Sulfide oxidation at halo-alkaline conditions in a fed-batch bioreactor

A biotechnological process is described to remove hydrogen sulfide (H(2)S) from high-pressure natural gas and sour gases produced in the petrochemical industry. The process operates at halo-alkaline conditions and combines an aerobic sulfide-oxidizing reactor with an anaerobic sulfate (SO(4) (2-)) and thiosulfate (S(2)O(3) (2-)) reducing reactor. The feasibility of biological H(2)S oxidation at pH around 10 and total sodium concentration of 2 mol L(-1) was studied in gas-lift bioreactors, using halo-alkaliphilic sulfur-oxidizing bacteria (HA-SOB). Reactor operation at different oxygen to sulfide (O(2):H(2)S) supply ratios resulted in a stable low redox potential that was directly related with the polysulfide (S(x) (2-)) and total sulfide concentration in the bioreactor. Selectivity for SO(4) (2-) formation decreased with increasing S(x) (2-) and total sulfide concentrations. At total sulfide concentrations above 0.25 mmol L(-1), selectivity for SO(4) (2-) formation approached zero and the end products of H(2)S oxidation were elemental sulfur (S(0)) and S(2)O(3) (2-). Maximum selectivity for S(0) formation (83.3+/-0.7%) during stable reactor operation was obtained at a molar O(2):H(2)S supply ratio of 0.65. Under these conditions, intermediary S(x) (2-) plays a major role in the process. Instead of dissolved sulfide (HS(-)), S(x) (2-) seemed to be the most important electron donor for HA-SOB under S(0) producing conditions. In addition, abiotic oxidation of S(x) (2-) was the main cause of undesirable formation of S(2)O(3) (2-). The observed biomass growth yield under SO(4) (2-) producing conditions was 0.86 g N mol(-1) H(2)S. When selectivity for SO(4) (2-) formation was below 5%, almost no biomass growth was observed.     

Hydrogen sulfide protects astrocytes against H(2)O(2)-induced neural injury via enhancing glutamate uptake

Excess extracellular glutamate, the main excitatory neurotransmitter, may result in excitotoxicity and neural injury. The present study was designed to study the effect of hydrogen sulfide (H(2)S), a novel neuromodulator, on hydrogen peroxide (H(2)O(2)) -induced glutamate uptake impairment and cellular injuries in primary cultured rat cortical astrocytes. We found that NaHS (an H(2)S donor, 0.1-1000 microM) reversed H(2)O(2)-induced cellular injury in a concentration-dependent manner. This effect was attenuated by L-trans-pyrrolidine-2,4-dicarboxylic (PDC), a specific glutamate uptake inhibitor. Moreover, NaHS significantly increased [(3)H]glutamate transport in astrocytes treated with H(2)O(2), suggesting that H(2)S may protect astrocytes via enhancing glutamate uptake function. NaHS also reversed H(2)O(2)-impaired glutathione (GSH) production. Blockade of glutamate uptake with PDC attenuated this effect, indicating that the effect of H(2)S on GSH production is secondary to the stimulation of glutamate uptake. In addition, it was also found that H(2)S may promote glutamate uptake activity via decreasing ROS generation, enhancing ATP production and suppressing ERK1/2 activation. In conclusion, our findings provide direct evidence that H(2)S has potential therapeutic value for oxidative stress-induced brain damage via a mechanism involving enhancing glutamate uptake function.     

Effects and mechanisms of H(2)O(2) on production of dicarboxylic acid.

The system of producing long chain dicarboxylic acid (DCA) by Candida tropicalis is an aerobic and viscous fermentation system. A method to overcome the gas-liquid transport resistance and to increase oxygen supply is by adding hydrogen peroxide (H(2)O(2)) to the fermentation system. Here we report that the H(2)O(2) not only can enhance the oxygen supply but also change the metabolism by inducing cytochrome P450, the key enzyme of a, o-oxidation. When C. tropicalis was cultivated in a 3-L bioreactor using the combination of aeration and H(2)O(2) feeding, DCA production rates increased by about 10% after a short period of decrease at the beginning. Furthermore, the experiments showed that the maximum activities of P450 could be induced at 2 mM H(2)O(2), and the inducible mechanisms are also discussed. Moreover, we suggest that alkane might be oxidized through the "peroxide shunt pathway" when H(2)O(2) is present. By adding H(2)O(2), the DCA yield in a 22-L bioreactor could increase by 25.3% and reach 153.9 g/L.     

Regulation of T-cell activation and T-cell growth factor (TCGF) production by hydrogen peroxide

Activated macrophages are known to release a variety of immunoregulatory substances including the low-molecular-weight substances hydrogen peroxide and lactate. We report here that lactate but not hydrogen peroxide is capable of supporting a substantial production of T-cell growth factor (TCGF) in cultures of accessory cell-depleted splenic T-cell populations after stimulation with concanavalin A. Hydrogen peroxide and its biosynthetic precursor superoxide anion (O2-) mediate, however, a strong augmentation of the TCGF production by accessory cell-depleted T-cell populations in the presence of lactate. Lactate inhibits the incorporation of [3H]thymidine in short-term cultures (18-26 hr) of accessory cell-depleted T cells. This confirms the rule that (optimal) production of T-cell growth factor requires a growth inhibitory signal. Concentrations of hydrogen peroxide which augment TCGF production most effectively (i.e., 1 X 10(-5) M) do not inhibit the incorporation of [3H]thymidine; and higher concentrations (3 X 10(-5)-1 X 10(-4) M) of hydrogen peroxide inhibit both the production of TCGF and the incorporation of [3H]thymidine. In agreement with the augmenting effect of hydrogen peroxide on TCGF production, it was observed that the proliferative response in mixed lymphocyte cultures is suppressed by catalase and augmented by 1 X 10(-5) M H2O2. Proliferative and cytotoxic responses in mixed lymphocyte cultures with an external source of interleukin 2 (IL-2) in contrast, are not augmented by 1 X 10(-5) M H2O2. The relatively high concentration of 1 X 10(-4) M hydrogen peroxide was found to inhibit the proliferative responses in mixed lymphocyte cultures with or without external IL-2 but not the cytotoxic response in the presence of IL-2. This indicates that CTL precursor cells may be relatively resistant against H2O2.     

Cells with dysfunctional telomeres are susceptible to reactive oxygen species hydrogen peroxide via generation of multichromosomal fusions and chromosomal fragments bearing telomeres

During genotoxic stress, reactive oxygen species hydrogen peroxide (H(2)O(2)) is a prime mediator of the DNA damage response. Telomeres function both to assist in DNA damage repair and to inhibit chromosomal end-to-end fusion. Here, we show that telomere dysfunction renders cells susceptible to H(2)O(2), via generation of multichromosomal fusion and chromosomal fragments. H(2)O(2) caused formation of multichromosomal end-to-end fusions involving more than three chromosomes, preferentially when telomeres were erosive. Interestingly, extensive chromosomal fragmentation (yielding small-sized fragments) occurred only in cells exhibiting such multichromosomal fusions. Telomeres were absent from fusion points, being rather present in the small fragments, indicating that H(2)O(2) cleaves chromosomal regions adjacent to telomeres. Restoration of telomere function or addition of the antioxidant N-acetylcysteine prevented development of chromosomal aberrations and rescued the observed hypersensitivity to H(2)O(2). Thus, chromosomal regions adjacent to telomeres become sensitive to reactive oxygen species hydrogen peroxide when telomeres are dysfunctional, and are cleaved to produce multichromosomal fusions and small chromosomal fragments bearing the telomeres.     

Urea hydrogen peroxide reduces the numbers of lactobacilli, nourishes yeast, and leaves no residues in the ethanol fermentation

Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five Lactobacillus spp. (Lactobacillus plantarum, L. paracasei, Lactobacillus sp. strain 3, L. rhamnosus, and L. fermentum) from approximately 10(7) to approximately 10(2) CFU/ml in a 2-h preincubation at 30 degrees C of normal-gravity wheat mash at approximately 21 g of dissolved solids per ml containing normal levels of suspended grain particles. Fermentation was completed 36 h after inoculation of Saccharomyces cerevisiae in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with L. paracasei at approximately 10(7) CFU/ml. There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added). However, the presence of L. paracasei at approximately 10(7) CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control. The bactericidal activity of UHP is greatly affected by the presence of particulate matter. In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present. No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30 degrees C whether the bactericidal agent was added as H(2)O(2) or as urea hydrogen peroxide. NADH peroxidase activity (involved in degrading H(2)O(2)) increased significantly (P = 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H(2)O(2). H(2)O(2)-resistant mutants were not expected or found when lethal levels of H(2)O(2) or UHP were used. Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca. 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast.     

Environmental influences on competitive hydrogen peroxide production in Streptococcus gordonii

Streptococcus gordonii is an important member of the oral biofilm. One of its phenotypic traits is the production of hydrogen peroxide (H2O2). H2O2 is an antimicrobial component produced by S. gordonii that is able to antagonize the growth of cariogenic Streptococcus mutans. Strategies that modulate H2O2 production in the oral cavity may be useful as a simple therapeutic mechanism to improve oral health, but little is known about the regulation of H2O2 production. The enzyme responsible for H2O2 production is pyruvate oxidase, encoded by spxB. The functional studies of spxB expression and SpxB abundance presented in this report demonstrate a strong dependence on environmental oxygen tension and carbohydrate availability. Carbon catabolite repression (CCR) modulates spxB expression carbohydrate dependently. Catabolite control protein A (CcpA) represses spxB expression by direct binding to the spxB promoter, as shown by electrophoretic mobility shift assays (EMSA). Promoter mutation studies revealed the requirement of two catabolite-responsive elements (CRE) for CcpA-dependent spxB regulation, as evaluated by spxB expression and phenotypic H2O2 production assays. Thus, molecular mechanisms for the control of S. gordonii spxB expression are presented for the first time, demonstrating the possibility of manipulating H2O2 production for increased competitive fitness.     

Green tea polyphenol blocks h(2)o(2)-induced interleukin-8 production from human alveolar epithelial cells

Reactive oxygen species (ROS) play crucial roles in ischemia-reperfusion (IR) injury of lung transplants. Reactive oxygen species may stimulate the production of neutrophil chemotactic factors such as interleukin-8 (IL-8), from alveolar epithelial cells, causing recruitment and activation of neutrophils in the reperfused tissue. Green tea polyphenol has potent anti-oxidative activities and anti-inflammatory effects by decreasing cytokine production. In the present study, we found that green tea polyphenol significantly inhibited IL-8 production induced by hydrogen peroxide (H(2)O(2)) in human lung alveolar epithelial cells (A549 line). It has been shown that mitogen activated protein kinases, such as Jun N-terminal kinase (JNK), p38 and p44/42, could mediate IL-8 production from a variety of cell types. We further investigated the effect of green tea polyphenol on these protein kinases, and demonstrated that H(2)O(2)-induced phosphorylation of JNK and p38 but not p44/42 was inhibited by green tea polyphenol in A549 cells. We speculate that green tea polyphenol may inhibit H(2)O(2)-induced IL-8 production from A549 cells through inactivation of JNK and p38.     

Factors contributing to hydrogen peroxide resistance in Streptococcus pneumoniae include pyruvate oxidase (SpxB) and avoidance of the toxic effects of the fenton reaction

Aerobic growth of Streptococcus pneumoniae results in production of amounts of hydrogen peroxide (H(2)O(2)) that may exceed 1 mM in the surrounding media. H(2)O(2) production by S. pneumoniae has been shown to kill or inhibit the growth of other respiratory tract flora, as well as to have cytotoxic effects on host cells and tissue. The mechanisms allowing S. pneumoniae, a catalase-deficient species, to survive endogenously generated concentrations of H(2)O(2) that are sufficient to kill other bacterial species is unknown. In the present study, pyruvate oxidase (SpxB), the enzyme responsible for endogenous H(2)O(2) production, was required for survival during exposure to high levels (20 mM) of exogenously added H(2)O(2). Pretreatment with H(2)O(2) did not increase H(2)O(2) resistance in the mutant, suggesting that SpxB activity itself is required, rather than an H(2)O(2)-inducible pathway. SpxB mutants synthesized 85% less acetyl-phosphate, a potential source of ATP. During H(2)O(2) exposure, ATP levels decreased more rapidly in spxB mutants than in wild-type cells, suggesting that the increased killing of spxB mutants was due to more rapid ATP depletion. Together, these data support the hypothesis that S. pneumoniae SpxB contributes to an H(2)O(2)-resistant energy source that maintains viability during oxidative stress. Thus, SpxB is required for resistance to the toxic by-product of its own activity. Although H(2)O(2)-dependent hydroxyl radical production and the intracellular concentration of free iron were similar to that of Escherichia coli, killing by H(2)O(2) was unaffected by iron chelators, suggesting that S. pneumoniae has a novel mechanism to avoid the toxic effects of the Fenton reaction.     

Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans

Biofilms are polymicrobial, with diverse bacterial species competing for limited space and nutrients. Under healthy conditions, the different species in biofilms maintain an ecological balance. This balance can be disturbed by environmental factors and interspecies interactions. These perturbations can enable dominant growth of certain species, leading to disease. To model clinically relevant interspecies antagonism, we studied three well-characterized and closely related oral species, Streptococcus gordonii, Streptococcus sanguinis, and cariogenic Streptococcus mutans. S. sanguinis and S. gordonii used oxygen availability and the differential production of hydrogen peroxide (H(2)O(2)) to compete effectively against S. mutans. Interspecies antagonism was influenced by glucose with reduced production of H(2)O(2). Furthermore, aerobic conditions stimulated the competence system and the expression of the bacteriocin mutacin IV of S. mutans, as well as the H(2)O(2)-dependent release of heterologous DNA from mixed cultures of S. sanguinis and S. gordonii. These data provide new insights into ecological factors that determine the outcome of competition between pioneer colonizing oral streptococci and the survival mechanisms of S. mutans in the oral biofilm.     

Hydrogen peroxide promotes injury-induced peripheral sensory axon regeneration in the zebrafish skin

Functional recovery from cutaneous injury requires not only the healing and regeneration of skin cells but also reinnervation of the skin by somatosensory peripheral axon endings. To investigate how sensory axon regeneration and wound healing are coordinated, we amputated the caudal fins of zebrafish larvae and imaged somatosensory axon behavior. Fin amputation strongly promoted the regeneration of nearby sensory axons, an effect that could be mimicked by ablating a few keratinocytes anywhere in the body. Since injury produces the reactive oxygen species hydrogen peroxide (H(2)O(2)) near wounds, we tested whether H(2)O(2) influences cutaneous axon regeneration. Exposure of zebrafish larvae to sublethal levels of exogenous H(2)O(2) promoted growth of severed axons in the absence of keratinocyte injury, and inhibiting H(2)O(2) production blocked the axon growth-promoting effects of fin amputation and keratinocyte ablation. Thus, H(2)O(2) signaling helps coordinate wound healing with peripheral sensory axon reinnervation of the skin.     

Electrochemical biofilm control: mechanism of action

Although it has been previously demonstrated that an electrical current can be used to control biofilm growth on metal surfaces, the literature results are conflicting and there is no accepted mechanism of action. One of the suggested mechanisms is the production of hydrogen peroxide (H(2)O(2)) on metal surfaces. However, there are literature studies in which H(2)O(2) could not be detected in the bulk solution. This is most likely because H(2)O(2) was produced at a low concentration near the surface and could not be detected in the bulk solution. The goals of this research were (1) to develop a well-controlled system to explain the mechanism of action of the bioelectrochemical effect on 316L stainless steel (SS) surfaces and (2) to test whether the produced H(2)O(2) can reduce cell growth on metal surfaces. It was found that H(2)O(2) was produced near 316L SS surfaces when a negative potential was applied. The H(2)O(2) concentration increased towards the surface, while the dissolved oxygen decreased when the SS surface was polarized to?-600 mV(Ag/AgCl). When polarized and non-polarized surfaces with identical Pseudomonas aeruginosa PAO1 biofilms were continuously fed with air-saturated growth medium, the polarized surfaces showed minimal biofilm growth while there was significant biofilm growth on the non-polarized surfaces. Although there was no detectable H(2)O(2) in the bulk solution, it was found that the surface concentration of H(2)O(2) was able to prevent biofilm growth.     

Superoxide, hydrogen peroxide and singlet oxygen in hematoporphyrin derivative-cysteine, -NADH and -light systems

Hematoporphyrin derivative and light in the presence of cysteine or glutathione were found to convert oxygen to superoxide and hydrogen peroxide at pH less than approx. 6.5, while at pH greater than 6.5 no superoxide or hydrogen peroxide production was observed. However, at pH values greater than 6.5 the rate of oxygen consumption increased. This rate paralleled the acid dissociation curve of the cysteine thiol group and is consistent with the chemical quenching of 1O2 by cysteine. The superoxide and hydrogen peroxide formation observed below pH 6.5 appeared not to be related to the singlet oxygen production of hematoporphyrin derivative. In addition, superoxide and hydrogen peroxide production was observed with hematoporphyrin derivative and light in the presence of NADH, both above and below pH 6.5. Direct detection of singlet oxygen luminescence at 1268 nm in the hematoporphyrin derivative-light system (2H2O as solvent) revealed an apparent linear increase in the singlet oxygen emission intensity as the p2H was raised from 7.0 to 10.0. Azide efficiently quenched this observed emission. In addition, at p2H 7.4, 1 mM cysteine resulted in a 40% reduction of the singlet oxygen luminescence, while at p2H 9.4 the signal was quenched by over 95% (under the experimental conditions employed). In total, we interpret these results as consistent with the chemical quenching of 1O2 by the ionized thiol group of cysteine.     

Cellular mechanism underlying the facilitation of contractile response of vas deferens smooth muscle by sodium orthovanadate

Hydrogen sulfide (H(2)S) has been shown to exert cardioprotective effects. However, the roles of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in H(2)S-induced cardioprotection have not been completely elucidated. In this study, cobalt chloride (CoCl(2)), a chemical hypoxia mimetic agent, was applied to treat H9c2 cells to establish a chemical hypoxia-induced cardiomyocyte injury model. The results showed that pretreatment with NaHS (a donor of H(2)S) before exposure to CoCl(2) attenuated the decreased cell viability, the increased apoptosis rate, the loss of mitochondrial membrane potential (ΔΨm), and the intracellular accumulation of reactive oxygen species (ROS) in H9c2 cells. Exposure of H9c2 cells to CoCl(2) or hydrogen peroxide (H(2)O(2)) upregulated expression of phosphorylated (p) ERK1/2, which was reduced by pretreatment with NaHS or N-acetyl-L-cysteine, a ROS scavenger. More importantly, U0126, a selective inhibitor of ERK1/2, mimicked the above cytoprotection of H(2)S against CoCl(2)-induced injury in H9c2 cells. In conclusion, these results indicate that H(2)S protects H9c2 cells against chemical hypoxia-induced injury partially by inhibiting ROS-mediated activation of ERK1/2.     

High hydrogen peroxide concentration in the feed-zone affects bioreactor cell productivity with liquid phase oxygen supply strategy

Liquid phase oxygen supply strategy (LPOS), in which hydrogen peroxide (H(2)O(2)) is used to supply oxygen to the bioreactor, leads to low cell productivity despite high specific productivities of relevant metabolites. We hypothesized that high H(2)O(2) concentrations in the feed-zone led to local cell death, which in turn, lead to lower cell productivity. To test the hypothesis, a mathematical model was developed. Bacillus subtilis 168 was used as the model system in this study. The model simulations of cell concentrations in the bioreactor-zone were verified with the experimental results. The feed-zone H(2)O(2) concentrations remained 12-14 times higher than bulk bioreactor concentrations. The high local concentrations are expected to cause local cell killing, which explains the decrease in overall cell production by 50% at 300 rpm compared to conventional cultivation. Further, among the four different feed strategies studied using the model, dissolved oxygen (DO) controlled H(2)O(2) feed strategy caused least local cell killing and improved overall cell production by 34%.     

ESR spin-trapping studies on the formation of thiosulfate and sulfide radical anions in aqueous solutions

The first spin-trapping evidence for the formation of thiosulfate (S2O3-.) and sulfide (S-.) radical anions from the reactions of hydrogen peroxide with thiosulphate and sulphide ions, respectively, was presented by electron spin resonance (ESR) spectroscopy using 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS, 1a) as a spin-trap in aqueous solutions. From the facts that the short-lived radical anions, S2O3-. and S-., could be detected during the oxidation with H2O2, it is suggested that these radical anions may become one of the candidates for the toxicity of sulfide ion in the living body.     

The contribution of thioredoxin-2 reductase and glutathione peroxidase to H(2)O(2) detoxification of rat brain mitochondria

Brain mitochondria are not only major producers of reactive oxygen species but they also considerably contribute to the removal of toxic hydrogen peroxide by the glutathione (GSH) and thioredoxin-2 (Trx2) antioxidant systems. In this work we estimated the relative contribution of both systems and catalase to the removal of intrinsically produced hydrogen peroxide (H(2)O(2)) by rat brain mitochondria. By using the specific inhibitors auranofin and 1-chloro-2,4-dinitrobenzene (DNCB), the contribution of Trx2- and GSH-systems to reactive oxygen species (ROS) detoxification in rat brain mitochondria was determined to be 60±20% and 20±15%, respectively. Catalase contributed to a non-significant extent only, as revealed by aminotriazole inhibition. In digitonin-treated rat hippocampal homogenates inhibition of Trx2- and GSH-systems affected mitochondrial hydrogen peroxide production rates to a much higher extent than the endogenous extramitochondrial hydrogen peroxide production, pointing to a strong compartmentation of ROS metabolism. Imaging experiments of hippocampal slice cultures showed on single cell level substantial heterogeneity of hydrogen peroxide detoxification reactions. The strongest effects of inhibition of hydrogen peroxide removal by auranofin or DNCB were detected in putative interneurons and microglial cells, while pyramidal cells and astrocytes showed lower effects. Thus, our data underline the important contribution of the Trx2-system to hydrogen peroxide detoxification in rat hippocampus. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Increased production of hydrogen peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon aeration: involvement of an NADH oxidase in oxidative stress

The growth of Lactobacillus delbrueckii subsp. bulgaricus (L. delbrueckii subsp. bulgaricus) on lactose was altered upon aerating the cultures by agitation. Aeration caused the bacteria to enter early into stationary phase, thus reducing markedly the biomass production but without modifying the maximum growth rate. The early entry into stationary phase of aerated cultures was probably related to the accumulation of hydrogen peroxide in the medium. Indeed, the concentration of hydrogen peroxide in aerated cultures was two to three times higher than in unaerated ones. Also, a similar shift from exponential to stationary phase could be induced in unaerated cultures by adding increasing concentrations of hydrogen peroxide. A significant fraction of the hydrogen peroxide produced by L. delbrueckii subsp. bulgaricus originated from the reduction of molecular oxygen by NADH catalyzed by an NADH:H(2)O(2) oxidase. The specific activity of this NADH oxidase was the same in aerated and unaerated cultures, suggesting that the amount of this enzyme was not directly regulated by oxygen. Aeration did not change the homolactic character of lactose fermentation by L. delbrueckii subsp. bulgaricus and most of the NADH was reoxidized by lactate dehydrogenase with pyruvate. This indicated that NADH oxidase had no (or a very small) energetic role and could be involved in eliminating oxygen.     

Biochemistry of the oxidation of lignin by Phanerochaete chrysosporium

The objective of this research was to identify the biochemical agents responsible for the oxidative degradation of lignin by the white-rot fungus Phanerochaete chrysosporium. We examined the hypothesis that activated oxygen species are involved, and we also sought the agent in ligninolytic cultures responsible for a specific oxidative degradative reaction in substructure model compounds. Results of studies of the production of activated oxygen species by cultures, of the effect of their removal on ligninolytic activity, and of their action on substructure model compounds support a role for hydrogen peroxide (H(2)O(2)) and possibly superoxide (O(2)(*)(-)) in lignin degradation. Involvement of hydroxyl radical (*OH) or singlet oxygen (1O(2)) is not supported by our data. The actual biochemical agent responsible for one important oxidative C-C bond cleavage reaction in non-phenolic lignin substructure model compounds, and in lignin itself, was found to be an enzyme. The enzyme is extracellular, has a molecular weight of 42,000 daltons, is azide-sensitive, and requires H(2)O(2) for activity.