Bioaugmentation potential of free and formulated 2,6-dichlorobenzamide (BAM) degrading Aminobacter sp. MSH1 in soil, sand and water.

Pesticides are used extensively worldwide, which has led to the unwanted contamination of soil and water resources. Former use of the herbicide 2,6-dichlorobenzonitrile (dichlobenil) has caused pollution of ground and surface water resources by the stable degradation product 2,6-dichlorobenzamide (BAM) in several parts of Europe, which has resulted in the costly closure of several drinking water wells. One strategy for preventing this in future is bioaugmentation using bacterial degraders. BAM-degrading Aminobacter sp. MSH1 was therefore formulated into dried beads and tests undertaken to establish their potential for use in the remediation of polluted soil, sand and water. The formulation procedure included freeze drying, combined with trehalose addition for cell wall protection, thus ensuring a high amount of viable cells following prolonged storage at room temperature. The beads were round-shaped pellets with a diameter of about 1.25 mm, a dry matter content of approximately 95 % and an average viable cell content of 4.4 × 10(9) cells/g bead. Formulated MSH1 cells led to a similar, and frequently even faster, BAM mineralisation (20-65 % (14)CO2 produced from (14)C-labelled BAM) in batch tests conducted with sand, water and different soil moisture contents compared to adding free cells. Furthermore, the beads were easy to handle and had a shelf life of several months.

Large-scale bioreactor production of the herbicide-degrading Aminobacter sp. strain MSH1.

The Aminobacter sp. strain MSH1 has potential for pesticide bioremediation because it degrades the herbicide metabolite 2,6-dichlorobenzamide (BAM). Production of the BAM-degrading bacterium using aerobic bioreactor fermentation was investigated. A mineral salt medium limited for carbon and with an element composition similar to the strain was generated. The optimal pH and temperature for strain growth were determined using shaker flasks and verified in bioreactors. Glucose, fructose, and glycerol were suitable carbon sources for MSH1 (µ = 0.1 h(-1)); slower growth was observed on succinate and acetic acid (µ = 0.01 h(-1)). Standard conditions for growth of the MSH1 strain were defined at pH 7 and 25 °C, with glucose as the carbon source. In bioreactors (1 and 5 L), the specific growth rate of MSH1 increased from µ = 0.1 h(-1) on traditional mineral salt medium to µ = 0.18 h(-1) on the optimized mineral salt medium. The biomass yield under standard conditions was 0.47 g dry weight biomass/g glucose consumed. An investigation of the catabolic capacity of MSH1 cells harvested in exponential and stationary growth phases showed a degradation activity per cell of about 3 × 10(-9) µg BAM h(-1). Thus, fast, efficient, large-scale production of herbicide-degrading Aminobacter was possible, bringing the use of this bacterium in bioaugmentation field remediation closer to reality.

Degradation of three benzonitrile herbicides by Aminobacter MSH1 versus soil microbial communities: pathways and kinetics.

BACKGROUND: The herbicide dichlobenil was banned in the European Union after its metabolite 2,6-dichlorobenzamide (BAM) was encountered in groundwater. Owing to structural similarities, bromoxynil and ioxynil might be converted to persistent metabolites in a similar manner. To examine this, we used an indigenous soil bacterium Aminobacter sp. MSH1 which is capable of mineralizing dichlobenil via BAM and 2,6-dichlorobenzoic acid (2,6-DCBA). RESULTS: Strain MSH1 converted bromoxynil and ioxynil to the corresponding aromatic metabolites, 3,5-dibromo-4-hydroxybenzoic acid (BrAC) and 3,5-diiodo-4-hydroxybenzoic acid (IAC) following Michaelis-Menten kinetics (adjusted R(2) between 0.907 and 0.999). However, in contrast to 2,6-DCBA, degradation of these metabolites was not detected in the pure-culture studies, suggesting that they might pose an environmental risk if similar partial degradation occurred in soil. By contrast, experiments with natural soils indicated 20-30% mineralization of ioxynil and bromoxynil within the first week. CONCLUSION: The degradation pathway of the three benzonitriles is initially driven by similar enzymes, after which more specific enzymes are responsible for further degradation. Ioxynil and bromoxynil mineralization in soil is not dependent on previous benzonitrile exposure. The accumulation of dead-end metabolites, as seen for dichlobenil, is not a major problem.

Pretreatment of the macroalgae Chaetomorpha linum for the production of bioethanol--comparison of five pretreatment technologies.

A qualified estimate for pretreatment of the macroalgae Chaetomorpha linum for ethanol production was given, based on the experience of pretreatment of land-based biomass. C. linum was subjected to hydrothermal pretreatment (HTT), wet oxidation (WO), steam explosion (STEX), plasma-assisted pretreatment (PAP) and ball milling (BM), to determine effects of the pretreatment methods on the conversion of C. linum into ethanol by simultaneous saccharification and fermentation (SSF). WO and BM showed the highest ethanol yield of 44 g ethanol/100g glucan, which was close to the theoretical ethanol yield of 57 g ethanol/100g glucan. A 64% higher ethanol yield, based on raw material, was reached after pretreatment with WO and BM compared with unpretreated C. linum, however 50% of the biomass was lost during WO. Results indicated that the right combination of pretreatment and marine macroalgae, containing high amounts of glucan and cleaned from salts, enhanced the ethanol yield significantly.

Examining the potential of plasma-assisted pretreated wheat straw for enzyme production by Trichoderma reesei.

Plasma-assisted pretreated wheat straw was investigated for cellulase and xylanase production by Trichoderma reesei fermentation. Fermentations were conducted with media containing washed and unwashed plasma-assisted pretreated wheat straw as carbon source which was sterilized by autoclavation. To account for any effects of autoclavation, a comparison was made with unsterilized media containing antibiotics. It was found that unsterilized washed plasma-assisted pretreated wheat straw (which contained antibiotics) was best suited for the production of xylanases (110 IU ml(-1)) and cellulases (0.5 filter paper units (FPU) ml(-1)). Addition of Avicel boosted enzyme titers with the highest cellulase titers (1.5 FPU ml(-1)) found with addition of 50 % w/w Avicel and with the highest xylanase production (350 IU ml(-1)) reached in the presence of 10 % w/w Avicel. Comparison with enzyme titers from other nonrefined feedstocks suggests that plasma pretreated wheat straw is a promising and suitable substrate for cellulase and hemicellulase production.

Plasma-assisted pretreatment of wheat straw for ethanol production.

The potential of wheat straw for ethanol production after pretreatment with O(3) generated in a plasma at atmospheric pressure and room temperature followed by fermentation was investigated. We found that cellulose and hemicellulose remained unaltered after ozonisation and a subsequent washing step, while lignin was degraded up to 95% by O(3). The loss of biomass after washing could be explained by the amount of lignin degraded. The washing water of pretreated samples (0-7 h) was analyzed for potential fermentation inhibitors. Approximately 30 lignin degradation products and a number of simple carboxylic acids and phenolic compounds were found, e.g., vanillic acid, acetic acid, and formic acid. Some components had the highest concentration at the beginning of the ozonisation process (0.5, 1 h), e.g., 4-hydroxybenzladehyde, while the concentration of others increased during the entire pretreatment (0-7 h), e.g., oxalic acid and acetovanillon. Interestingly, washing had no effect on the ethanol production with pretreatment times up to 1 h. Washing improved the glucose availability with pretreatment times of more than 2 h. One hour of ozonisation was found to be optimal for the use of washed and unwashed wheat straw for ethanol production (maximum ethanol yield, 52%). O(3) cost estimations were made for the production of ethanol at standard conditions.

Plasma-assisted pretreatment of wheat straw.

O3 generated in a plasma at atmospheric pressure and room temperature, fed with dried air (or oxygen-enriched dried air), has been used for the degradation of lignin in wheat straw to optimize the enzymatic hydrolysis and to get more fermentable sugars. A fixed bed reactor was used combined with a CO2 detector and an online technique for O3 measurement in the fed and exhaust gas allowing continuous measurement of the consumption of O3. This rendered it possible for us to determine the progress of the pretreatment in real time (online analysis). The process time can be adjusted to produce wheat straw with desired lignin content because of the online analysis. The O3 consumption of wheat straw and its polymeric components, i.e., cellulose, hemicellulose, and lignin, as well as a mixture of these, dry as well as with 50% water, were studied. Furthermore, the process parameters dry matter content and milled particle size (the extent to which the wheat straw was milled) were investigated and optimized. The developed methodology offered the advantage of a simple and relatively fast (0.5-2 h) pretreatment allowing a dry matter concentration of 45-60%. FTIR measurements did not suggest any structural effects on cellulose and hemicellulose by the O3 treatment. The cost and the energy consumption for lignin degradation of 100 g of wheat straw were calculated.