In recent years, it has become clear that the rapidly acidifying chemical oxygen demand (RACOD) content of the waste water and the surface tension of the reactor liquid contribute to the phenomenon of granular growth in upflow anaerobic sludge-blanket reactors (UASB). By adding 20% of directly soluble RACOD, in the form of a sucrose/starch mixture, on top of the original COD load and by adjusting the reactor liquid surface tension below 50 mN m−1 with linear alkylbenzenesulphonate, granular growth and sludge-bed stability could be enhanced significantly within 40 days. Carrot pulp, a waste product having a high short-chain fatty acid precursor potential, was applied as an alternative fibrous RACOD source. Best results were obtained when adding the carrot pulp freshly to the laboratory-scale UASB reactor in an in-recycle liquefying chamber. This concept of adding carrot pulp waste product as a granular growth supplement by means of an in-recycle liquefying chamber therefore merits testing in practice.

The mutant Pol6 of Penicillium occitanis is an interesting strain for producing cellulases and hemicellulases. The nitrogen source and substrate that regulate cellulase production were evaluated in shake-flask and fermentor (batch and fed-batch) culture. The nature of the nitrogen source and the C/N ratio markedly affected cellulase production by P. occitanis. When nitrate was used in Mandels and Weber's basal growth medium with a C/N ratio below 20.2, it resulted in more cellulase production than from urea or ammonium sulphate. Crude substrates such as wheat bran and wheat flour residues, used in combination with a local cellulose esparto grass paper pulp as an alternative nitrogen source and cellulose substrates, also gave high cellulase yields. Greatest cellulase yields and productivity were obtained by fed-batch cultivation [23 filter-paper activity units (FPU)/ml and 168 FPUI−1h−1].

Bacillus stearothermophilus T-6 produces an extracellular thermostable xylanase that can bleach paper pulp optimally at 60°C and pH 9.0. We developed an efficient method for purifying the enzyme from the cell broth by using successive steps of batch adsorption on the cellulosic cation exchanger SE-52. The optimal pH values for adsorption and elution are 5.5 and 9 respectively. The conductivity of the cell broth should remain below 7 mS/cm2 and the suitable temperature range for adsorption is 15–60°C. The adsorption parameters are: maximum capacity, 118 mg enzyme/g adsorbent; dissociation constant, 0.6 mg/ml; partition coefficient 0.988. On the basis of these results, a large-scale (12 000 l) purification process was carried out resulting in a 55% final yield and over 95% pure enzyme.

Enzymatic treatment of pine and birch kraft pulps with a xylanase preparation from a thermophilic anaerobic bacterium Dictyoglomus sp. strain B1 was studied in order to improve pulp bleachability. Maximal solubilization of pulp xylan was obtained at 90° C and pH 6.0–7.0. The enzyme was also active in the alkaline pH range; at pH 9.0 xylan hydrolysis was decreased by only 18% from the maximum at pH 7.0. The positive effect of xylanase pretreatment at 80° C and pH 6.0 or 8.0 on bleachability of pine kraft pulp was demonstrated. The brightness was increased by two ISO units in one-stage peroxide delignification, which corresponds well to values obtained with other enzymes at lower temperatures and pH values. Thus, the Dictyoglomus xylanase is well suited for pulp treatments at elevated temperatures in neutral and alkaline conditions.

Palm-oil mill fiber (POMF) is a fibrous, natural hard material discharged in enormous amounts from palm-oil mills in tropical plantations; therefore, research to find microorganisms that decompose POMF was conducted. As the result of screening, a new thermophilic fungus, Chaetomium sp. nov. MS-017, exhibiting rapid growth on POMF was isolated from rotted wood. Based on partial characterization of the decomposition of POMF, it was shown that MS-017 preferentially assimilates polysaccharides, especially hemicelluloses such as xylan. A preliminary composting study indicated that MS-017 produced 855 g of decomposed product from 1,000 g of intact POMF in 12 days under optimized solid-culture conditions. The decomposition rate of POMF was 23% (w/w), and the cell yield calculated from consumed POMF was as high as 36% (w/w). These results indicate that MS-017 has a very high potential to decompose POMF and that it is suitable for economical production of compost to recycle by-product biomass from oil-palm plantations.

High-molecular-weight lignin was methylated with diazomethane. The lignin (i.e., phenolic lignin) and methylated lignin (i.e., non-phenolic lignin) were mixed with fully bleached softwood pulp. Degradation of the lignin preparations by the white rot fungus Pycnoporus cinnabarinus was studied. After a 3-month incubation with the fungus, over 40% of the non-phenolic lignin and about 70% the phenolic lignin were degraded. The presence of phenolic hydroxyl groups in lignin greatly enhanced the degradation rate of lignin. This study reveals that P. cinnabarinus, an exclusively laccase-producing fungus, is capable of oxidatively degrading both phenolic and non-phenolic lignins. The ability of the fungus to degrade non-phenolic lignin suggests that a laccase/mediator system is involved in the complete degradation of lignin. After the fungal degradation of lignins, the content of carboxylic acids substantially increased for both phenolic and non-phenolic lignins.

Ethanol-precipitated substances after fermentation of various agro-industrial wastes by Aureobasidium pullulans were examined for their pullulan content. Grape skin pulp extract, starch waste, olive oil waste effluents and molasses served as substrates for the fermentation. A glucose-based defined medium was used for comparison purposes. Samples were analysed by an enzyme-coupled assay method and by high-performance anion-exchange chromatography with pulsed amperometric detection after enzymic hydrolysis with pullulanase. Fermentation of grape skin pulp extract gave 22.3 g l−1 ethanol precipitate, which was relatively pure pullulan (97.4% w/w) as assessed by the coupled-enzyme assay. Hydrolysed starch gave only 12.9 g l−1 ethanol precipitate, which increased to 30.8 g l−1 when the medium was supplemented with NH4NO3 and K2HPO4; this again was relatively pure pullulan (88.6% w/w). Molasses and olive oil wastes produced heterogeneous ethanol-precipitated substances containing small amounts of pullulan, even when supplemented with nitrogen and phosphate. Overall, grape skin pulp should be considered as the best substrate for pullulan production. Starch waste requires several hydrolyis steps to provide a usable carbon source, which reduces its economic attraction as an industrial process.

Triglycerides, a major class of wood extractives, contribute to the colloidal pitch that initiates pitch deposits. Because industrial or pilot-scale treatments with lipolytic enzymes to reduce triglyceride concentrations in pulp have not been successful in North America, we investigated such treatments at a laboratory scale. Different batches of industrial softwood chemithermomechanical pulps (CTMP) were treated with a range of concentrations of two commercial lipases: Resinase A 2X (Novo Nordisk AG) and Lipidase 10 000 (American Laboratories Inc.). A pilot-scale thermomechanical pulp (TMP) made from the same wood as the CTMP, but without the sodium hydrosulfite used in the CTMP, was also treated with the lipases. While triglycerides decreased in all the pulp treatments, the extent of their hydrolysis varied according to the ratio of triglyceride to the fatty/resin acid fraction. As this ratio can vary significantly in softwood TMP and CTMP, the success of industrial treatments of softwood mechanical pulps by commercial lipases may be related to variations in this ratio. Supporting this, adding linoleic acid to an extractives-free pulp that was spiked with olive oil reduced lipase activity by up to 55%.

A number of hydroxamic acids have been synthesized and investigated as laccase-mediators for pulp bleaching. As compared with N-hydroxyacetanilide (NHA), one of the most effective laccase-mediators reported so far, N-(4-cyanophenyl)acetohydroxamic acid (NCPA), resulted in the highest brightness and lowest kappa number of hardwood kraft pulp of all the laccase-mediators studied. The bleaching efficacy of a laccase/7-cyano-4-hydroxy-2H-1,4-benzoxazin-3-one system was also comparable with that of a laccase/NHA system. A laccase/NCPA system was further studied for the bleaching of unbleached softwood kraft pulp. The effects of pulp consistency, laccase dosage, NCPA dosage, incubation time, and oxygen pressure on the bleaching efficacy of a laccase/NCPA system were studied.

The fungal solubilization of cell wall components of sugar-beet pulp, during solid-state fermentation of Thermoascus aurantiacus, is reported here. The extracellular fungal enzyme activities related to the substrate degradation were also studied. In 120 h, more than 60% of the main sugar-beet pulp polysaccharides, i.e. pectins, arabinose- and glucose-containing polysaccharides, were rapidly brought into solution by the fungus. The slow accumulation of monosaccharides compared to the fast degradation of the polysaccharides suggested that most of the released sugars were consumed by the fungus. The analysis of the enzymes present in the water extracts of the solid-state cultures proved that the fungus was able to synthesize a complete enzymatic system required for the hydrolysis of the main sugar-beet pulp polysaccharides. The highest enzyme activities measured were β-glucosidase and α-L-arabinofuranosidase.

The production of cellulase and xylanase was investigated with a newly isolated strain of Trichoderma viride BT 2169. The medium composition was optimized on a shake-flask scale using the Graeco-Latin square technique. The temperature and time for optimal growth and production of the enzymes in shake cultures were optimized using a central composite design. The temperature optima for maximal production of filter paper cellulase (FPase), xylanase and β-gluosidase were 32.8°, 34.7° and 31.1° C, respectively, and the optimum times for production of these enzymes were found to be 144, 158 and 170 h, respectively. The optimized culture medium and conditions (33° C) gave 0.55 unit of FPase, 188.1 units of xylanase and 3.37 units of β-glucosidase per milliliter of culture filtrate at 144 h of shake culture. Among different carbon sources tested, the maximum enzyme activities were produced with sulphite pulp and all three enzymes were produced irrespective of the carbon sources used. Batch fermentation in a laboratory fermentor using 2% sulphite pulp allowed the production of 0.61 unit of FPase, 145.0 units of xylanase and 2.72 units of β-glucosidase. In a fed-batch fermentation on 6% final Avicel concentration FPase and β-glucosidase were 3.0 and 2.4 times higher respectively than those in batch fermentation on 2% Avicel. The pH and temperature optima as well as pH and temperature stabilities of T. viride enzymes were found to be comparable to T. reesei and some other fungal enzymes.

A total of 272 strains of filamentous fungi were isolated from soil, leaves of coffee plants and coffee cherries collected in coffee-growing areas of Mexico on three semi-synthetic culture media containing coffee extract, coffee extract with sucrose and coffee pulp extract. The isolated strains were purified by conventional techniques and identified by microscopic examination. Strains were selected on the basis of their caffeine-degrading ability in well-defined liquid medium containing caffeine. Most of the isolated microorganisms belong to Aspergillus, Penicillium, Trichoderma, Fusarium, and Humicola genera. Five strains belonging to Aspergillus species and two strains belonging to Penicillium species had the ability to degrade almost 100% of the caffeine in liquid medium. A comparative study on the evaluation of natural microflora present in coffee pulp and coffee husk revealed the presence of a wide variety of microorganisms. The percentage distribution of fungi, bacteria and yeast was almost similar in all the samples, except in coffee husk where the fungal population was slightly higher than in the other two samples. The yeast population was predominant when the coffee pulp was lyophilized immediately after pulping. However, there was a wide diversity in the microbial population with respect to selective media containing functional nutritional groups like cellulose, starch and pectin.

Degradation products from the addition of extracellular enzymes from Thermomonospora fusca BD25 to ball-milled wheat straw, oat spelt xylan and solubilised kraft pulps were characterised by HPLC and TLC. Overall, a high percentage hydrolysis of oat spelt xylan (28.9%) occurred after 26 h incubation. However, the rates of hydrolysis of ball-milled wheat straw and kraft pulp were approximately 4-6-fold less than xylan hydrolysis, although the total percentage hydrolysis of available substrate was similar (22.2% and 25.9% respectively). Incubation of kraft pulp and ball-milled wheat straw by crude extracellular enzymes of T. fusca BD25 resulted in the detection of aromatic compounds at concentrations of 0.6 microg ml(-1) and 8.7 microg ml(-1), respectively. Hydrolysis of oat spelt xylan by T. fusca BD25 extracellular enzymes yielded a mixture of xylose, xylotriose and putative substituted-xylotriose, while the products of ball-milled wheat straw hydrolysis were xylose, glucose and a small oligomer present in the digest. The results highlight the ability of culture supernatant from T. fusca to release both simple sugars and aromatic compounds from lignocellulosic substrates and suggest a role for this organism in the biobleaching of pulp.

Two compost piles were prepared, using two ventilation systems: forced ventilation and ventilation through mechanical turning. The material to compost was a mixture of orange waste, olive pomace, and grass clippings (2:1:1 v/v). During the composting period (375 days), samples were periodically taken from both piles, and the enumeration of fungi, actinomycetes, and heterotrophic bacteria was carried out. All studied microorganisms were incubated at 25 and 55 °C after inoculation in appropriate growth media. Fungi were dominant in the early stages of both composting processes; heterotrophic bacteria proliferated mainly during the thermophilic stage, and actinomycetes were more abundant in the final stage of the composting process. Our results showed that the physical and chemical parameters: temperature, pH, moisture, and aeration influenced the variation of the microbial population along the composting process. This study demonstrated that composting of these types of wastes, despite the prolonged mesophilic stage, provided an expected microbial variation.

The production of ethanol from carob pods by Saccharomyces cerevisiae in solid-state fermentation was investigated. The maximal ethanol concentration (160±3 g/kg dry pods), ethanol productivity (6.7 ± 0.2 g/kg per hour), ethanol yield (40 ± 1.8%), biomass concentration (7.5 ± 0.4 x 108 cells/g carob pulp) and fermentation efficiency (80 ± 2%) were obtained at an inoculum amount of 3%, a particle size of 0.5 mm, a moisture level of 70%, a pH of 4.5 and a temperature of 30°C. Under the same fermentation conditions both sterilized and non-sterilized carob pods pulp gave the same maximum ethanol concentration.

Clostridium beijerinckii DSM 6423 was studied using different continuous production methods to give maximum and stable production of isopropanol and n-butanol. In a single-stage continuous culture, when wood pulp was added as a cell holding material, we could increase the solvent productivity from 0.47 to 5.52 g L−1 h−1 with the yield of 54% from glucose. The overall solvent concentration of 7.51 g L−1 (39.4% isopropanol and 60.6% n-butanol) with the maximum solvent productivity of 0.84 g L−1 h−1 was obtained with two-stage continuous culture. We were able to run the process for more than 48 overall retention times without losing the ability to produce solvents.

In order to develop a method for producing fuel ethanol from cassava pulp using cell surface engineering (arming) technology, an arming yeast co-displaying α-amylase (α-AM), glucoamylase, endoglucanase, cellobiohydrase, and β-glucosidase on the surface of the yeast cells was constructed. The novel yeast strain, possessing the activities of all enzymes, was able to produce ethanol directly from soluble starch, barley β-glucan, and acid-treated Avicel. Cassava is a major crop in Southeast Asia and used mainly for starch production. In the starch manufacturing process, large amounts of solid wastes, called cassava pulp, are produced. The major components of cassava pulp are starch (approximately 60%) and cellulose fiber (approximately 30%). We attempted simultaneous saccharification and ethanol fermentation of cassava pulp with this arming yeast. During fermentation, ethanol concentration increased as the starch and cellulose fiber substrates contained in the cassava pulp decreased. The results clearly showed that the arming yeast was able to produce ethanol directly from cassava pulp without addition of any hydrolytic enzymes.

Chlorogenate hydrolase (EC 3.1.1.42, CHase) was highly induced in mycelia of Aspergillus sojae AKU 3312 grown in Czapek medium containing either instant coffee powder or coffee pulp as inducer. No CHase formation was observed in the mycelia when cultivated without the inducer. CHase was purified readily from CHase-induced mycelia to high homogeneity, and the purified CHase revealed the molecular weight of 180,000 consisting of two identical subunits of 88 kDa. Equimolar quinate (QA) and caffeate (CA) were confirmed on hydrolysis of chlorogenate (CGA). The purified CHase was only useful for a laboratory scale hydrolysis of CGA. For practical QA and CA production using scaled up hydrolysis of vegetable extracts of natural CGA resources, the enzyme activity of purified CHase decreased and denatured irreversibly. Preparation of coffee pulp koji and its application to QA and CA production were proposed instead of purified CHase. When coffee pulp koji was heated at 60 degrees C for 30 min, CHase survived without any appreciable loss of enzyme activity while vegetative mycelial growth and spore germination were terminated. The heated coffee pulp koji thus prepared was effective itself as stable immobilized catalyst of CHase for QA and CA production from vegetable CGA resources such as coffee powders, coffee pulp, and others.

Natural carbon sources, such as those present in cane sugar molasses and grape juice, promote the synthesis of astaxanthin in different Phaffia rhodozyma yeasts. One of these, coconut milk, has a very rich nutrient composition. The aim of this work was to investigate the utility of coconut milk as sole source of energy for astaxanthin pigment production by P. rhodozyma strains. Currently, coconut pulp is widely used in industrial processes in Mexico for the production of shampoos, candies, food, etc. However, coconut milk is a waste product. We show that coconut milk enhances astaxanthin production. The fermentation yielded 850 microg/g yeast with the NRRL-10921 wild-type strain and 1850 microg/g yeast with the mutated R1 strain. Production was better than reported results employing other natural carbon sources.

Treating kraft pulps with the crude xylanase from Streptomyces roseiscleroticus followed by alkali extraction reduces the kappa number in a linear manner with enzyme doses up to about 3 IU/gm of oven-dry pulp. The enzyme complex consists of four isoenzymes designated Xyl1, Xyl2, Xyl3 and Xyl4. Each can release chromophores when used alone and each can facilitate alkali extraction to reduce the kappa number, but their relative abilities are different. Of the four isozymes, Xyl4 releases the least color and 237-nm-absorbing material whereas Xyl3 releases the most. Xyl4 best enhances the ability of alkali to reduce the kappa number. The UV absorption spectrum of the material released by alkali extraction differs significantly from the spectral characteristics of that released during enzyme treatment. The alkali-solubilized material has a maximum absorptivity at 265 nm and relatively little absorptivity at 237 nm. The material released during enzyme treatment absorbs strongly at 205 and 237 nm. UV/VIS spectroscopy of the enzyme- or alkali-released material does not show a characteristics lignin peak at 280 nm, nor does it reveal any notable peaks in the visible region. Analysis of the material released by enzyme treatment revealed more than 40 product peaks after fractionation by reversed-phase HPLC. We observed many products with strong UV absorption. These were relatively hydrophilic. Fewer products absorbed in the visible region. These were more hydrophobic. All four isoenzymes exhibit endo-action patterns; none forms xylose from oat-spelt xylan. The action patterns fell into two groups: endo-1 enzymes (Xyl1 and Xyl3) formed xylotriose (X3) and other lower oligosaccharides as the predominant products; endo-2 enzymes (Xyl2 and Xyl4) formed roughly equimolar amounts of X3, xylotetraose (X4), and xylopentaose (X5), and tended to leave larger amounts of undigested higher oligosaccharides.

The biological upgrading of wheat straw with Streptomyces cyaneus was examined through the analysis of chemical and structural changes of the transformed substrate during solid-state fermentation. Analysis of enzymes produced during the growth of S. cyaneus showed that phenol oxidase was the predominant enzyme. The reduction in Klason lignin content (16.4%) in the transformed substrate indicated the ability of this strain to delignify lignocellulose residues and suggests a role for phenol oxidase in the bacterial delignification process. Microscopic examination of the transformed substrate showed that the initial attack occurred at the less lignified cell walls (phloem and parenchyma), while xylem and sclerenchyma were slowly degraded. The pattern of degradation of sclerenchymatic tissues by S. cyaneus showed delamination between primary and secondary walls and between S1 and S2 due to partial removal of lignin. In the later stages of the decay a disorganization of the secondary walls was detected on account of fibrillation of this layer. A comparison of the properties of the pulp from wheat straw transformed by S. cyaneus with untreated wheat straw showed that pretreatment improved the characteristics that determine the quality of pulp. This was indicated by an increase in pulp brightness and by a decrease in the kappa number. These changes occurred without significantly affecting the viscosity, a measure of the quality of the cellulose fibres. These results support the potential application of this organism or its oxidative enzymes in biopulping.

Potato pulp, one of the agricultural waste products obtained in high quantities during starch production, contains starch, cellulose, hemicelluloses, pectin, proteins, free amino acids and salts. It exhibits physical and physicochemical properties of a typical colloid. It is mainly used, in a dried and pelleted form, as cattle feed. Its autochthonic microbial flora (bacteria, fungi) was identified and studied with a view towards the degradative potential of the microorganisms and ways of conserving the pulp for subsequent technical applications; 33 isolates (28 bacteria, 4 fungi, 1 yeast), belonging to 15 genera were characterized. Biological conservation was possible at very low oxygen pressure, brought about by the autochthonic anaerobic microorganisms causing acidification. Chemical conservation was achieved with sorbic aicd. By treatment with hot water vapour under pressure (autoclaving), followed by a pressure release procedure, intact cells in the pulp (both potato cells and microorganisms, not spores) were destroyed, and their contents and wall fragments were set free. This process resulted in low drying costs and was a prerequisite for the production of a powder that can be used as glue or as animal feed.

Optimization of batch pyrite bioleaching with Sulfolobus acidocaldarius was performed using statistical modelling and experimental design. First a screening design was made followed by response surface modelling. The dominating factors identified were pH, pulp density and particle size. The highest batch leaching rate after optimization was 270 mg iron·l−1·h−1 for 6% (w/v) pulp density, pH = 1.5 and particle size

The influence of the growth of three higher fungi on the composition of wheat straw was investigated. Pleurotus pulmonarius, P. sajor-caju and Lentinus edodes grew very well on lignocellulosic substrates, breaking down a considerable amount of lignin. The initial lignin concentration of straw was halved after 12 weeks of fungal growth, doubling the enzymic digestibility. Together with lignin, the higher fungi consumed half of the amount of hemicellulose (i.e. 15%), leaving cellulose fairly intact, which should remain as an energy source for ruminants.

Brevibacterium sp. strain R312 has an endocellular β-glucosidase, a nitrile hydratase and an amidase that can break down some cyanoglucosides. Nonsterile cassava pulp suspensions were fermented using this strain and 70%–80% reduction of nitrile compounds, in particular cyanoglucosides and α-hydroxynitriles, was observed. This type of nitrile-hydratase-active microorganism could be a solution for the detoxification of cassava. Experiments conducted with the yeast Candida molischiana and C. wickkerhamii showed no improvement in detoxification.

Culture conditions affecting lignin degradation of an unbleached hardwood kraft pulp by Phanerochaete chrysosporium have been examined. Optimum pH and temperature for lignin degradation (about 33%) were 3.5 and 38°C, respectively. Optimum fungal growth was at a pH of 4.5 and a temperature of around 32°C. Addition of exogeneous glucose to the cultures lessened the degradation of pulp carbohydrates. Lignin degradation was stimulated by oxygen atmosphere and non-agitated cultures. Increased surface to volume ratio (decreased culture depth) enhanced lignin degradation (about 56% at a depth of 1.2 cm). Finally, the correlations: pulp yield vs. residual glucose, ligninase activity vs. mycelium, and extent of delignification vs. residual extracellular H2O2 were discussed in light of recent findings of ligninases responsible for ligninolysis.

An experiment was conducted to find out the correct seeding (inoculum) material for the start up of anaerobic digestion using coffee pulp as substrate. Three different inocula viz., slurry from cowdung biogas plant, sewage sludge and rumen fluid were tried for their efficiency in producing maximum methanogenic activities. The result revealed that the sludge from a biogas plant is the best source of inoculum, which enhanced microbial activities, substrate utilization and gas yield. The various physico-chemical changes that occurred during fermentation were also discussed in detail.