Effect of permeate drag force on the development of a biofouling layer in a pressure-driven membrane separation system.

The effect of permeate flux on the development of a biofouling layer on cross-flow separation membranes was studied by using a bench-scale system consisting of two replicate 100-molecular-weight-cutoff tubular ultrafiltration membrane modules, one that allowed flow of permeate and one that did not (control). The system was inoculated with Pseudomonas putida S-12 tagged with a red fluorescent protein and was operated using a laminar flow regimen under sterile conditions with a constant feed of diluted (1:75) Luria-Bertani medium. Biofilm development was studied by using field emission scanning electron microscopy and confocal scanning laser microscopy and was subsequently quantified by image analysis, as well as by determining live counts and by permeate flux monitoring. Biofilm development was highly enhanced in the presence of permeate flow, which resulted in the buildup of complex three-dimensional structures on the membrane. Bacterial transport toward the membrane by permeate drag was found to be a mechanism by which cross-flow filtration contributes to the buildup of a biofouling layer that was more dominant than transport of nutrients. Cellular viability was found to be not essential for transport and adhesion under cross-flow conditions, since the permeate drag overcame the effect of bacterial motility.

Biological granulated activated carbon fluidized bed reactor for atrazine remediation.

To show that an adsorbing biofilm carrier (GAC) can be advantageous for atrazine bioremediation over a non-adsorbing carrier, fluidized bed (FB) reactors were operated under atrazine limiting concentrations using Pseudomonas sp. strain ADP as the atrazine degrading bacteria. The following interrelated subjects were investigated: 1) atrazine adsorption to GAC under conditions of atrazine partial penetration in the biofilm, 2) differences in atrazine degradation rates and 3) stability of atrazine biodegradation under non-sterile anoxic conditions in the GAC reactor versus a reactor with a non-adsorbing biofilm carrier. Results from batch adsorption tests together with modeling best described the biofilm as patchy in nature with covered and non-biofilm covered areas. Under conditions of atrazine partial penetration in the biofilm, atrazine adsorption occurs in the non-covered areas and is consequently desorbed at the base of the biofilm substantially increasing the active biofilm surface area. The double flux of atrazine to the biofilm in the GAC reactor results in lower effluent atrazine concentrations as compared to a FB reactor with a non-adsorbing carrier. Moreover, under non-sterile denitrification conditions, atrazine degradation stability was found to be much higher (several months) using GAC as a biofilm carrier while non-adsorbing carrier reactors showed sharp deterioration within 30 days due to contamination of non-atrazine degrading bacteria.

Atrazine degradation under denitrifying conditions in continuous culture of Pseudomonas ADP.

The simultaneous removal of atrazine and nitrate in continuous culture under denitrifying conditions using Pseudomonas sp. strain ADP was investigated. Under all operational conditions the nitrate removal efficiency was always higher than 90%, while atrazine degradation deteriorated with time due to contamination by foreign denitrifying bacteria, lacking the ability to degrade atrazine. Recovery of atrazine degradation ability was achieved by applying aerobic conditions with atrazine as the sole nitrogen source.

Inactivation of lignin peroxidase during oxidation of the highly reactive substrate ferulic acid.

Ferulic acid (4-hydroxy-3-methoxycinnamic acid) (FA) was found to be a highly reactive substrate for lignin peroxidase (LIP), exhibiting a k(cat) of 41.7 s(-1). Despite the high reactivity, two modes of inactivation prevailed during the oxidation of FA. The first, H(2)O(2)-dependent inactivation, was evidenced by incomplete substrate oxidation and accumulation of LIP compound III (LIPIII), even at relatively low H(2)O(2) concentrations. This was attributed to the high turnover rate along with the inability of FA to revert LIPIII to the native state, as evidenced by pre-steady-state kinetics. H(2)O(2)-dependent inactivation could be avoided by inclusion of veratryl alcohol (VA), which efficiently reverts LIPIII to the native state. However, VA also mediated FA oxidation, and significantly decreased the reaction rate, which is unlike for previously reported VA-mediated reactions. The second mechanism of LIP inactivation was attributed to binding of phenoxy radicals or oxidation products to the enzyme and its extent directly correlated with the amount of FA consumed. This inactivation could be considerably suppressed by inclusion of gelatin. Therefore, during the oxidation of highly reactive phenolics, different kinds of protectors are required for efficient oxidation and maintaining LIP activity over time. This is of importance when considering emerging biotechnological applications for LIP.

Initial steps of ferulic acid polymerization by lignin peroxidase.

The major products of the initial steps of ferulic acid polymerization by lignin peroxidase included three dehydrodimers resulting from beta-5' and beta-beta'coupling and two trimers resulting from the addition of ferulic acid moieties to decarboxylated derivatives of beta-O-4'- and beta-5'-coupled dehydrodimers. This is the first time that trimers have been identified from peroxidase-catalyzed oxidation of ferulic acid, and their formation appears to be favored by decarboxylation of dehydrodimer intermediates. After initial oxidation, the coupling reactions appear to be determined by the chemistry of ferulic acid phenoxy radicals, regardless of the enzyme and of whether the reaction is performed in vitro or in vivo. This claim is supported by our finding that horseradish peroxidase provides a similar product profile. Furthermore, two of the dehydrodimers were the two products obtained from laccase-catalyzed oxidation (Tatsumi, K. S., Freyer, A., Minard, R. D., and Bollag, J.-M. (1994) Environ. Sci. Technol. 28, 210-215), and the most abundant dehydrodimer is the most prominent in grass cell walls (Ralph, J., Quideau, S., Grabber, J. H., and Hatfield, R. D. (1994) J. Chem. Soc. Perkin Trans. 1, 3485-3498). Our results also indicate that the dehydrodimers and trimers are further oxidized by lignin peroxidase, suggesting that they are only intermediates in the polymerization of ferulic acid. The extent of polymerization appears to be dependent on the ionization potential of formed intermediates, H(2)O(2) concentration, and, probably, enzyme stability.

Manganese deficiency can replace high oxygen levels needed for lignin peroxidase formation by Phanerochaete chrysosporium.

The combined effects of Mn and oxygen on lignin peroxidase (LIP) activity and isozyme composition in Phanerochaete chrysosporium were studied by using shallow stationary cultures grown in the presence of limited or excess N. When no Mn was added, LIP was formed in both N-limited and N-excess cultures exposed to air, but no LIP activity was observed at Mn concentrations greater than 13 mg/liter. In oxygen-flushed, N-excess cultures, LIP was formed at all Mn concentrations, and the peak LIP activity values in the extracellular fluid were nearly identical in the presence of Mn concentrations ranging from 3 to 1,500 mg/liter. When the availability of oxygen to cultures exposed to air was increased by growing the fungus under nonimmersed liquid conditions, higher levels of Mn were needed to suppress LIP formation compared with the levels needed in shallow stationary cultures. The composition of LIP isozymes was affected by the levels of N and Mn. Addition of veratryl alcohol to cultures exposed to air did not eliminate the suppressive effect of Mn on LIP formation. A deficiency of Mn in N-excess cultures resulted in lower biomass and a lower rate of glucose consumption than in the presence of Mn. In addition, almost no activity of the antioxidant enzyme Mn superoxide dismutase was observed in Mn-deficient, N-excess cultures, but the activity of this enzyme increased as the Mn concentration increased from 3 to 13 mg/liter. No Zn/Cu superoxide dismutase activity was observed in N-excess cultures regardless of the Mn concentration.

Lignin Peroxidase Isozymes from Phanerochaete chrysosporium Can Be Enzymatically Dephosphorylated.

The extracellular lignin peroxidase (LIP) protein profile of the fungus Phanerochaete chrysosporium, grown in nonimmersed liquid culture under conditions of excess nitrogen, changed markedly with culture age. At peak LIP activity (day 4), the heme-protein profile in the extracellular fluid, analyzed by anion-exchange high-pressure liquid chromatography, was characterized by a predominance of the LIP isozymes H1 and H2, small amounts of H6 and H8, and other minor peaks, designated Ha and Hb. On day 5, the level of H1 increased and it became the dominant isozyme, with a corresponding decrease in the level of H2. Moreover, the relative levels of H6 and H8 decreased with corresponding increases in Ha and Hb levels. This change in LIP profile occurred extracellularly and resulted from the enzymatic dephosphorylation of LIP isozymes. An enzymatic fraction responsible for LIP isozyme dephosphorylation, termed LIP dephosphorylating (LpD) fraction, was partially purified from the culture fluid. Incubation of the LpD fraction with (sup32)P-labeled H2, H6, H8, and H10 isozymes separated from nitrogen-limited cultures resulted in the formation of the dephosphorylated isozymes H1, Ha, Hb, and Hc, respectively. Dephosphorylation did not significantly change the catalytic properties of the LIP isozymes with veratryl alcohol as a substrate. LIP dephosphorylation is therefore suggested to be a posttranslational modification process catalyzed extracellularly by the LpD activity.

Extracellular proteases produced by the wood-degrading fungus Phanerochaete chrysosporium under ligninolytic and non-ligninolytic conditions.

When subjected to nitrogen limitation, the wood-degrading fungus Phanerochaete chrysosporium produces two groups of secondary metabolic, extracellular isoenzymes that depolymerize lignin in wood: lignin peroxidases and manganese peroxidases. We have shown earlier the turnover in activity of the lignin peroxidases to be due in part to extracellular proteolytic activity. This paper reports the electrophoretic characterization of two sets of acidic extracellular proteases produced by submerged cultures of P. chrysosporium. The protease activity seen on day 2 of incubation, during primary growth when nitrogen levels are not known to be limiting, consisted of at least six proteolytic bands ranging in size from 82 to 22 kDa. The activity of this primary protease was strongly reduced in the presence of SDS. Following the day 2, when nitrogen levels are known to become limiting and cultures become ligninolytic, the main protease activity (secondary protease) consisted of a major proteolytic band of 76 kDa and a minor band of 25 kDa. The major and minor secondary protease activities were inhibited by phenylmethyl-sulfonyl fluoride and pepstatin A, respectively. When cultures were grown in the presence of excess nitrogen (non-ligninolytic condition), the primary protease remained the principal protease throughout the culture period. These results identify and characterize a specific proteolytic activity associated with conditions that promote lignin degradation.

Overproduction of lignin peroxidase by Phanerochaete chrysosporium (BKM-F-1767) under nonlimiting nutrient conditions.

The ligninolytic enzymes synthesized by Phanerochaete chrysosporium BKM-F-1767 immobilized on polyurethane foam were characterized under limiting, sufficient, and excess nutrient conditions. The fungus was grown in a nonimmersed liquid culture system under conditions close to those occurring in nature, with nitrogen concentrations ranging from 2.4 to 60 mM. This nonimmersed liquid culture system consisted of fungal mycelium immobilized on porous pieces of polyurethane foam saturated with liquid medium and highly exposed to gaseous oxygen. Lignin peroxidase (LIP) activity decreased to almost undetectable levels as the initial NH4+ levels were increased over the range from 2.4 to 14 mM and then increased with additional increases in initial NH4+ concentration. At 45 mM NH4+, LIP was overproduced, reaching levels of 800 U/liter. In addition, almost simultaneous secretion of LIP and secretion of manganese-dependent lignin peroxidase were observed on the third day of incubation. Manganese-dependent lignin peroxidase activity was maximal under nitrogen limitation conditions (2.4 mM NH4+) and then decreased to 40 to 50% of the maximal level in the presence of sufficient or excess initial NH4+ concentrations. Overproduction of LIP in the presence of a sufficient nitrogen level (24 mM NH4+) and excess nitrogen levels (45 to 60 mM NH4+) seemed to occur as a response to carbon starvation after rapid glucose depletion. The NH4+ in the extracellular fluid reappeared as soon as glucose was depleted, and an almost complete loss of CO2 was observed, suggesting that an alternative energy source was generated by self-proteolysis of cell proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium.

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.

Effect of Environmental Conditions on Extracellular Protease Activity in Lignolytic Cultures of Phanerochaete chrysosporium.

Two different types of extracellular protease activity were identified in the culture fluid of Phanerochaete chrysosporium wild-type BKM-F grown in submerged batch culture on N-limited media. The first activity, which appears to be inherent to the active growth phase, displayed a maximum on day 2 and decreased to a very low level on day 4. The second activity, which appeared at day 8 following the peak of ligninase activity, seems to be characteristic of late secondary metabolism and is stimulated by carbon starvation. Cultures started with half the amount of glucose of other cultures showed a remarkably earlier development of secondary activity. In contrast, the fed-batch addition of glucose started when ligninase activity was at a maximum (day 6) completely repressed secondary protease activity and enhanced ligninase production. The addition of exogenous veratryl alcohol increased the level of secondary protease activity, whereas the oxygen supply pattern significantly affected both the time course and the level of overall proteolytic activity. The addition of phenylmethylsulfonyl fluoride to growing cultures (0, 1, or 6 days) diminished overall protease activity, while it significantly enhanced ligninase activity. In all cases, the time courses of protease and ligninase activities were negatively correlated, indicating that protease activity promotes the decline of ligninase activity in batch culture.