John M. Harkin

Degradation of the lignin model compount springgylglycol-β-guaiacyl ether by Polyporus versicolor and Stereum frustulatum☆

Abstract 1. 1. Model compounds were used to test the hypothesis that phenol-oxidizing enzymes of white-rot fungi can effect depolymerization of lignin. Studies were made of the effects of syringlyglycol-β-guaiacyl ether of (i) whole cultures of Polyporus versicolor and Stereum frustulatum , (ii) culture filtrates of the former, and (iii) a p -diphenol oxidase ( p -diphenol:O 2 oxidoreductase, EC 1.10.3.2, formerly known as laccase) purified from such filtrates. The fungi were known from previous studies to decompose lignin in wood, but to differ markedly in their production of phenol-oxidizing enzymes. 2. 2. In culture filtrates of P. versicolor , the benzyl alcohol group of syringlyglycol-β-guaiacyl ether was oxidized to a carbonyl group, giving α-guaiacoxyacetosyringone; the same transformation occured in whole cutlures of S. frustulatum . The alkyl-phenyl carbon-to-carbon bond in both syringlyglycol-β-guaiacyl ether and α-guaiacoxyacetosyringone was cleaved by culture filtrates of P. versicolor with formation of guaiacoxyacetaldehyde and guaiacoxyacetic acid, respectively. The syringyl moieties of both parent compounds were converted to 2,6-dimethoxy- p -benzoquinone by culture filtrates of P. versicolor and by whole cultures of S. frustulatum . p -Diphenol oxidase purified from cultures filtrates of P. versicolor effected all of the above reaction. Since neither the filtrates nor the p -diphenol oxidase had any effect on syringlyglycol-β-guaiacyl ether or α-guaiacoxyacetosyringone in a nitrogen atmosphere, the above alkyl-phenyl cleavage reactions of these compounds apparently were oxidative. 3. 3. Whole cultures of P. versicolor and S. frustulatum reduced guaiacoxyacetaldehyde and guaiacoxyacetic acid to 2-guaiacoxyethanol, which accumulated in the cultures and was resistant to further alteration by either organism. The ethanol derivative was also produced from syringlyglycol-β-guaiacyl ether and α-guaiacoxyacetosyringone by both fungi, indicating that the above alkyl-phenyl cleavages also were affected by whole cultures. 4. 4. These experiments indicate that oxidative splitting of the alkyl-phenyl bond in certain phenylpropanoid monomers by phenol oxidases may be important in the depolymerization of lignin by white-rot fungi. A schematic model for sprucelignin was used to estimate that approx. 41% of the phenylpropanoid units in spruce lignin theoretically are vulnerable to the types of cleavage described in this paper.

Mammalian mitochondria as in vitro monitors of water quality.

To measure acute toxicity effectively, a system must provide a simple, sensitive, and rapid measurement of physiological parameters which are indicative of overall organism (bacteria) or organelle (mitochondria) viability. To detect a broad spectrum of toxicants, the parameters should be associated with a major metabolic process controlled by interdependent enzyme systems. In the bioassay reported here, phosphorylating submitochondrial particles (SMP), prepared by sonic disruption of the heavy fraction of intact bovine heart mitochondria serve as in vitro monitors of aquatic toxicity. The bioassay is based on the phenomenon of energy-coupled reverse electron transfer (RET), discovered in plant mitochondria, and later explored using SMP preparations from mammalian sources. RET responses permit rapid, simple, and sensitive measurement of acute toxicity by spectrophotometric recording of the rate of NAD reduction. The criterion of toxicity in this test is inhibition of NAD reduction in the presence of toxic substances. The RET reaction was chosen over other methods because responses are easily quantifiable and the energy-coupled RET reaction is competent in reconstituted freeze-dried submitochondrial particles. Thus, SMP - a stable biological preparation- can be distributed to other laboratories. This feature is important in selecting a routine bioassay.

Assessment of chemical toxicity using mammalian mitochondrial electron transport particles

New spectrophotometric bioassay procedures have been developed for evaluating chemical toxicity, using electron transport particles isolated from bovine heart mitochondria, based on the ability of many toxic chemicals to interfere with the integrated function of electron transport enzymes. The sensitivity of the mitochondrial assays is compared to published sensitivities of otherin vivo andin vitro toxicity testing methods. Regression analysis of logarithmically transformed toxicity values for 42 chemicals, including 8 pesticides, 5 drugs, 6 metals, 8 alcohols, 5 respiratory inhibitors, 4 phenols, and 2 phthalates, indicates excellent correlation between the sensitivity of the new assays and the sensitivity of mammalian cytotoxicity studies (r2 =0.86). Data from aquatic exposure toxicity tests conducted in fish are also highly correlated with the mitochondrial assay results (r2=0.79). However, correlation of data from these methods with median lethal dose studies conducted in rats is not as good because of the inability ofin vitro and aquatic exposure analyses to account for the gastrointestinal absorption, hepatic metabolism, and excretion processes which modify toxic responses following oral administration.

Use of submitochondrial particles for prediction of chemical toxicity in man

The mitochondrial tests are good indicators of the relative toxicity of many xenobiotics to cells and fish. The current investigation studies the ability of these procedures to predict in vivo tissue concentrations associated with clinical illness in man

Transport modelling of biological tracers from septic systems

The potential of contamination of individual ground water supplies from septic systems was investigated. Chloride (Cl) and electrical conductivity (Ce) as tracers of chemicals and indicator bacteria—total coliforms (TC), fecal coliforms (FC) and fecal streptococci (FS)—were determined for 7 months in septic tank effluent and ground water samples collected downgradient from 17 new or replacement septic systems. Indicator bacteria were also determined occasionally in ground water samples from wells near the systems for a continuing period of 2 years. Following 5 years of system operation a last round of analyses was conducted. Water table fluctuations in the vicinity of the septic systems were monitored for 2 years. As a model for viruses, poliovirus in feces from babies who had received the trivalent Sabin oral poliovirus vaccine was introduced into one septic system as a single dose by flushing stools down the toilet. Effluent and ground water were assayed for poliovirus over a period of approx. 6 months. The data were evaluated statistically and a biological contaminant transport model was tested to simulate ground water contamination by the tracers of pathogens. No tracer bacteria reached ground water from any of the 17 septic systems. Presumably, the bacteria were removed by the soil under the seepage bed; mean transport was < 1 count/100 ml for each bacterial type, in keeping with U.S. drinking water regulations. Poliovirus entered and spread in ground water from the one septic system tested, even though this system was functioning properly. Of 3.3 × 108 PFU of poliovirus inoculated into the septic tank, a mean of 70 PFU/100 ml escaped from the tank with the effluent and an overall mean of 62 PFU/100 ml were transported to ground water. Septic systems had little influence on water table fluctuations in their vicinity, compared to the seasonal climatic factors.

Methyl isothiocyanate volatilization from fields treated with metam-sodium

Emission of methyl isothiocyanate (MITC) from fields treated with metam-sodium (sodium N-methyldithiocarbamate) is a potential environmental and human safety hazard. Concentrations of MITC at three heights above four arable fields were measured following the application of metam- sodium at a rate of 480 litre ha ˇ1 (166 kg AI ha ˇ1 ). Two of these fields were treated by injection into a center-pivot irrigation system (chemigation), while in the other two fields the fumigant was applied through injection directly into the soil. Generally, higher MITC air concentrations were observed above chemigated than above injected fields. Maximum MITC air concentrations were 11.2 and 7.4 mg m ˇ3 recorded 10cm above ground 6-8 h following application and the minimum concentrations were 0.7 and 0.2 m gm ˇ 3 observed at 200 cm 30-35h after application above chemigated and injected fields, respectively. The estimated MITC respiratory exposure a worker might encounter during the re-entry period ranged between 1.37 and 0.03 mg day ˇ1 in chemigated fields and between 0.35 and 0.02mg day ˇ1 in the injected fields. These results suggest that application of the fumigant through injection reduced MITC volatilization losses in comparison with the chemigation method, thus posing a relatively lower risk of exposure to MITC emissions. # 2000 Society of Chemical Industry

Competitive touchdown PCR for estimation of Escherichia coli DNA recovery in soil DNA extraction.

Competitive approaches have shown promise for overcoming some of the difficulties in the use of PCR for assessment of specific bacterial species in soil. A competitive touchdown PCR (cTD-PCR) protocol specific for the rrsB gene of Escherichia coli was developed for tracking the organism in environments impacted by human wastes. Regression of product ratios from co-amplification of varying amounts of analyte and competitor DNA templates was linear. To test the robustness of the method, reactions were titrated with an extract of sterilized soil; no significant effect was detected. The cTD-PCR was used to assay recovery of E. coli DNA from soil. Stock DNA was spiked onto two sterilized soils during extraction, and the purified extracts were analyzed by cTD-PCR. Recovery of DNA spiked at a rate of 180 ng g(-1) was 34+/-7% (mean+/-S.D.) for an agricultural silt loam. DNA spiked at 1.8 pg g(-1) was recovered at a mean rate of 6.1+/-1.3%. DNA in these extracts was not directly quantifiable by image analysis. The cTD-PCR method provides a useful means of quantifying small amounts of E. coli DNA, and could be modified for other specific targets in a mixture of DNA from a variety of organisms.

Leaching of Methyl Isothiocyanate in Plainfield Sand Chemigated with Metam-Sodium

Soil column studies were undertaken to investigate the influence of soil water content and irrigation on leaching, distribution and persistence of methyl isothiocyanate (MITC) in a sandy soil chemigated with the soil fumigant metam-sodium. No leaching was obtained from columns at low water content (0.042 or 0.074 cm 3 cm -3 ). However, 8.4 (±2.8), 34.2 (±7.4) and 119.4 (±8.3) μg of MITC leached from columns at 0.105, 0.137 and 0.168 cm 3 cm -3 water content, respectively. Increased leaching resulted from sprinkler application of 25-mm of water to columns at 0.137 cm 3 cm -3 water content. Leaching of MITC constituted only a small fraction of the amount applied even in the worst case. Methyl isothiocyanate persisted in soil for 15 days at 2°C in varying amounts under the different water regimes. Relatively high amounts of MITC residues (8-12 mg kg -1 soil) were detected in the top 25-cm layer of all the soil columns. Degradation was the major pathway of dissipation for the chemical despite the soil water regime.

Recovery of fermentable sugars from process vegetable wastewaters

Abstract The U.S. fruit and vegetable processing industry annually generates approximately 11 million t of byproduct wastes along with over 430 × 109 l of effluent wastewaters. Appropriate disposal of these wasteflows is a costly burden to the generating industries. By selective processing of certain of these wasteflows through reverse osmosis (RO) facilities, approximately 1.42 million t of fermentable sugars could be recovered as a 20% sugar concentrate suitable for bioconversion to useful liquid fuels. The annual fuel alcohol potential of these sugars is between 750 and 900 million l. Economic analysis indicates that overall alcohol production costs should average only 40% of current FOB prices for U.S. bulk fuel alcohol. The coincident major reduction in wastewater BOD and associated disposal cost, and the recovery of over 43 × 109 l of reusable RO permeate water afford added incentives for industrial participation in the production of sugar concentrates from fruit and vegetable byproduct wasteflows.

Influence of detergent formulation on nutrient movement through sand columns simulating mound and conventional septic system drainfields

Abstract The effects of phosphate (P) and zeolite (Z) -built detergents on leaching of N and P through sand columns simulating septic system drainfields were examined in laboratory columns. To simulate mound septic system drainfields, paired sets of columns were dosed intermittently with septic tank effluent from households using P- or Z-built detergent. Two other paired sets of columns were flooded with P- or Z-effluent to simulate new conventional septic system drainfields; after clogging mats or “crusts” developed at infiltration surface, the subsurfaces of the columns were aerated to simulate mature (crusted) conventional septic system drainfields. NO 3 loading in leachate was 1.1 times higher and ortho-P loading was 4.3 times lower when columns were dosed with Z- than with P-effluent. Dosed columns removed P poorly; total phosphorus (TP) loading in leachate was 81 and 19 g m −2 yr −1 with P- and Z-effluent, respectively. In flooded columns 1.3, 2.0 and 1.8 times more NH 4 , organic nitrogen (ON) and total nitrogen (TN) respectively, were leached with Z- than with P-effluent; NO 3 leaching was similar. Flooded columns removed P efficiently; TP leached through flooded systems was 2.5 and 1.4 g m −2 yr −1 with P- and Z effluent, respectively. Crusted columns fed Z-effluent leached 1.2, 2.6, 1.4 and 2.1 times more NH 4 , NO 3 , ON and TN, respectively, than those with P-effluent but 1.8 times less TP. Crusted columns removed P satisfactorily: 8.2 and 4.6 g m −2 yr −1 TP with P- and Z-effluent, respectively. The P-built detergent substantially improves the efficiency of N removal with satisfactory P removal in columns simulating conventional septic system drainfield. Simultaneous removal of N and P under flooded conditions might be explained by precipitation of struvite-type minerals. Dosed system drainfields were less efficient in removing N and P compared to flooded and crusted system drainfelds.