Removing chiral contamination of lactate solutions by selective metabolism of the D-enantiomer.

OBJECTIVE: A bio-based process is appealing for purification of L-lactic acid, the major enantiomer of polylactic acid syrup, generated by thermochemical processes at the end of life of PLA-based plastics, from its chiral impurity, D-lactic acid, before reuse. RESULTS: Polylactic acid (PLA), a renewable alternative to petroleum-derived plastics, contains a mixture of L- and D-lactic acid (LA) isomers with the L-isomer dominating (up to 95 %). A novel bio-based process was developed to produce chirally pure L-LA from syrup produced during recycling of PLA-plastics. This process utilizes an engineered Escherichia coli (strain DC1001) containing novel gene deletions (lld, ykg) that eliminated the oxidative metabolism of L-lactate, leaving the membrane-bound D-lactate dehydrogenases to selectively metabolize the D-isomer. Strain DC1001 removed 8.7 g D-lactate l(-1) from a PLA-syrup containing 135 g total lactic acid l(-1) in 24 h. Average rates of removal of D-lactic acid were 0.25 g D-lactate h(-1) (g cell dry weight)(-1) and 0.36 g D-lactate l(-1) h(-1). CONCLUSION: Bio-based purification of PLA-syrup utilizing E. coli strain DC1001 is an attractive process step during recycling of PLA-plastics. This selective oxidation process can also be used to remove chiral contamination of L-lactate in medical applications.

Comparison of thermophilic and mesophilic one-stage, batch, high-solids anaerobic digestion.

The concept of starting up a batch, high-solids anaerobic digester by simply flooding the bed with a pH-buffer solution was tested using a mixture of vegetable waste and wood chips as feedstock at mesophilic (38 degrees C) and thermophilic (55 degrees C) conditions. At both temperatures stable and balanced methanogenesis was rapidly established within four days and was sustained until substrate was exhausted. Methanogenesis was more rapidly initiated in the thermophilic digester than in the mesophilic digester. Acetic, propionic and butyric acids accumulated in the leachate of both digesters during the start-up of digestion of uninoculated batch of waste. Thereafter all acids were degraded; which was quicker in the thermophilic digester. The accumulation and degradation of these acids was slower in the mesophilic digester. These studies showed that inoculum for carrying out thermophilic and mesophilic anaerobic digestion is readily available within the waste and its activity for complete mineralization of organic matter can be enhanced and sustained by providing adequate alkalinity. By employing a process in which anaerobic digestion of subsequent batches of waste was carried out by flooding with leachate drained from the digestion of a previous batch of waste, the volatile organic acid accumulation was maintained low and 95% of the methane yield potential of the waste was produced in 11 days under thermophilic conditions as opposed to 27 days under mesophilic conditions.

Anaerobic digestion of space mission wastes.

The technical feasibility of applying leachbed high-solids anaerobic digestion for reduction and stabilization of the organic fraction of solid wastes generated during space missions was investigated. This process has the advantages of not requiring oxygen or high temperature and pressure while producing methane, carbon dioxide, nutrients, and compost as valuable products. Anaerobic biochemical methane potential assays run on several waste feedstocks expected during space missions resulted in ultimate methane yields ranging from 0.23 to 0.30 L g-1 VS added. Modifications for operation of a leachbed anaerobic digestion process in space environments were incorporated into a new design, which included; (1) flooded operation to force leachate through densified feedstock beds; and (2) separation of biogas from leachate in a gas collection reservoir. This mode of operation resulted in stable performance with 85% conversion of a typical space solid waste blend, and a methane yield of 0.3 Lg per g VS added after a retention time of 15 days. These results were reproduced in a full-scale prototype system. A detailed analysis of this process was conducted to design the system sized for a space mission with a six-person crew. Anaerobic digestion compared favorably with other technologies for solid waste stabilization.

Enhanced degradation of waste grass clippings in one and two stage anaerobic systems.

The present work investigated the use of a simple rumen-fluid-inoculated anaerobic treatment system for the degradation of organic waste. Fresh rumen fluid collected from a fistulated sheep was used as the inoculum and fresh grass clippings were used as the waste material for treatment. Studies were carried out on both a one-stage system where the ligno-cellulosic fraction breaks down into a mixture of soluble products including volatile fatty acids and a two- stage system where these products are subsequently mineralised to biogas. In the one stage system about 70% of the organic waste was solubilized and in the two stage system about 60% waste material was solubilized in a week. About 50% of the degradation was achieved in a 4 day period, showing that a 4 day solids retention time would be a suitable operating regime. The maximum volatile fatty acid production rate was 327 mg COD l(-1) h(-1). A higher loading rate of 30 g l(-1) d(-1) was achieved in these systems compared to anaerobic digesters. Microbiological studies showed an increase in the number of fungal spores as well as a decrease in the number of protozoa in the treatment system. These numbers attained stable values over the duration of the experiments. The system developed is superior to conventional composting or anaerobic digestion and can be applied for the treatment of ligno-cellulosic agricultural residues.

The effect of citrate, oxalate, acetate, silicate and phosphate on stability of synthetic arsenic-loaded ferrihydrite and Al-ferrihydrite.

The effect of the organic species oxalate, citrate and acetate and the inorganic species silicate and phosphate on release of As(V) from synthetic arsenic-loaded ferrihydrite and Al-ferrihydrite in the pH range 4-8 was investigated. For ferrihydrite, the organic species increased %As(V) extraction in the order: acetatePO4(3-). Competitive adsorption of the investigated organic and inorganic species on ferrihydrite and Al-ferrihydrite could explain the enhanced release of As(V) into solution. However, disproportionate adsorption of other species relative to amount of As(V) release occurs, suggesting that mechanisms other than competitive adsorption are also operative. The results could best be explained in terms of combination of competitive adsorption and processes involving pH-dependent dissolution of ferrihydrite/Al-ferrihydrite, formation of various complex ionic species involving Al(III), Fe(III), As(V), SiO3(2-), PO4(3-), OH-, H+ and organic species: acetate, oxalate and citrate.

Effect of mixing on biomethanation of cattle-manure slurry.

The benefits and extent of mixing required during biomethanation of cattle-manure slurry was studied by investigating the effect of: 1) continuous and intermittent mixing, 2) agitator impeller speed and position; 3) not providing assisted mixing; 4) mixing on production of extracellular polymeric substances; and 5) mixing on the ultimate anaerobic biodegradability. Biomethanation was not adversely affected: during intermittent mixing; or when only sufficient mixing was provided to maintain off-bottom suspension of digester contents; or by doubling impeller speed. In fact continuous digestion of cattle-manure slurry without mechanical stirring was superior in terms of gas production. This can be attributed to increased loss of active volatile solids during stirring. Moreover, long-term batch digestion studies showed that the rate of biomethanation in a continuously stirred digester was inferior to that of a non-stirred one. Mixing was found to decrease production of extracellular polymeric substances (EPS). The presence of an increased level of EPS during the quiescent state could indicate increased attachment of cells to each other, resulting in larger agglomerates with better settling properties thus increasing biomass retention time.

Influence of co-substrates on structure of microbial aggregates in long-chain fatty acid-fed anaerobic digesters.

AIMS: The purpose of this study was to investigate the influence of co-substrates, such as glucose and cysteine, on the structure of microbial aggregates in anaerobic digesters treating oleate, a long-chain fatty acid (LCFA). METHODS AND RESULTS: Transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) were used to examine the structure of microbial aggregates. Fluorescence in situ hybridization (FISH) techniques were also used to characterize and localize the different trophic groups present in the aggregates. Oleate was found to inhibit the methanogenic activity and formation of granular biomass in digesters. The addition of co-substrates, such as glucose and cysteine either singly or in combination, increased the methanogenic activity and formation of granular biomass. Glucose was more effective than cysteine in reducing the inhibition by oleate on the methanogenic bacteria and in enhancing the formation of granules. CONCLUSIONS: The addition of nutrient substrate, such as glucose and cysteine could decrease the toxicity of LCFA on anaerobic granulation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the addition of other substrates might decrease the toxicity of LCFA on the granulation of biomass in anaerobic digesters and enhance methanogenic activity. A combination of TEM, CLSM and FISH techniques provides a better tool for visualizing microbial aggregates and for differentiating and localizing different microbial groups within these aggregates.

Cellulolytic activity in leachate during leach-bed anaerobic digestion of municipal solid waste.

The degradation of municipal solid waste (MSW) under mesophilic conditions can be enhanced by exchanging leachate between fresh waste and stabilised waste. The optimum point in time when leachate from an anaerobically digesting waste bed can be used to initiate degradation of another waste bed might occur when the leachate of the digesting waste bed is highly active with cellulolytic and methanogenic bacteria. In this study, the cellulolytic activity of the leachate was measured using the cellulose-azure assay. As products of hydrolysis are soluble compounds, the rate of generation of these compounds was estimated based on a soluble chemical oxygen demand (SCOD) balance around the fresh waste bed. It was found that once the readily soluble material present in MSW was washed out there was very little generation of SCOD without the production of methane, indicating that flushing leachate from a stabilised waste bed resulted in a balanced inoculation of the fresh waste bed. With the onset of sustained methanogenesis, the rate of SCOD generation equalled the SCOD released from the digester as methane. The experimental findings also showed that cellulolytic activities of the leachate samples closely followed the trend of SCOD generation. reserved.

Stable performance of anaerobic digestion in the presence of a high concentration of propionic acid.

An automatically controlled, glucose-fed, anaerobic digester was deliberately inhibited by addition of phenol. To overcome the phenol inhibition the feed dilution rate was lowered in such a way that the methane yield from glucose was kept the same as that under normal conditions. The concentrations of acetic and butyric acids remained below 100 mg/l, however, propionic acid accumulated to 2,750 mg/l. Phenol apparently inhibited all tropic groups of organisms and it was shown that the propionic acid was formed from the metabolism of phenol. From the nature of the operating strategy, it was deduced that the digester continued to convert all the glucose that was supplied to methane showing that propionic acid accumulation did not inhibit conversion of glucose to methane. Therefore, propionic acid accumulation may be an effect and not a cause of inhibition of the anaerobic digestion process.

Protein degradation during anaerobic wastewater treatment: derivation of stoichiometry.

The stoichiometry of reactions that describe protein degradation in anaerobic treatment systems were investigated. A methodology was developed to describe protein degradation to organic acids using a single reaction step. The reactions for individual amino acid fermentation and their mediating organisms were reviewed. The dominant fermentation pathways were selected based on a number of assumptions. Using the amino acid content of a model protein, it was then possible to determine stoichiometric coefficients for each major organic acid product in the overall degradation of the protein. The theoretical coefficients were then compared to those determined from two experimental runs on a continuously-fed, well-mixed, laboratory-scale anaerobic wastewater treatment system. In general, the coefficients compared well thus validating the use of a single reaction step for the overall catabolic reaction of protein degradation to organic acids. Furthermore, even when the protein concentration in feed or the feed flow rate was doubled, the amino acid fermentation pathways were found to occur predominantly by only one pathway. Although the choice of Stickland reactions over uncoupled degradation provided good comparisons, an electron balance showed that only about 40% of the amino acids could have proceeded coupled to other amino acid reactions. Uncoupled degradation of the remaining amino acids must have relied on the uptake of hydrogen produced from these reactions by hydrogen-consuming methane bacteria.

Microbial ecology of the leach bed anaerobic digestion of unsorted municipal solid waste.

The microbial ecology of the sequential, leach-bed, mesophilic anaerobic digestion of unsorted, coarse municipal solid waste (MSW) was examined over 80 days. The methane yield was approximately 75% of the ultimate biochemical methane potential (BMP) of the waste loaded into the digesters. The operational strategy involved a sequence of two digesters containing fresh and anaerobically stabilised MSW respectively. Cell wall phospholipid fatty acid (PLFA) and ether lipid (PLEL) analysis was used to monitor changes in microbial biomass. Both Bacterial and Archaeal biomass were heavily influenced by pH during the two-week start up period. Archaeal biomass peaked just before the methane production rate reached a maximum whereas Bacterial biomass peaked at a later stage. Changes in the phylogenetic diversity of the population were monitored by denaturing gradient gel electrophoresis (DGGE). An analysis of the changes in DGGE banding patterns suggested that rapid start-up of a new reactor was effected by inoculation as well as the provision of buffering capacity from the mature reactor leachate.

Expert system for control of anaerobic digesters.

Continuous anaerobic digesters are systems that present challenging control problems including the possibility that an unmeasured disturbance can change the sign of the steady-state process gain. An expert system is developed that recognizes changes in the sign of process gain and implements appropriate control laws. The sole on-line measured variable is the methane production rate, and the manipulated input is the dilution rate. The expert system changes the dilution rate according to one of four possible strategies: a constrained conventional set-point control law, a constant yield control law (CYCL) that is nearly optimal for the most common cause of change in the sign of the process gain, batch operation, or constant dilution rate. The algorithm uses a t test for determining when to switch to the CYCL and returns to the conventional set-point control law with bumpless transfer. The expert system has proved successful in several experimental tests: severe overload; mild, moderate, and severe underload; and addition of phenol in low and high levels. Phenol is an inhibitor that in high concentrations changes the sign of the process gain.

Avoiding digester imbalance through real-time expert system control of dilution rate.

Process control of anaerobic digesters is a particularly challenging problem because of the diversity of possible causes that can lead to digester imbalance. Conventional control schemes can fail in consequence of a reversal in the sign of the steady-state gain caused by some type of disturbance. In this work we present an expert system approach that takes into account the particularity of this process. The developed algorithm is demonstrated to compensate successfully for changes in the digester feed medium when simulated against a model for a continuous anaerobic digester.