Bioprocessing of Two Crop Residues for Animal Feeding into a High-Yield Lovastatin Feed Supplement.

This work aimed to evaluate the lovastatin (Lv) production by solid-state fermentation (SSF) from selected crop residues, considering the post-fermented residues as feed supplements for ruminants. The SSF was performed with two substrates (wheat bran and oat straw) and two A. terreus strains (CDBB H-194 and CDBB H-1976). The Lv yield, proximate analysis, and organic compounds by GC-MS in the post-fermented residues were assessed. The combination of the CDBB H-194 strain with oat straw at 16 d of incubation time showed the highest Lv yield (23.8 mg/g DM fed) and the corresponding degradation efficiency of hemicellulose + cellulose was low to moderate (24.1%). The other three treatments showed final Lv concentrations in decreasing order of 9.1, 6.8, and 5.67 mg/g DM fed for the oat straw + CDBB H-1976, wheat bran + CDBB H-194, and wheat bran + CDBB H-1976, respectively. An analysis of variance of the 22 factorial experiment of Lv showed a strong significant interaction between the strain and substrate factors. The kinetic of Lv production adequately fitted a zero-order model in the four treatments. GC-MS analysis identified only a couple of compounds from the residues fermented by A. terreus CDBB H-194 (1,3-dipalmitin trimethylsilyl ether in the fermented oat straw and stearic acid hydrazide in the fermented wheat bran) that could negatively affect ruminal bacteria and fungi. Solid-state fermentation of oat straw with CDBB H-194 deserves further investigation due to its high yield of Lv; low dietary proportions of this post-fermented oat straw could be used as an Lv-carrier supplement for rumen methane mitigation.

Fermentation performance of a Mexican native Clavispora lusitaniae strain for xylitol and ethanol production from xylose, glucose and cellobiose.

Lignocellulose hydrolysates are rich in fermentable sugars such as xylose, cellobiose and glucose, with high potential in the biotechnology industry to obtain bioproducts of higher economic value. Thus, it is important to search for and study new yeast strains that co-consume these sugars to achieve better yields and productivity in the processes. The yeast Clavispora lusitaniae CDBB-L-2031, a native strain isolated from mezcal must, was studied under various culture conditions to potentially produce ethanol and xylitol due to its ability to assimilate xylose, cellobiose and glucose. This yeast produced ethanol under microaerobic conditions with yields of 0.451 gethanol/gglucose and 0.344 gethanol/gcellobiose, when grown on 1% glucose or cellobiose, respectively. In mixtures (0.5% each) of glucose:xylose and glucose:xylose:cellobiose the yields were 0.367 gethanol/gGX and 0. 380 gethanol/gGXC, respectively. Likewise, in identical conditions, C. lusitaniae produced xylitol from xylose with a yield of 0.421 gxylitol/gxylose. In 5% glucose or xylose, this yeast had better ethanol and xylitol titers and yields, respectively. However, glucose negatively affected xylitol production in the mixture of both sugars (3% each), producing only ethanol. Xylose reductase (XR) and xylitol dehydrogenase (XDH) activities were evaluated in cultures growing on xylose or glucose, obtaining the highest values in cultures on xylose at 8 h (25.9 and 6.22 mU/mg, respectively). While in glucose cultures, XR and XDH activities were detected once this substrate was consumed (4.06 and 3.32 mU/mg, respectively). Finally, the XYL1 and XYL2 genes encoding xylose reductase and xylitol dehydrogenase, respectively, were up-regulated by xylose, whereas glucose down-regulated their expression.

Lovastatin as a supplement to mitigate rumen methanogenesis: an overview.

Methane from enteric fermentation is the gas with the greatest environmental impact emitted by ruminants. Lovastatin (Lv) addition to feedstocks could be a strategy to mitigate rumen methane emissions via decreasing the population of methanogenic archaea (MA). Thus, this paper provides the first overview of the effects of Lv supplementation, focusing on the inhibition of methane production, rumen microbiota, and ruminal fermentation. Results indicated that Lv treatment had a strong anti-methanogenic effect on pure strains of MA. However, there are uncertainties from in vitro rumen fermentation trials with complex substrates and rumen inoculum.Solid-state fermentation (SSF) has emerged as a cost-effective option to produce Lv. In this way, SSF of agricultural residues as an Lv-carrier supplement in sheep and goats demonstrated a consistent decrease in ruminal methane emissions. The experimental evidence for in vitro conditions showed that Lv did not affect the volatile fatty acids (VFA). However, in vivo experiments demonstrated that the production of VFA was decreased. Lv did not negatively affect the digestibility of dry matter during in vitro and in vivo methods, and there is even evidence that it can induce an increase in digestibility. Regarding the rumen microbiota, populations of MA were reduced, and no differences were detected in alpha and beta diversity associated with Lv treatment. However, some changes in the relative abundance of the microbiota were induced. Further studies are recommended on: (i) Lv biodegradation products and stability, as well as its adsorption onto the solid matter in the rumen, to gain more insight on the "available" or effective Lv concentration; and (ii) to determine whether the effect of Lv on ruminal fermentation also depends on the feed composition and different ruminants.

Biological synthesis of iron nanoparticles using hydrolysates from a waste-based biorefinery.

The purpose of this work was to produce iron nanoparticles (Fe-NP) by microbial pathway from anaerobic bacteria grown in anaerobic fluidized bed reactors (AnFBRs) that constitute a new stage of a waste-based biorefinery. Bioparticles from biological fluidized bed reactors from a biorefinery of organic fraction of municipal solid wastes (that produces hydrolysates rich in reducing sugars) were nanodecorated (embedded nanobioparticle or nanodecorated bioparticle, ENBP) by biological reduction of iron salts. Factors "origin of bioparticles" (either from hydrogenogenic or methanogenic fluidized bed reactor) and "type of iron precursor salt" (iron chloride or iron citrate) were explored. SEM and high-resolution transmission electron microscopy (HRTEM) showed amorphous distribution of nanoparticles (NP) on the bioparticles surface, although small structures that are nanoparticle-like could be seen in the SEM micrographs. Some agglomeration of NPs was confirmed by DLS. Average NP size was lower in general for NP in ENBP-M than ENBP-H according to HRTEM. The factors did not have a significant influence on the specific surface area of NPs, which was high and in the range 490 to 650 m2 g-1. Analysis by EDS displayed consistent iron concentration 60-65% iron in nanoparticles present in ENBP-M (bioparticles previously grown in methanogenic bioreactor), whereas the iron concentration in NPs present in ENBP-H (bioparticles previously grown in hydrogenogenic bioreactor) was more variable in a range from 8.5 to 62%, depending on the iron salt. X-ray diffraction patterns showed the typical peaks for magnetite at 35° (3 1 1), 43° (4 0 0), and 62° (4 0 0); moreover, siderite diffraction pattern was found at 26° (0 1 2), 38° (1 1 0), and 42° (1 1 3). Results of infrared analysis of ENBP in our work were congruent with presence of magnetite and occasionally siderite determined by XRD analysis as well as presence of both Fe+2 and F+3 (and selected satellite signal peaks) observed by XPS. Our results on the ENBPs hold promise for water treatment, since iron NPs are commonly used in wastewater technologies that treat a wide variety of pollutants. Finally, the biological production of ENBP coupled to a biorefinery could become an environmentally friendly platform for nanomaterial biosynthesis as well as an additional source of revenues for a waste-based biorefinery.

A soft sensor based on online biomass measurements for the glucose estimation and control of fed-batch cultures of Bacillus thuringiensis.

On bioprocess engineering, experimental measurements are always a costly part of the modeling effort; therefore, there is a constant need to develop cheaper, simpler, and more efficient methodologies to exploit the information available. The aim of the present work was to develop a soft sensor with the capacity to perform reliable substrate predictions and control in microbial cultures of the fed-batch type, using mainly microbial growth data. This objective was achieved using dielectric spectroscopy technology for online monitoring of microbial growth and hybrid neural networks for online prediction of substrate concentration. The glucose estimator was integrated to a fuzzy logic controller to control the substrate concentration in a fed-batch experiment. Dielectric spectroscopy is a technology sensitive to the air volume fraction in the culture media and the turbulence generated by the agitation; however, the introduction of a polynomial function for the calibration of the permittivity signal allowed biomass estimations with an approximation error of 2%. The methodology presented in this work was successfully implemented for the glucose prediction and control of a fed-batch culture of Bacillus thuringiensis with an approximation error of 6%.

Expression of a codon-optimized ß-glucosidase from Cellulomonas flavigena PR-22 in Saccharomyces cerevisiae for bioethanol production from cellobiose.

Bioethanol is one of the main biofuels produced from the fermentation of saccharified agricultural waste; however, this technology needs to be optimized for profitability. Because the commonly used ethanologenic yeast strains are unable to assimilate cellobiose, several efforts have been made to express cellulose hydrolytic enzymes in these yeasts to produce ethanol from lignocellulose. The C. flavigenabglA gene encoding ß-glucosidase catalytic subunit was optimized for preferential codon usage in S. cerevisiae. The optimized gene, cloned into the episomal vector pRGP-1, was expressed, which led to the secretion of an active ß-glucosidase in transformants of the S. cerevisiae diploid strain 2-24D. The volumetric and specific extracellular enzymatic activities using pNPG as substrate were 155 IU L-1 and 222 IU g-1, respectively, as detected in the supernatant of the cultures of the S. cerevisiae RP2-BGL transformant strain growing in cellobiose (20 g L-1) as the sole carbon source for 48 h. Ethanol production was 5 g L-1 after 96 h of culture, which represented a yield of 0.41 g g-1 of substrate consumed (12 g L-1), equivalent to 76% of the theoretical yield. The S. cerevisiae RP2-BGL strain expressed the ß-glucosidase extracellularly and produced ethanol from cellobiose, which makes this microorganism suitable for application in ethanol production processes with saccharified lignocellulose.

Effect of sudden addition of PCE and bioreactor coupling to ZVI filters on performance of fluidized bed bioreactors operated in simultaneous electron acceptor modes.

The present work evaluated the effects of (i) feeding a water contaminated with 80 mg/L PCE to bioreactors seeded with inoculum not acclimated to PCE, (ii) coupling ZVI side filters to bioreactors, and (iii) working in different biological regimes, i.e., simultaneous methanogenic aeration and simultaneous methanogenic-denitrifying regimes, on fluidized bed bioreactor performance. Simultaneous electron acceptors refer to the simultaneous presence of two compounds operating as final electron acceptors in the biological respiratory chain (e.g., use of either O2 or NO3- in combination with a methanogenic environment) in a bioreactor or environmental niche. Four lab-scale, mesophilic, fluidized bed bioreactors (bioreactors) were implemented. Two bioreactors were operated as simultaneous methanogenic-denitrifying (MD) units, whereas the other two were operated in partially aerated methanogenic (PAM) mode. In the first period, all bioreactors received a wastewater with 1 g chemical oxygen demand of methanol per liter (COD-methanol/L). In a second period, all the bioreactors received the wastewater plus 80 mg perchloroethylene (PCE)/L; at the start of period 2, one MD and one PAM were coupled to side sand-zero valent iron filters (ZVI). All bioreactors were inoculated with a microbial consortium not acclimated to PCE. In this work, the performance of the full period 1 and the first 60 days of period 2 is reported and discussed. The COD removal efficiency and the nitrate removal efficiency of the bioreactors essentially did not change between period 1 and period 2, i.e., upon PCE addition. On the contrary, specific methanogenic activity in PAM bioreactors (both with and without coupled ZVI filter) significantly decreased. This was consistent with a sharp fall of methane productivity in those bioreactors in period 2. During period 2, PCE removals in the range 86 to 97 % were generally observed; the highest removal corresponded to PAM bioreactors along with the highest dehalogenation efficiency (94 %). Principal component analysis as well as cluster analysis confirmed the trends mentioned above, i.e., the better performance of PAM over MD, and the unexpected no effect of the ZVI side filters on PCE removal and dehalogenation efficiencies. To the best of our knowledge, this is the first report on the combined treatment ZVI-biological of a water polluted with PCE, where the biological operation relied on simultaneous electron acceptors.

Bioenergy and bioproducts from municipal organic waste as alternative to landfilling: a comparative life cycle assessment with prospective application to Mexico.

A life cycle assessment (LCA) of a four-stage biorefinery concept, coined H-M-Z-S, that converts 1 t of organic fraction of municipal solid waste (OFMSW) into bioenergy and bioproducts was performed in order to determine whether it could be an alternative to common disposal of OFMSW in landfills in the Mexican reality. The OFMSW is first fermented for hydrogen production, then the fermentates are distributed 40 % to the methane production, 40 % to enzyme production, and 20 % to the saccharification stage. From hydrogen and methane, up to 267 MJ and 204 kWh of gross heat and electricity were produced. The biorefinery proved to be self-sustainable in terms of power (95 kWh net power), but it presented a deficit of energy for heating services (-155 MJ), which was partially alleviated by digesting the wastes from the bioproducts stages (-84 MJ). Compared to landfill, biorefinery showed lower environmental impacts in global warming (down to -128 kg CO2-eq), ozone layer depletion (2.96 × 10-6 kg CFC11-eq), and photochemical oxidation potentials (0.011 kg C2H4-eq). The landfarming of the digestates increased significantly the eutrophication impacts, up to 20 % below the eutrophication from landfilling (1.425 kg PO4-eq). These results suggest that H-M-Z-S biorefinery could be an attractive alternative compared to conventional landfilling for the management of municipal solid wastes, although new alternatives and uses of co-products and wastes should be explored and tested. Moreover, the biorefinery system would benefit from the integration into the market chain of the bioproducts, i.e., enzymes and hydrolysates among others.

Long-term response on growth, antioxidant enzymes, and secondary metabolites in salicylic acid pre-treated Uncaria tomentosa microplants.

OBJECTIVE: To obtain micro propagated Uncaria tomentosa plantlets with enhanced secondary metabolites production, long-term responses to salicylic acid (SA) pre-treatments at 1 and 100 µM were evaluated after propagation of the plantlets in a SA-free medium. RESULTS: SA pre-treatments of single node cuttings OF U. tomentosa produced long-term responses in microplants grown for 75 days in a SA-free medium. Reduction in survival rate, root formation, and stem elongation were observed only with 100 µM SA pre-treatments with respect to the control (0 + DMSO).Both pre-treatments enhanced H2O2 and inhibited superoxide dismutase and catalase activities, while guaiacol peroxidase was increased only with 1 µM SA. Also, both pre-treatments increased total monoterpenoid oxindole alkaloids by ca. 55 % (16.5 mg g(-1) DW), including isopteropodine, speciophylline, mitraphylline, isomitraphylline, rhynchopylline, and isorhynchopylline; and flavonoids by ca. 21 % (914 µg g(-1) DW), whereas phenolic compounds were increased 80 % (599 µg g(-1) DW) at 1 µM and 8.2 % (359 µg g(-1) DW) at 100 µM SA. CONCLUSION: Pre-treatment with 1 µM SA of U.tomentosa microplants preserved the survival rate and increased oxindole alkaloids, flavonoids, and phenolic compounds in correlation with H2O2 and peroxidase activity enhancements, offering biotechnological advantages over non-treated microplants.

The influence of total solids content and initial pH on batch biohydrogen production by solid substrate fermentation of agroindustrial wastes.

Hydrogen is a valuable clean energy source, and its production by biological processes is attractive and environmentally sound and friendly. In México 5 million tons/yr of agroindustrial wastes are generated; these residues are rich in fermentable organic matter that can be used for hydrogen production. On the other hand, batch, intermittently vented, solid substrate fermentation of organic waste has attracted interest in the last 10 years. Thus the objective of our work was to determine the effect of initial total solids content and initial pH on H2 production in batch fermentation of a substrate that consisted of a mixture of sugarcane bagasse, pineapple peelings, and waste activated sludge. The experiment was a response surface based on 2(2) factorial with central and axial points with initial TS (15-35%) and initial pH (6.5-7.5) as factors. Fermentation was carried out at 35 °C, with intermittent venting of minireactors and periodic flushing with inert N2 gas. Up to 5 cycles of H2 production were observed; the best treatment in our work showed cumulative H2 productions (ca. 3 mmol H2/gds) with 18% and 6.65 initial TS and pH, respectively. There was a significant effect of TS on production of hydrogen, the latter decreased with initial TS increase from 18% onwards. Cumulative H2 productions achieved in this work were higher than those reported for organic fraction of municipal solid waste (OFMSW) and mixtures of OFMSW and fruit peels waste from fruit juice industry, using the same process. Specific energetic potential due to H2 in our work was attractive and fell in the high side of the range of reported results in the open literature. Batch dark fermentation of agrowastes as practiced in our work could be useful for future biorefineries that generate biohydrogen as a first step and could influence the management of this type of agricultural wastes in México and other countries and regions as well.

Influence of discontinuing feeding degradable cosubstrate on the performance of a fluidized bed bioreactor treating a mixture of trichlorophenol and phenol.

The purpose of our research was to evaluate the effect of eliminating supplementation of sucrose to the reactor influent on the performance of a lab scale partially-aerated methanogenic fluidized bed bioreactor (PAM-FBBR). Two operational stages were distinguished: in the first stage the influent contained a mixture of 120/30/1000 mg/L of 2,4,6-trichlorophenol/phenol/COD-sucrose (TCP/Phe/COD-sucrose); in the second stage only the xenobiotic concentrations were the same 120/30 mg/L of TCP/Phe whereas sucrose addition was discontinued. Removal efficiencies of TCP, Phe, and COD were very high and close for both stages; i.e., η(TCP): 99.9 and 99.9%; η(Phe): 99.9 and 99.9%; η(COD) = 96.46 and 97.48% for stage 1 and stage 2, respectively. Traces of 2,4,6 dichlorophenol (0.05 mg/L) and 4-chlorophenol (0.07-0.26 mg/L) were found during the first 15 days of operation of the second stage, probably due to the adaptation to no co-substrate conditions. Net increase of chloride anion Cl(-) in effluent ranged between 59.5 and 61.5 mg Cl(-)/L that was very close to the maximum theoretical concentration of 62.8 mg Cl(-)/L. PCR-DGGE analysis revealed a richness decrease of eubacterial domain posterior to sucrose elimination from the influent whereas archaeal richness remained almost the same. However, the bioreactor performance was not negatively affected by discontinuing the addition of co-substrate sucrose. Our results indicate that the application of PAM-FBBR to the treatment of groundwaters polluted with chlorophenols and characterized by the lack of easily degradable co-substrates, is a promising alternative for on site bioremediation.

Re-fermentation of washed spent solids from batch hydrogenogenic fermentation for additional production of biohydrogen from the organic fraction of municipal solid waste.

In the first batch solid substrate anaerobic hydrogenogenic fermentation with intermittent venting (SSAHF-IV) of the organic fraction of municipal solid waste (OFMSW), a cumulative production of 16.6 mmol H(2)/reactor was obtained. Releases of hydrogen partial pressure first by intermittent venting and afterward by flushing headspace of reactors with inert gas N(2) allowed for further hydrogen production in a second to fourth incubation cycle, with no new inoculum nor substrate nor inhibitor added. After the fourth cycle, no more H(2) could be harvested. Interestingly, accumulated hydrogen in 4 cycles was 100% higher than that produced in the first cycle alone. At the end of incubation, partial pressure of H(2) was near zero whereas high concentrations of organic acids and solvents remained in the spent solids. So, since approximate mass balances indicated that there was still a moderate amount of biodegradable matter in the spent solids we hypothesized that the organic metabolites imposed some kind of inhibition on further fermentation of digestates. Spent solids were washed to eliminate organic metabolites and they were used in a second SSAHF-IV. Two more cycles of H(2) production were obtained, with a cumulative production of ca. 2.4 mmol H(2)/mini-reactor. As a conclusion, washing of spent solids of a previous SSAHF-IV allowed for an increase of hydrogen production by 15% in a second run of SSAHF-IV, leading to the validation of our hypothesis.

Treatment of mezcal vinasses: a review.

Mexican distilleries produce near eight million liters of mezcal per year, and generate about 90 million liters of mezcal vinasses (MV). This acidic liquid waste is very aggressive to the environment because of its high content of toxic and recalcitrant organic matter. As a result, treatment is necessary before discharge to water bodies. It is interesting, yet disturbing; verify that there is a significant gap on the treatment of MV. However, there is an abundant body of research on treatment of other recalcitrant toxic effluents that bear some similarity to MV, for example, wine vinasse, vinasses from the sugar industry, olive oil, and industrial pulp and paper wastewaters. The objective of this review is to critically organize the treatment alternatives of MV, assess their relative advantages and disadvantages, and finally detect the trends for future research and development. Experience with treatment of this set of residuals, indicates the following trends: (i) anaerobic digestion, complemented by oxidative chemical treatments (e.g. ozonation) are usually placed as pretreatments, (ii) aerobic treatment alone and combined with ozone which have been directed to remove phenolic compounds and color have been successfully applied, (iii) physico-chemical treatments such as Fenton, electro-oxidation, oxidants and so on., which are now mostly at lab scale stage, have demonstrated a significant removal of recalcitrant organic compounds, (iv) fungal pretreatment with chemical treatment followed by oxidative (O(3)) or anaerobic digestion, this combination seems to give attractive results, (v) vinasses can be co-composted with solid organic wastes, particularly with those from agricultural activities and agro-industies; in addition to soil amenders with fertilizing value to improve soil quality in typical arid lands where agave is cultivated, it seems to be a low cost technology very well suited for rural regions in underdeveloped countries where more sophisticated technologies are difficult to adopt, due to high costs and requirements of skilled personnel.

Small microcapsules of crystal proteins and spores of Bacillus thuringiensis by an emulsification/internal gelation method.

This paper describes a microencapsulation process of a spore crystal aggregate produced by Bacillus thuringiensis var. kurstaki HD-1. The methodology is based on the emulsification/internal gelation method, and was implemented to produce microcapsules of small diameter (< 10 µm) with the capacity to protect the spore crystal aggregate from extreme ultraviolet radiation. The diameter of microcapsules was in the range of 3.1 ± 0.2-6.8 ± 0.4 µm, which is considered adequate for biological control purposes. The protective effect of the alginate coat was verified by the remaining 60 ± 2% and 40 ± 1% of spore viability and protein activity, respectively, after UV-B radiation of 236 J, and with bioassays with Spodoptera frugiperda. It is expected that the protective effect of the alginate coat will improve the effectiveness of the Bt-HD1 formulated as small diameter microcapsules, and their yield, once they are released into the environment, will also be improved.

Production of cellulases and xylanases under catabolic repression conditions from mutant PR-22 of Cellulomonas flavigena.

Derepressed mutant PR-22 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenic treatment of Cellulomonas flavigena PN-120. This mutant improved its xylanolytic activity from 26.9 to 40 U mg(-1) and cellulolytic activity from 1.9 to 4 U mg(-1); this represented rates almost 2 and 1.5 times higher, respectively, compared to its parent strain growing in sugarcane bagasse. Either glucose or cellobiose was added to cultures of C. flavigena PN-120 and mutant PR-22 induced with sugarcane bagasse in batch culture. The inhibitory effect of glucose on xylanase activity was more noticeable for parent strain PN-120 than for mutant PR-22. When 20 mM glucose was added, the xylanolytic activity decreased 41% compared to the culture grown without glucose in mutant PR-22, whereas in the PN-120 strain the xylanolytic activity decreased by 49% at the same conditions compared to its own control. Addition of 10 and 15 mM of glucose did not adversely affect CMCase activity in PR-22, but glucose at 20 mM inhibited the enzymatic activity by 28%. The CMCase activity of the PN-120 strain was more sensitive to glucose than PR-22, with a reduction of CMCase activity in the range of 20-32%. Cellobiose had a more significant effect on xylanase and CMCase activities than glucose did in the mutant PR-22 and parent strain. Nevertheless, the activities under both conditions were always higher in the mutant PR-22 than in the PN-120 strain. Enzymatic saccharification experiments showed that it is possible to accumulate up to 10 g l(-1) of total soluble sugars from pretreated sugarcane bagasse with the concentrated enzymatic crude extract from mutant PR-22.

Design of a new rotating drum bioreactor operated at atmospheric pressure on the bioremediation of a polluted soil.

This paper reports the effect of the operation and design characteristics of rotating drum bioreactors (RDBs) aerated by natural convection and applied to the treatment of a soil highly polluted with weathered total petroleum hydrocarbons (TPH) (55,000 +/- 2,600 mg/kg). The parameters studied were length to diameter ratio (L/D), rotating speed (N) and lifter type. The highest TPH removal (59.6 +/- 0.7%) was obtained with the RDB of the lowest L/D ratio (1.5). Removals diminished by 27, 36 and 56%, with a ratio increment of 2.1, 3.1 and 5.1, respectively. Increment of the N, at an optimal value and lifter change from straight to helicoidal showed an improvement on the TPH removal of 20 and 30%, respectively. According to these results, slurry surface renewal through the variation of the N and the change of slurry flow was able to improve TPH removal in RDBs operated by natural convection.

Fungal post-treatment of pulp mill effluents for the removal of recalcitrant pollutants.

The objective of this work was to evaluate the post-treatment of an anaerobic recalcitrant effluent (anaerobically-treated weak black liquor, AnE) in an aerobic, upflow reactor packed with "biocubes" of Trametes versicolor immobilized onto small cubes of holm oak wood. The treated effluent (named anaerobic effluent; AnE) from an anaerobic fluidized bed reactor was fed to an up-flow aerobic fungal packed bed reactor (PBR). Two HRT were tested in this unit, namely 5 and 2.5days; the PBR operated 60days at 5-day HRT and 35days at 2.5-day HRT. The aerobic packed bench scale reactor was a glass column 1.5L total geometric volume containing 0.75L biocubes of T. versicolor immobilized onto holm oak wood small cubes of 5mm side. The reactor was operated at 25 degrees C. The pH of the AnE was adjusted to 4.5 before feeding; no carbohydrates or other soluble carbon source was supplemented. The fungal packed bed bioreactor averaged organic matter removals of 30% and 32% COD basis, during an experimental run of 60days at 5-day HRT and 35days at 2.5-day HRT, respectively. Colour and ligninoids contents were removed at higher percentages (69% and 54% respectively, average of both HRT). There was no significant difference between reactor performance at 5- and 2.5-day HRT, so, operation at 2.5-day HRT is recommended since reactor throughput is double. Activity of manganese peroxidase and laccase was found during the entire operation of the fungal PBR whereas lignin peroxidase activity practically disappeared in the second operation period. In general, enzyme activities were higher in the first period of operation (5-day HRT) than at 2.5-day HRT. To the best of our knowledge, this is one of the few works that demonstrated extended performance (3months) of a fungal bioreactor for the treatment of a recalcitrant wastewater with no supplementation of glucose or other expensive, soluble carbohydrate.