The effect of drying and size reduction pretreatments on recovery of inorganic crop nutrients from inedible wheat residues.

Inorganic nutrients can be easily recovered from ALS crop residue solid wastes by aqueous leaching. However, oven drying and milling pretreatment of these residues has been frequently required to accommodate crop scientists and facility storage limitations. As part of a research study that will compare three different bioreactor technologies for processing these wastes, we realized that different drying and size-reduction pretreatments had been utilized for each technology. This paper compares the effects of residue pretreatment on recovery of nutrients by leaching. Pretreatments included three drying methods [fresh, oven-dried (70 degrees C overnight), and freeze-dried] and two size reduction methods [chopped (2 cm length) and milled (2 mm diameter)]. Determination of mass balances (dry weight and ash content of solids) before and after leaching indicated solubilization was least for fresh residues (23% dry weight loss and 50% for ash loss), and most for freeze-dried residues (41-47% dry weight loss and nearly 100% for ash loss). Mineral recovery of major elements (NO3, PO4, K, Ca, and Mg) in leachates was poorest for fresh residues. P and K recovery in leachates were best for oven-dried residues and Ca, Mg, and N recovery best for freeze-dried residues. The differences in recovery for N, P, and K in leachates were minimal between chopping and milling and slightly better for Ca and Mg from milled residues.

Recovery of resources for advanced life support space applications: effect of retention time on biodegradation of two crop residues in a fed-batch, continuous stirred tank reactor.

Bioreactor retention time is a key process variable that will influence costs that are relevant to long distance space travel or long duration space habitation. However. little is known about the effects of this parameter on the microbiological treatment options that are being proposed for Advanced Life Support (ALS) systems. Two bioreactor studies were designed to examine this variable. In the first one, six retention times ranging from 1.3 to 21.3 days--were run in duplicate, 81 working-volume continuous stirred tank reactors (CSTR) that were fed ALS wheat residues. Ash-free dry weight loss, carbon mineralization, soluble TOC reduction, changes in fiber content (cellulose, hemicellulose, and lignin), bacterial numbers, and mineral recoveries were monitored. At short retention times--1.33 days--biodegradation was poor (total: 16-20%, cellulose - 12%, hemicellulose - 28%) but soluble TOC was decreased by 75-80% and recovery of major crop inorganic nutrients was adequate, except for phosphorus. A high proportion of the total bacteria (ca. 83%) was actively respiring. At the longest retention time tested, 21.3 days, biodegradation was good (total: 55-60%, cellulose ca. 70%, hemicellulose - ca. 55%) and soluble TOC was decreased by 80%. Recovery of major nutrients, except phosphorus, remained adequate. A very low proportion of total bacteria was actively respiring (ca. 16%). The second bioreactor study used potato residue to determine if even shorter retention times could be used (range 0.25-2.0 days). Although overall biodegradation deteriorated, the degradation of soluble TOC continued to be ca. 75%. We conclude that if the goal of ALS bioprocessing is maximal degradation of crop residues, including cellulose, then retention times of 10 days or longer will be needed. If the goal is to provide inorganic nutrients with the smallest volume/weight bioreactor possible, then a retention time of 1 day (or less) is sufficient.

An overview: recycling nutrients from crop residues for space applications.

Without some form of regenerative life support system, long duration space habitation or travel will be limited severely by the prohibitive costs of resupplying air, water, and food from Earth. Components under consideration for inclusion in a regenerative life support system are based on either physicochemical or biological processes. Physicochemical systems would use filtration and elemental phase changes to convert waste materials into usable products, while biological systems would use higher plants and bioreactors to supply crew needs. Neither a purely biological nor strictly a physicochemical approach can supply all crew needs, thus, the best each approach can offer will be combined into a hybrid regenerative life support system. Researchers at Kennedy Space Center (KSC) Advanced Life Support Breadboard Project have taken the lead on bioregenerative aspects of space life support. The major focus has been on utilization of higher plants for production of food, oxygen, and clean water. However, a key to any regenerative life support system is recycling and recovery of resources (wastes). In keeping with the emphasis at KSC on bioregenerative systems and with the focus on plants, this paper focuses on research with biologically-based options for resource recovery from inedible crop residues.

Integration of waste processing and biomass production systems as part of the KSC Breadboard project.

After initial emphasis on large-scale baseline crop tests, the Kennedy Space Center (KSC) Breadboard project has begun to evaluate long-term operation of the biomass production system with increasing material closure. Our goal is to define the minimum biological processing necessary to make waste streams compatible with plant growth in hydroponic systems, thereby recycling nutrients into plant biomass and recovering water via atmospheric condensate. Initial small and intermediate-scale studies focused on the recycling of nutrients contained in inedible plant biomass. Studies conducted between 1989-1992 indicated that the majority of nutrients could be rapidly solubilized in water, but the direct use of this crop "leachate" was deleterious to plant growth due to the presence of soluble organic compounds. Subsequent studies at both the intermediate scale and in the large-scale Biomass Production Chamber (BPC) have indicated that aerobic microbiological processing of crop residue prior to incorporation into recirculating hydroponic solutions eliminated any phytotoxic effect, even when the majority of the plant nutrient demand was provided from recycled biomass during long term studies (i.e. up to 418 days). Current and future studies are focused on optimizing biological processing of both plant and human waste streams.

Evaluation of an anaerobic digestion system for processing CELSS crop residues for resource recovery.

Three bioreactors, connected in series, were used to process CELSS potato residues for recovery of resources. The first stage was an anaerobic digestor (8 L working volume; cow rumen contents inoculum; fed-batch; 8 day retention time; feed rate 25 gdw day-1) that converted 33% of feed (dry weight loss) to CO2 and "volatile fatty acids" (vfa, 83:8:8 mmolar ratio acetic:propionic:butyric). High nitrate-N in the potato residue feed was absent in the anaerobic effluent, with a high portion converted to NH4(+)-N and the remainder unaccounted and probably lost to denitrification and NH4+ volatilization. Liquid anaerobic effluent was fed to an aerobic, yeast biomass production vessel (2 L volume; Candida ingens inoculum; batch [pellicle] growth; 2 day retention time) where the VFAs and some NH4(+)-N were converted into yeast biomass. Yeast yields accounted for up to 8% of potato residue fed into the anaerobic bioreactor. The third bioreactor (0.5 L liquid working volume; commercial nitrifier inoculum; packed-bed biofilm; continuous yeast effluent feed; recirculating; constant volume; 23 day hydraulic retention time) was used to convert successfully the remaining NH4(+)-N into nitrate-N (preferred form of N for CELSS crop production) and to remove the remaining degradable soluble organic carbon. Effluents from the last two stages were used for partial replenishment of minerals for hydroponic potato production.

Effects of bioreactor retention time on aerobic microbial decomposition of CELSS crop residues.

The focus of resource recovery research at the KSC-CELSS Breadboard Project has been the evaluation of microbiologically mediated biodegradation of crop residues by manipulation of bioreactor process and environmental variables. We will present results from over 3 years of studies that used laboratory- and breadboard-scale (8 and 120 L working volumes, respectively) aerobic, fed-batch, continuous stirred tank reactors (CSTR) for recovery of carbon and minerals from breadboard grown wheat and white potato residues. The paper will focus on the effects of a key process variable--bioreactor retention time--on response variables indicative of bioreactor performance. The goal is to determine the shortest retention time that is feasible for processing CELSS crop residues, thereby reducing bioreactor volume and weight requirements. Pushing the lower limits of bioreactor retention times will provide useful data for engineers who need to compare biological and physicochemical components. Bioreactor retention times were manipulated to range between 0.25 and 48 days. Results indicate that increases in retention time lead to a 4-fold increase in crop residue biodegradation, as measured by both dry weight losses and CO2 production. A similar overall trend was also observed for crop residue fiber (cellulose and hemicellulose), with a noticeable jump in cellulose degradation between the 5.3 day and 10.7 day retention times. Water-soluble organic compounds (measured as soluble TOC) were appreciably reduced by more than 4-fold at all retention times tested. Results from a study of even shorter retention times (down to 0.25 days), in progress, will also be presented.

Comparison of aerobically-treated and untreated crop residue as a source of recycled nutrients in a recirculating hydroponic system.

This study compared the growth of potato plants on nutrients recycled from inedible potato biomass. Plants were grown for 105 days in recirculating, thin-film hydroponic systems containing four separate nutrient solution treatments: (1) modified half-strength Hoagland's (control), 2) liquid effluent from a bioreactor containing inedible potato biomass, 3) filtered (0.2 micrometer) effluent, and 4) the water soluble fraction of inedible potato biomass (leachate). Approximately 50% of the total nutrient requirement in treatments 2-4 were provided (recycled) from the potato biomass. Leachate had an inhibitory effect on leaf conductance, photosynthetic rate, and growth (50% reduction in plant height and 60% reduction in tuber yield). Plants grown on bioreactor effluent (filtered or unfiltered) were similar to the control plants. These results indicated that rapidly degraded, water soluble organic material contained in the inedible biomass, i.e., material in leachate, brought about phytotoxicity in the hydroponic culture of potato. Recalcitrant, water soluble organic material accumulated in all nutrient recycling treatments (650% increase after 105 days), but no increase in rhizosphere microbial numbers was observed.

Dynamics of microorganism populations in recirculating nutrient solutions.

This overview covers the basic microbial ecology of recirculating hydroponic solutions. Examples from NASA and Soviet CELSS tests and the commercial hydroponic industry will be used. The sources of microorganisms in nutrient solutions include air, water, seeds, plant containers and plumbing, biological vectors, and personnel. Microbial fates include growth, death, and emigration. Important microbial habitats within nutrient delivery systems are root surfaces, hardware surfaces (biofilms), and solution suspension. Numbers of bacteria on root surfaces usually exceed those from the other habitats by several orders of magnitude. Gram negative bacteria dominate the microflora with fungal counts usually much lower. Trends typically show a decrease in counts with increasing time unless stressed plants increase root exudates. Important microbial activities include carbon mineralization and nitrogen transformations. Important detrimental interactions include competition with plants, and human and plant pathogenesis.

Reduced sulfur in ashes and slags from the gasification of coals: availability for chemical and microbial oxidation.

This study was initiated to determine if reduced sulfur contained in coal gasifier ash and slag was available for microbial and chemical oxidation because eventual large-quantity landfill disposal of these solid wastes is expected. Continuous application of distilled water to a column containing a high-sulfur-content (4% [wt/wt]) gasifier slag yielded leachates with high sulfate levels (1,300 mg of sulfate liter) and low pH values (4.2). At the end of the experiment, a three-tube most-probable-number analysis indicated that the waste contained 1.3 x 10 thiosulfate-oxidizing bacteria per g. Slag samples obtained aseptically from the column produced sulfate under both abiotic and biotic conditions when incubated in a mineral nutrient solution. Both microbial and chemical sulfate syntheses were greatly stimulated by the addition of thiosulfate to the slag-mineral nutrient solution. These results led to a test of microbial versus chemical sulfur oxidation in ashes and slags from five gasification processes. Sulfate production was measured in sterile (autoclaved) and nonsterile suspensions of the solid wastes in a mineral nutrient solution. These ashes and slags varied in sulfur content from 0.3 to 4.0% (wt/wt). Four of these wastes demonstrated both chemical (2.0 to 27 mug of sulfate g day) and microbial (3.1 to 114 mug of sulfate g day) sulfur oxidation. Obvious relationships between sulfur oxidation rate and either sulfur content or particle size distribution of the wastes were not immediately evident. We conclude that the sulfur contained in all but one waste is available for oxidation to sulfuric acid and that microorganisms play a partial role in this process.

Method for measuring dissolved hydrogen in anaerobic ecosystems: application to the rumen.

A method of transferring dissolved H2 to a CO2 headspace and then absorbing out the CO2 to concentrate the H2 before gas chromatographic analysis was developed to measure low concentrations of dissolved H2. A detection limit of 10 pmol of H2 ml-1 of water was achieved. When used ot monitor H2 changes in a bovine rumen, a 10-fold increase in H2 was noted 1 h after feeding and then declined rapidly to the normal steady-state concentration of 1 microM.

Kinetic parameters of the conversion of methane precursors to methane in a hypereutrophic lake sediment.

The kinetic parameters K(m), V(max), T(t) (turnover time), and v (natural velocity) were determined for H(2) and acetate conversion to methane by Wintergreen Lake sediment, using short-term (a few hours) methods and incubation temperatures of 10 to 14 degrees C. Estimates of the Michaelis-Menten constant, K(m), for both the consumption of hydrogen and the conversion of hydrogen to methane by sediment microflora averaged about 0.024 mumol g of dry sediment. The maximal velocity, V(max), averaged 4.8 mumol of H(2) g h for hydrogen consumption and 0.64 mumol of CH(4) g h for the conversion of hydrogen to methane during the winter. Estimated natural rates of hydrogen consumption and hydrogen conversion to methane could be calculated from the Michaelis-Menten equation and estimates of K(m), V(max), and the in situ dissolved-hydrogen concentration. These results indicate that methane may not be the only fate of hydrogen in the sediment. Among several potential hydrogen donors tested, only formate stimulated the rate of sediment methanogenesis. Formate conversion to methane was so rapid that an accurate estimate of kinetic parameters was not possible. Kinetic experiments using [2-C]acetate and sediments collected in the summer indicated that acetate was being converted to methane at or near the maximal rate. A minimum natural rate of acetate conversion to methane was estimated to be about 110 nmol of CH(4) g h, which was 66% of the V(max) (163 nmol of CH(4) g h). A 15-min preincubation of sediment with 5.0 x 10 atm of hydrogen had a pronounced effect on the kinetic parameters for the conversion of acetate to methane. The acetate pool size, expressed as the term K(m) + S(n) (S(n) is in situ substrate concentration), decreased by 37% and T(t) decreased by 43%. The V(max) remained relatively constant. A preincubation with hydrogen also caused a 37% decrease in the amount of labeled carbon dioxide produced from the metabolism of [U-C]valine by sediment heterotrophs.

Application of the fluorescent-antibody technique to the study of a methanogenic bacterium in lake sediments.

Fluorescent antibody (FA) was prepared for a methanogenic bacterium isolated from Wintergreen Lake pelagic sediment. The isolate resembles Methanobacterium formicicum. The FA did not cross-react with 9 other methanogens, including M. formicicum strains, or 24 heterotrophs, 18 of which had been isolated from Wintergreen Lake sediment. FA-reacting methanogens were detected in heat-fixed smears of several different lake sediments and anaerobic sewage sludge. Pretreatment of all samples with either rhodamine-conjugated geletin or bovine serum albumin adequately controlled nonspecific absorption of the FA. Autofluorescent particles were observed in the sediment samples but, with experience, they could easily be distinguished from FA-reacting bacteria. FA direct counts of the specific methanogen in Wintergreen Lake sediments were made on four different sampling dates and compared with five-tube most-probable-number estimates of the total methanogenic population that was present in the same samples. The FA counts ranged from 3.1 X 10(6) to 1.4 X 10(7)/g of dry sediment. The highest most-probable-number estimates were at least an order ofmagnitude lower.