Removal of phenolic inhibitors from lignocellulose hydrolysates using laccases for the production of fuels and chemicals.

Lignocellulose is the most abundant biopolymer in the biosphere. It is inexpensive and therefore considered an attractive feedstock to produce biofuels and other biochemicals. Thermochemical and/or enzymatic pretreatment is used to release fermentable monomeric sugars. However, a variety of inhibitory by-products such as weak acids, furans, and phenolics that inhibit cell growth and fermentation are also released. Phenolic compounds are among the most toxic components in lignocellulosic hydrolysates and slurries derived from lignin decomposition, affecting overall fermentation processes and production yields and productivity. Ligninolytic enzymes have been shown to lower inhibitor concentrations in these hydrolysates, thereby enhancing their fermentability into valuable products. Among them, laccases, which are capable of oxidizing lignin and a variety of phenolic compounds in an environmentally benign manner, have been used for biomass delignification and detoxification of lignocellulose hydrolysates with promising results. This review discusses the state of the art of different enzymatic approaches to hydrolysate detoxification. In particular, laccases are used in separate or in situ detoxification steps, namely in free enzyme processes or immobilized by cell surface display technology to improve the efficiency of the fermentative process and consequently the production of second-generation biofuels and bio-based chemicals.

[Relevance of Surface-Exposed Lysine Residues Designed for Functionalization of Laccase].

Fungal laccases are oxidoreductases with low-specificity for substrates. The characterization of laccase's surface is a prerequisite used to obtain hybrid catalysts with new properties. Surface-exposed lysine residues are targets in immobilization reactions. In this work, LAC3-K0, an enzyme devoid of lysine, was used as a platform to detect potential surface-exposed sites suitable for replacement with a lysine residue. Seven sites were selected from a LAC3-K0 3-D model, and single lysine mutants (UNIKn, n = residue number) were obtained by site-directed mutagenesis. All mutants were expressed in Saccharomyces cerevisiae W303-1A and detected as functional secreted proteins by their ability to oxidize guaiacol or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) on agar plates. All variants were active at acidic pH but presented no activity at neutral pH, as expected. Likewise, variants were stable a temperature between 15-55°C, and were completely inactivated at 70°C. Oxidation assays revealed that the replacement of one or two surface residues with lysine greatly affected enzyme activity and substrate specificity. The catalytic; parameters (KM^(app) and kcat^(app)) determined with ABTS were found to be different among the variants; Vmax^(app) was 1.5-2 fold higher in UNIK269 and triple mutant, with a KM^(app) of 0.27 and 0.30, respectively; kcat^(app )was 30.25 in UNIK238 and 32.34 in the triple mutant. The role of hydrophobic patches detected on the surface of LAC3-K0 was determined to be a favorable factor to be considered in the interaction of hybrid materials. All variants with uniquely surface located lysine created in this work can be in demand for obtaining laccases with a certain substrate specificity in the design of hybrid materials.

Bacterial succession and co-occurrence patterns of an enriched marine microbial community during light crude oil degradation in a batch reactor.

AIMS: The aim of this study was to investigate the dynamic changes in the bacterial structure and potential interactions of an acclimatized marine microbial community during a light crude oil degradation experiment. METHODS AND RESULTS: The bacterial community effectively removed 76·49% of total petroleum hydrocarbons after 30 days, as evidenced by GC-FID and GC-MS analyses. Short-chain alkanes and specific aromatic compounds were completely degraded within the first 6 days. High-throughput sequencing of 16S rRNA gene indicated that the starting bacterial community was mainly composed by Marinobacter and more than 30 non-dominant genera. Bacterial succession was dependent on the hydrocarbon uptake with Alcanivorax becoming dominant during the highest degradation period. Sparse correlations for compositional data algorithm revealed one operational taxonomic unit (OTU) of Muricauda and an assembly of six OTUs of Alcanivorax dieselolei and Alcanivorax hongdengensis as critical keystone components for the consortium network maintenance and stability. CONCLUSIONS: This work exhibits a stabilized marine bacterial consortium with the capability to efficiently degrade light crude oil in 6 days, under laboratory conditions. Successional and interaction patterns were observed in response to hydrocarbon consumption, highlighting potential interactions between Alcanivorax and keystone non-dominant OTUs over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results contribute to the understanding of interactions and potential roles of specific members of hydrocarbonoclastic marine bacterial communities, which will be useful for further bioaugmentation studies concerning the associations between indigenous and introduced micro-organisms.

Production of laccases by Pleurotus ostreatus in submerged fermentation in co-culture with Trichoderma viride.

AIMS: To evaluate the production and stability of laccases by Pleurotus ostreatus in liquid co-cultures with Trichoderma viride as a function of infection time and agitation rate. METHODS AND RESULTS: Pleurotus ostreatus cultures were infected with T. viride spores at 30 and 48 h. Maximal laccase volumetric activity was seen after 48 h (control cultures) or 72 h (co-cultures) of cultivation time. Only the cultures infected at 30 h showed an increased laccase volumetric activity compared to control cultures. After maximal laccase volumetric activity value was reached, a sharp decrease in it was observed in control cultures. Co-cultures exhibited a comparatively lower loss of activity. The influence of P. ostreatus and/or T. viride on the stability of laccase volumetric activity and isoenzyme pattern was evaluated. Trichoderma viride induced changes in the laccase isoenzyme pattern. Agitated cultures increased biomass growth and specific productivity threefold and sevenfold, respectively, to the static cultures. CONCLUSIONS: The laccase volumetric activity is very likely the result of the balance between biosynthesis and degradation/biotransformation rates occurring during the cultures. The individual presence of P. ostreatus or T. viride in the culture negatively affected the volumetric laccase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The evaluation of culture parameters that could influence Trichoderma-basidomycetes interaction and laccase production during submerged fermentation has not been reported. This study showed how laccase production in co-cultures of P. ostreatus and T. viride was influenced by the infection time and agitation/oxygenation conditions.

Selection of Trichoderma strains capable of increasing laccase production by Pleurotus ostreatus and Agaricus bisporus in dual cultures.

AIMS: To select Trichoderma strains for enhanced laccase production in Pleurotus ostreatus or Agaricus bisporus cultures. METHODS AND RESULTS: Laccase production by P. ostreatus and A. bisporus was evaluated in liquid (axenic) and solid (dual cultures) malt extract medium. Oxidation of ABTS, DMP and syringaldazine was evaluated in order to assess the potential of Trichoderma strains to enhance laccase production by basidiomycetes. Selected Pleurotus-Trichoderma interactions yielded higher increases in laccase volumetric activity and an additional laccase isoform was produced. By contrast, Agaricus-Trichoderma interactions lead to smaller increases on laccase volumetric activity, probably as result of repression (or degradation) towards one of the laccases isoforms. CONCLUSIONS: The strains of P. ostreatus and A. bisporus assessed in this work showed good potential as laccase producers. The Trichoderma-mediated biological stimulation of laccase production by P. ostreatus and A. bisporus is relevant in order to develop highly productive processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Extracellular laccases from basidiomycetes are produced only in small amounts. It is therefore important to increase process productivity for potential industrial applications. The results from this study enable the selection Trichoderma strains capable of increasing laccase production by P. ostreatus or A. bisporus in dual cultures.

Slurry-phase biodegradation of weathered oily sludge waste.

We assessed the biodegradation of a typical oily sludge waste (PB401) in Mexico using several regimes of indigenous microbial consortium and relevant bioremediation strategies in slurry-phase system. Abiotic loss of total petroleum hydrocarbons (TPH) in the PB401 was insignificant, and degradation rates under the various treatment conditions ranged between 666.9 and 2168.7 mg kg(-1) day(-1) over a 15 days reaction period, while viable cell count peaked at between log(10)5.7 and log(10)7.4 cfu g(-1). Biostimulation with a commercial fertilizer resulted in 24% biodegradation of the TPH in the oily waste and a corresponding peak cell density of log(10)7.4 cfu g(-1). Addition of non-indigenous adapted consortium did not appear to enhance the removal of TPH from the oily waste. It would appear that the complexities of the components of the alkylaromatic fraction of the waste limited biodegradation rate even in a slurry system.

Biodegradation of heavy crude oil Maya using spent compost and sugar cane bagasse wastes.

Experiments were carried out to evaluate the use of some agroindustrial wastes as supports in solid state cultures for the biodegradation of crude oil Maya in static column reactors over 15-20 days periods. Spent compost and cane bagasse wastes showed superior qualities over peat moss waste as support candidates with the advantage that they contain appreciable densities of autochthonous microorganisms in the order of 10(2) cfu g(-1). Mercuric chloride (2%) was able to completely inhibit growth of these microfloras. Biodegradation was enhanced in the presence of the IMP consortium and highest when microflora from cane bagasse only was the bioaugmentation partner (180.7 mg kg(-1) day(-1)). Combination of these waste materials (3:1 ratio, respectively) was observed to significantly biodegrade the crude oil by approximately 40% in 15 days from an initial concentration of 10,000 mg kg(-1) with a four order of magnitude increase in microbial density during this period. Spent compost and cane bagasse wastes are veritable solid support candidates for use in the biodegradation of crude oil polluted systems.

Potential of Burkholderia cepacia RQ1 in the biodegradation of heavy crude oil.

The potential of Burkholderia cepacia strain RQ1 in the biodegradation of heavy crude oil (Maya) was assessed to develop an active indigenous bacterial consortium for the bioremediation of crude oil-polluted systems in Nigeria. The heavy crude oil (Maya) was utilized as sole source of carbon, attaining maximum cell densities of 10(8) cfu ml(-1) from an initial 10(5) cfu ml(-1) in 15 days. Biomass also increased with oil concentrations up to 0.8% (w/v). Growth rates ranged from 0.028 h(-1) to 0.036 h(-1) and degradation rates decreased with increasing concentrations of oil from 0.009 day(-1) to 0.004 day(-1). The quantity of oil metabolized increased significantly (P < 0.05) with increasing concentrations of oil. However, the growth of the bacterium was inhibited at crude oil concentrations beyond 6% (w/v). The pH of the culture media also dropped significantly (P < 0.05) during the 15-day test period, while the non-asphaltic fractions of the oil were significantly reduced (by about 89%) during the same period. The bacterium harbours a plasmid of about 10 kb that lacks restriction sites for the endonucleases Asp718, BamHI and PstI.