Jim A. Field

Screening for ligninolytic fungi applicable to the biodegradation of xenobiotics

Woody tissues are composed mainly of three biopoly- mers: cellulose; hemicellulose; and lignin. Lignin, a highly irregular aromatic polymer which serves to pro- vide strength and structure to the tissue, is synthesized in plants by a random peroxidase-catalysed polym- erization of substituted p-hydroxy-cinnamyl alcohols. Only a few groups of microorganisms are capable of degrading complex lignin polymers, and they are best exemplified by the white-rot fungi, which cause the greatest degree of mineralization. The white-rot fungus

Enhanced biodegradation of aromatic pollutants in cocultures of anaerobic and aerobic bacterial consortia

Toxic aromatic pollutants, concentrated in industrial wastes and contaminated sites, can potentially be eliminated by low cost bioremediation systems. Most commonly, the goal of these treatment systems is directed at providing optimum environmental conditions for the mineralization of the pollutants by naturally occurring microflora. Electrophilic aromatic pollutants with multiple chloro, nitro and azo groups have proven to be persistent to biodegradation by aerobic bacteria. These compounds are readily reduced by anaerobic consortia to lower chlorinated aromatics or aromatic amines but are not mineralized further. The reduction increases the susceptibility of the aromatic molecule for oxygenolytic attack. Sequencing anaerobic and aerobic biotreatment steps provide enhanced mineralization of many electrophilic aromatic pollutants. The combined activity of anaerobic and aerobic bacteria can also be obtained in a single treatment step if the bacteria are immobilized in particulate matrices (e.g. biofilm, soil aggregate, etc.). Due to the rapid uptake of oxygen by aerobes and facultative bacteria compared to the slow diffusion of oxygen, oxygen penetration into active biofilms seldom exceeds several hundred micrometers. The anaerobic microniches established inside the biofilms can be applied to the reduction of electron withdrawing functional groups in order to prepare recalcitrant aromatic compounds for further mineralization in the aerobic outer layer of the biofilm.Aside from mineralization, polyhydroxylated and chlorinated phenols as well as nitroaromatics and aromatic amines are susceptible to polymerization in aerobic environments. Consequently, an alternative approach for bioremediation systems can be directed towards incorporating these aromatic pollutants into detoxified humic-like substances. The activation of aromatic pollutants for polymerization can potentially be encouraged by an anaerobic pretreatment step prior to oxidation. Anaerobic bacteria can modify aromatic pollutants by demethylating methoxy groups and reducing nitro groups. The resulting phenols and aromatic amines are readily polymerized in a subsequent aerobic step.

Azo dye decolourisation by anaerobic granular sludge.

The decolourisation of 20 selected azo dyes by granular sludge from an upward-flow anaerobic sludge bed (UASB) reactor was assayed. Complete reduction was found for all azo dyes tested, generally yielding colourless products. The reactions followed first-order kinetics and reaction rates varied greatly between dyes: half-life times ranged from 1 to about 100 h. The slowest reaction rates were found for reactive dyes with a triazine reactive group. There was no correlation between a dyes half-life time and its molecular weight, indicating that cell penetration was probably not an important factor. Since granular sludge contains sulphide, eight dyes were also monitored for direct chemical decolourisation by sulphide. All these dyes were reduced chemically albeit at slower rates than in the presence of sludge at comparable sulphide levels. Increasing sulphide concentrations, even when present in huge excess, stimulated the azo reduction rate. The results indicate that granular sludge can decolourise a broad spectrum of azo dye structures due to non-specific extracellular reactions. Reducing agents (e.g., sulphide) in sludge play an important role. The presence of anaerobic biomass is probably beneficial for maintaining the pools of these reduced compounds.

Biodegradation of selected azo dyes under methanogenic conditions

Biological treatment of wastewaters discharged by the textile industry could potentially be problematic due to the high toxicity and recalcitrance of the commonly-used azo dye compounds. In the present report, the fate of two azo dyes under methanogenic conditions was studied. Mordant Orange 1 (MO1) and Azodisalicylate (ADS) were completely reduced and decolorised in continuous UASB reactors in the presence of cosubstrates. In the MO1 reactor, both 5-aminosalicylic acid (5-ASA) and 1,4-phenylenediamine were identified as products of azo cleavage. After long adaptation periods, 5-ASA was detected at trace levels, indicating further mineralization. ADS, a pharmaceutical azo dye constructed from two 5-ASA units, was completely mineralized even in the absence of cosubstrate, indicating that the metabolism of 5-ASA could provide the reducing equivalents needed for the azo reduction. Batch experiments confirmed the ADS mineralization. These results demonstrate that some azo dyes could serve as a carbon, energy, and nitrogen source for anaerobic bacteria.

Increasing ligninolytic enzyme activities in several white-rot basidiomycetes by nitrogen-sufficient media

Ligninolytic enzyme activities were monitored in five white-rot fungi cultivated on nitrogen (N)-limited glucose-BIII medium and were compared with the activities obtained in media supplemented with 56 mm peptone-N. Only Phanerochaete chrysosporium and two Bjerkandera strains produced detectable lignin peroxidase (LiP) activity. LiP was stimulated by either N-limitation or N-sufficiency in P. chrysosporium and Bjerkandera spp., respectively. All of the fungal strains tested produced manganese-dependent peroxidase (MnP) activity, which was consistently stimulated by the peptone supplementation. Both the manganese-independent peroxidase (MiP) activities, which were detected only in Bjerkandera spp., and the laccase activities, which were detected only in Lentinula edodes and Pleurotus ostreatus, were also enhanced by peptone. In several fungal strains, the activity of ligninolytic enzymes was likewise stimulated by supplying 56 mm NH+4-N at an initial pH of 7·3. This study indicates that several commercially important and commonly occurring white-rot fungi produce higher ligninolytic enzyme activities in response to a nitrogen-rich medium, in contrast to the physiological model proposed for P. chrysosporium.

Advanced anaerobic wastewater treatment in the near future

New insights into the anaerobic degradation of very different categories of compounds, and into process and reactor technology will lead to very promising new generations of anaerobic treatment system, such as ‘Expanded Granular Sludge Bed’ (EGSB) and ‘Staged Multi-Phase Anaerobic’ (MPSA) reactor systems. These concepts will provide a higher efficiency at higher loading rates, are applicable for extreme environmental conditions (e.g. low and high temperatures) and to inhibitory compounds. Moreover, by integrating the anaerobic process with other biological methods (sulphate reduction, micro-aerophilic organisms) and with physical-chemical methods, a complete treatment of the wastewater can be accomplished at very low costs, while at the same time valuable components can be recovered for reuse.

Continuous detoxification, transformation, and degradation of nitrophenols in upflow anaerobic sludge blanket (UASB) reactors.

The anaerobic transformation and degradation of nitrophenols by granular sludge was investigated in upflow anaerobic sludge blanket (UASB) reactors continuously fed with a volatile fatty acid (VFA) mixture as the primary substrate. During the start‐up, subtoxic concentrations of 2‐nitrophenol (2‐NP), 4‐nitrophenol (4‐NP), and 2, 4‐dinitrophenol (2, 4‐DNP) were utilized. 4‐NP and 2, 4‐DNP were readily converted to the corresponding aromatic amine; whereas 2‐NP was converted to nonaromatic products via intermediate formation of 2‐aminophenol (2‐AP). These conversions led to a dramatic detoxification of the mononitrophenols because the reactors treated the nitrophenolics at the concentrations which were over 25 times higher than those that caused severe inhibition. VFA removal efficiencies greater than 99% were achieved in both reactors at loading rates greater than 11.4 g COD per liter of reactor volume per day even at volumetric loading of mononitrophenols up to 910 mg/L · d.

Biodegradation of azo dyes in cocultures of anaerobic granular sludge with aerobic aromatic amine degrading enrichment cultures

Abstract A prerequisite for the mineralization (complete biodegradation) of many azo dyes is a combination of reductive and oxidative steps. In this study, the biodegradation of two azo dyes, 4-phenylazophenol (4-PAP) and Mordant Yellow 10 (4-sulfophenylazo-salicylic acid; MY10), was evaluated in batch experiments where anaerobic and aerobic conditions were integrated by exposing anaerobic granular sludge to oxygen. Under these conditions, the azo dyes were reduced, resulting in a temporal accumulation of aromatic amines. 4-Aminophenol (4-AP) and aniline were detected from the reduction of 4-PAP. 5-Aminosalicylic acid (5-ASA) and sulfanilic acid (SA) were detected from the reduction of MY10. Subsequently, aniline was degraded further in the presence of oxygen by the facultative aerobic bacteria present in the anaerobic granular sludge. 5-ASA and SA were also degraded, if inocula from aerobic enrichment cultures were added to the batch experiments. Due to rapid autoxidation of 4-AP, no enrichment culture could be established for this compound. The results of this study indicate that aerobic enrichment cultures developed on aromatic amines combined with oxygen-tolerant anaerobic granular sludge can potentially be used to completely biodegrade azo dyes under integrated anaerobic/aerobic conditions.

Fate and biodegradability of sulfonated aromatic amines

Ten sulfonated aromatic amines were tested for their aerobic and anaerobic biodegradability and toxicity potential in a variety of environmental inocula. Of all the compounds tested, only two aminobenzenesulfonic acid (ABS) isomers, 2- and 4-ABS, were degraded. The observed degradation occurred only under aerobic conditions with inocula sources that were historically polluted with sulfonated aromatic amines. Bioreactor experiments, with non-sterile synthetic wastewater, confirmed the results from the aerobic batch degradation experiments. Both ABS isomers were degraded in long-term continuous experiment by a␣bioaugmented enrichment culture. The maximum degradation rate in the aerobic bioreactor was 1.6–1.8 gl−1 d−1 for 2-ABS and a somewhat lower value for 4-ABS at hydraulic retention times (HRT) of 2.8–3.3h. Evidence for extensive mineralization of 2- and 4-ABS was based on oxygen uptake and carbon dioxide production during the batch experiments and the high levels of chemical oxygen demand (COD) removal in the bioreactor. Furthermore, mineralization of the sulfonate group was demonstrated by high recovery of sulfate. The sulfonated aromatic amines did not show any toxic effects on the aerobic and anaerobic bacterial populations tested. The poor biodegradability of sulfonated aromatic amines indicated under the laboratory conditions of this study suggests that these compounds may not be adequately removed during biological wastewater treatment.

Detoxification and partial mineralization of the azo dye mordant orange 1 in a continuous upflow anaerobic sludge-blanket reactor

Abstract In batch toxicity assays, azo dye compounds were found to be many times more toxic than their cleavage products (aromatic amines) towards methanogenic activity in anaerobic granular sludge. Considering the ability of anaerobic microorganisms to reduce azo groups, detoxification of azo compounds towards methanogens can be expected to occur during anaerobic wastewater treatment. In order to test this hypothesis, the anaerobic degradation of one azo dye compound, Mordant orange 1 (MO1), by granular sludge was investigated in three separate continuous upflow anaerobic sludge-blanket reactors. One reactor, receiving no cosubstrate, failed after 50 days presumably because of a lack of reducing equivalents. However, the two reactors receiving either glucose or a volatile fatty acids (acetate, propionate, butyrate) mixture, could eliminate the dye during operation for 217 days. The azo dye was reductively cleaved to less toxic aromatic amines (1,4-phenylenediamine and 5-aminosalicylic acid) making the treatment of MO1 feasible at influent concentrations that were over 25 times higher than their 50% inhibitory concentrations. In the reactor receiving glucose as cosubstrate, 5-aminosalicylic acid could only be detected at trace levels in the effluent after day 189 of operation. Batch biodegradability assays with the sludge sampled from this reactor confirmed the mineralization of 5-aminosalicylic acid to methane.