Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate ...

INSTITUTO POLITÉCNICO NACIONAL

A PCR-based method for the quantitative detection of Lentinus edodes and Trametes versicolor, two ligninolytic fungi applied for wastewater treatment and bioremediation, was developed. Genomic DNA was used to optimize a PCR method targeting the conserved copper-binding sequence of laccase genes. The method allowed the quantitative detection and differentiation of these fungi in single and defined-mixed cultures after fractionation of the PCR products by electrophoresis in agarose gels. Amplified products of about 150 bp for L. edodes, and about 200 bp for T. versicolor were purified and cloned. The PCR method showed a linear detection response in the 1.0 microg-1 ng range. The same method was tested with genomic DNA from a third fungus (Phanerochaete chrysosporium), yielding a fragment of about 400 bp. Southern-blot and DNA sequence analysis indicated that a specific PCR product was amplified from each genome, and that these corresponded to sequences of laccase genes. This PCR protocol permits the detection and differentiation of three ligninolytic fungi by amplifying DNA fragments of different sizes using a single pair of primers, without further enzymatic restriction of the PCR products. This method has potential use in the monitoring, evaluation, and improvement of fungal cultures used in wastewater treatment processes.

A PCR method for the detection and differentiation of Lentinus edodes and Trametes versicolor in defined-mixed cultures used for wastewater treatment.

A fluidized bed bioreactor (FBBR) was operated for more than 1000 days under two regimes, Methanogenic (M) and Methanogenic-Aerobic (M-A), to remove 2,4,6-trichlorophenol (TCP) and phenol (Phe) from a synthetic wastewater, containing different amounts of TCP and Phe, using different aeration flow-rates (0, 2.13, and 1.06 NL O(2)/L.day). M conditions (80:20 mg/L of TCP:Phe, 0 NL O(2)/L.day) showed similar TCP and Phe removal (>95%). Nevertheless accumulation of 4-chlorophenol (4CP) up to 16 mg/L and Phe up to 4 mg/L was observed, while in M-A conditions (80:20 mg/L of TCP:Phe, 2.13 NL O(2)/L.day) TCP and Phe removal achieved 99.9(+)% and after 70 days no accumulation of intermediates were detected. The increase of TCP and Phe in the influent under M-A conditions from 80:20 to 120:30 mg/L of TCP:Phe did not negatively affect the removal of TCP, intermediates and Phe; in fact, they were similar to those in previous M-A conditions. The decrease in the oxygen flow rate from 2.13 to 1.06 NL O(2)/L.day had no negative effect on pollutant removals, which were as high as in previous two M-A conditions. The specific methanogenic activity of bioparticles of the fluidized bed decreased with long-term partial aeration, starting from 1.097 mmol CH(4)/h.g(TKN) in the M regime (day 60) to <0.02 mmolCH(4)/h.g(TKN) at day 1050, suggesting aerobic regime in the bioreactor rather than an M-A regime. In conclusion, complete removal of TCP and less chlorinated intermediates could be achieved in an initially methanogenic FBBR under conditions of partial aeration, although long-term operation seemed to negatively affect the methanogenic activity of biomass. It is also likely that after extended aeration the microbial community was finally enriched with strains with the ability to attack 2,4,6-TCP under aerobic conditions. This report represents the first evidence of a long exposure to oxygen of an anaerobic microbial consortium that efficiently remove TCP.

Impact of long-term partial aeration on the removal of 2,4,6-trichlorophenol in an initially methanogenic fluidized bed bioreactor.

In March 2009, public health surveillance detected increased numbers of influenza-like illness presenting to hospitals in Mexico City. The aetiological agent was subsequently determined to be a novel influenza A (H1N1) triple reassortant, which has spread worldwide. As a consequence the World Health Organisation has declared the first Influenza pandemic of the 21st century.

Identification of influenza A pandemic (H1N1) 2009 variants during the first 2009 influenza outbreak in Mexico City.

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.

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

Cysteine proteases (CPs) from the C1 family, which are similar to papain, can be found in animals and plants, as well as some viruses and prokaryotes. These enzymes have diverse physiological functions and are thus very attractive for science and industry. Jacaratia mexicana, a member of the Caricaceae plant family, contains several CPs, the principal being mexicain, found to favorably compete against papain for many industrial applications due to its high stability and specific activity. In this study, leaves of J. mexicana were used to isolate a CP-coding gene, similar to those that code for mexicain and chymomexicain. By using rapid amplification of cDNA ends (RACE) as well as oligonucleotide design from papain-like conserved amino acids (aa), a sequence of 1404 bp consisting of a 5' terminal untranslated region (UTR) of 153 bp, a 3' terminal UTR of 131 bp, with a polyadenylation (poly(A)) signal sequence and a poly(A) tail, and an open reading frame (ORF) of 1046 bp, was obtained by overlapping three partial sequences. Two full-length cDNA sequences that encode for mexicain-like proteases were cloned from mRNA (JmCP4 and JmCP5). JmCP4 is predicted to have an ORF of 1044 bp, which codifies for polypeptides that have a 26 aa signal peptide region, a 108 aa propeptide region and a mature enzyme of 214 aa. A 969 bp fragment (JmCP5) encodes for a partial sequence of a CP gene, without the signal peptide region but with a full-length propeptide region. The sequence analysis showed that this protease presented a high similarity to other plant CPs from J. mexicana, Vasconcellea cundinamarcensis, Vasconcellea stipulata, and Carica papaya, among others, mainly at the conserved catalytic site. Obtaining the sequence of this CP gene from J. mexicana provides an alternative for production in a standard system and could be an initial step towards the commercialization of this enzyme.

Isolation of cDNA from Jacaratia mexicana encoding a mexicain-like cysteine protease gene.

Two-phase partitioning bioreactors (TPPBs) are based on the addition of an organic phase, often called vector, to a bioreactor in order to increase mass transfer of oxygen or gaseous substrates from the gaseous phase to the aqueous phase. In TPPBs, like in any other reactor design, the characterization of the bioprocess is often required for design, control, and operation purposes. Pulse respirometry is a method that allows for microbial processes characterization through the determination of several stoichiometric and kinetic parameters with relatively little experimental effort. Despite its interest and its previous application in countless applications, pulse respirometry has never been applied in TPPBs. In this work, pulse respirometry was assessed in a model TPPB degrading terephthalic acid and using Elvax™ as solid vector to enhance oxygen transfer. The results indicated that the addition of 10 to 20% Elvax increased oxygen transfer by up to 97%, compared to control with no vector. Pulse respirometry was successfully applied and allowed for the determination of the growth yield, the substrate affinity constant, and the maximum growth rate, within other. It is concluded that pulse respirometry is a useful method, not only for the characterization of processes in TPPBs but also for the selection of a vector within several brands commercially available.

Pulse respirometry in two-phase partitioning bioreactors: case study of terephthalic acid biodegradation.

β-(1,3)(1,6)-D-glucan is fungal cell wall component that has demonstrated immunomodulatory and anti-cancer effects. The (1,3)-β-glucan synthase is one of the main enzymes involved in its biosynthesis.

Claudio Garibay-Orijel

Bioprocesses Department,
Laboratory of Environmental Biotechnology,
Unidad Profesional Interdisciplinaria de Biotecnología,
Bioprocesses Department, Laboratory of Environmental Biotechnology, Unidad Profesional Interdisciplinaria de Biotecnología

INSTITUTO POLITÉCNICO NACIONAL

Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor