Microbial biomass yield and turnover in soil biodegradation tests: carbon substrate effects

Most of the standardized biodegradation tests used to assess the ultimate biodegradation of environmentally degradable polymers are based solely on the determination of net evolved carbon dioxide. However, under aerobic conditions, it has to be considered that heterotrophic microbial consortia metabolize carbon substrates both to carbon dioxide and in the production of new cell biomass. It is generally accepted that in the relatively short term, 50% of the carbon content of most organic substrates is converted to CO₂, with the remaining carbon being assimilated as biomass or incorporated into humus. The latter is particularly important when the metabolism of the organic matter occurs in a soil environment. A straightforward relationship between the free-energy content of a carbon substrate (expressed as the standard free-energy of combustion) and its propensity for conversion to new microbial biomass rather than mineralization to CO₂ has been established. This can potentially lead to underestimation of biodegradation levels of test compounds, especially when they consist of carbon in a fairly low formal oxidation state and relatively high free-energy content. In the present work, the metabolism of different kind of carbon substrates, especially in soil, is reviewed and compared with our own experimental results from respirometric tests. The results show that conversion of highly oxidized materials, such as the commonly used reference materials, cellulose or starch, to CO₂ may be significantly overestimated. The addition of glucosidic material to soil leads to greatly increased respiration and is accompanied by a very low conversion to biomass or humic substances. In contrast, relatively less oxidized substrates metabolize more slowly to give both CO₂ and biomass to an extent which may be significantly underestimated if glucosidic materials are used as the reference. The need for an overall carbon balance taking into account both the carbon immobilized as biomass and that volatized as CO₂ must be considered in standard respirometric procedures for assessing the biodegradability of slowly degrading macromolecules.     

Assessing the biodegradation potential of polymers in screening- and long-term test systems

This paper presents a test scheme for assessing the biodegradation potential of polymers, starting with aquatic screening systems (aerobic and anaerobic) and continuing to long-term systems. At the end of the scheme the material has to prove its behavior under the relevant disposal conditions. Aerobic screening was performed mainly under aquatic conditions, but also in soil, using BOD-respirometry. Carbon balances were performed to obtain a better evaluation of the biodegradation potential. Under anaerobic conditions, biodegradation in an aquatic medium was followed by measuring CH₄ and CO₂ production. Polymers not fully degraded in the screening systems were tested in aquarium systems for at least 1 year. Biodegradation was followed by monitoring the DOC released in the water, mass loss, and microbial growth on the samples and in the water as well as via FTIR spectroscopy and SEM pictures. Results are presented for the polymers PHB, PHBV, PCL, Mater-Bi AI05H and ZF03U, and Bioceta. By combining the data from the screening with the aquarium system, a good picture of the degradation behavior of the polymers is obtained.Paper presented at the Bio/Environmentally Degradable Polymer Society—Third National Meeting, June 6–8, 1994, Boston, Massachusetts.     

Effect of the composting substrate on biodegradation of solid materials under controlled composting conditions

Biodegradability under composting conditions is assessed by test methods, such as ASTM D 5338-92, based on the measurement of CO₂ released by test materials when mixed with mature compost and maintained in a controlled composting environment. However, in real composting, biodegradation occurs in fresh waste. To clarify this point, the biodegradation of paper and of a starch-based biodegradable thermoplastic material, Mater-Bi ZI01U, was followed by measuring the weight loss of samples introduced either into a mature compost or into a synthetic waste. The weight loss in mature compost was higher at the beginning but tended to decrease; in synthetic waste a first lag phase was followed by an exponential phase. Complete degradation of paper was noticed simultaneously in the two substrates (after 25 days). The bulkier Mater-Bi samples were fully degraded after 20 days in fresh waste, but after 45 days in mature compost. Therefore, the test methods using mature compost as a substrate can possibly underestimate the biodegradation rate occurring in fresh waste, i.e., in real composting plants, and have to be considered as conservative test methods. The test procedure described in this paper seems very suitable as a screening method to verify the compostability of plastic materials in a composting environment.     

Kinetics of Aerobic Polymer Degradation in Soil by Means of the ASTM D 5988-96 Standard Method

The standard test method ASTM D 5988-96 for determining the degree and rate of aerobic biodegradation of plastic materials in contact with soil was applied to poly(3-hydroxybutyrate) and poly(-caprolactone). The method proved to be reliable and supplied reproducible measurements of CO₂ production, provided potassium (instead of barium) hydroxide was used as a trapping solution. The trends of CO₂ evolution, as a function of time, observed for the degradation of polymer powders in soil are similar to those predicted by simple first order kinetics in solution. The experimental data are described by a Michaelis–Menten type model, which accounts for the heterogeneity of the polymer-soil system. The kinetic equation deduced predicts the degradation rate to the proportional to the exposed polymer surface area.     

Aerobic biometer analysis of glucose and starch biodegradation

The ASTM D5210-91 protocol for evaluating the biodegradability of a polymer was examined. The reactor design was modified not only to account for the total CO₂ evolved but also to allow for the simultaneous carbon assessment in microbes, soluble products, and solid samples. Improvements in the test procedure were implemented such as (1) refining the CO₂ pretrap and posttrap design, (2) optimizing the carbon dioxide removal efficiency, (3) accounting for the total polymeric carbon, (4) standardizing the inoculum, and (5) revising the nutrient medium. By growing the sludge on a suitable substrate prior to polymeric exposure, a constant microbial density was obtained. The modified ASTM method provides an assessment of the polymeric carbon degradation at any given time. The results of this work have specific significance to the behavior of polymers in a sewage waste treatment plant, where sludge is continuously being acrated, and also for aerobic biodegradation in general.     

Temperature Effects on Soil Mineralization of Polylactic Acid Plastic in Laboratory Respirometers

A respirometric system was used to analyze the biodegradation of high molecular weight (120,000 to 200,000 g mol^–1) polylactic acid (PLA) plastic films in soil under laboratory conditions. The respirometric system consisted of air-conditioning pretraps, a soil reactor, and a carbon dioxide (CO₂) posttrap. A 200-g homogeneous soil mixture of all-purpose potting soil : manure soil : sand [1 : 1 : 1 (w/w)] and 1.5 g of PLA plastic films in 1 × 1-cm² squares was added to each bottle. The respirometers were placed in a 28, 40, or 55°C water bath for 182 days. Treatments (three replicates) included native corn starch (positive control), polyethylene (Glad Cling Wrap; negative control), and three PLA films: Ca-I (Cargill Dow Polymers LLC, monolayer), GII (Cargill Dow Polymers LLC, Generation II), and Ch-I (Chronopol; monolayer). The degree of polymer mineralization was indicated by the cumulative CO₂ liberated from each respirometer. The initial average mineralization rate and total percentage mineralized of the PLA plastic films at 28, 40, and 55°C was 24.3, 41.5, and 76.9 mg/day with a 27, 45, and 70% carbon loss, respectively. No decrease in soil pH was observed after 182 days of mineralization. Hence, increase in soil temperature drastically enhanced the biodegradation of PLA plastic films in soil under laboratory conditions (P < 0.0001).     

Biodegradation of Coir and Sisal Applied in the Automotive Industry

This paper discusses the results of biodegradability tests of natural fibers used by the automotive industry, namely: coir, coir with latex, and sisal. The biodegradation of coir, coir with latex, and of sisal fibers was determined by monitoring the production of carbon dioxide (CO₂) (IBAMA—E.1.1.2, 1988) and fungal growth (DIN 53739, 1984). The contents of total extractives, lignin, holocellulose, ashes, carbon, nitrogen and hydrogen of the fibers under study were determined in order to ascertain their actual content and to understand the results of the biodegradation tests. The production of CO₂ indicated low biodegradation, i.e., about 10% in mass, for all the materials after 45 days of testing; in other words, no material inhibited glucose degradation. However, the percentage of sisal fiber degradation was fourfold higher than that of coir with latex in the same period of aging. The fungal growth test showed a higher growth rate on sisal fibers, followed by coir without latex. In the case of coir with latex, we believe the fungal growth was not intense, because natural latex produces a bactericide or fungicide for its preservation during bleeding [1]. An evaluation of the materials after 90 days of aging tests revealed breaking of the fibers, particularly sisal and coir without latex, indicating fungal attack and biodegradation processes.     

Feasibility of greenhouse gas emissions offsets for natural source zone depletion of petroleum hydrocarbons

Hydrocarbon biodegradation is an important process for remediating petroleum hydrocarbons and managing large sites. However, this biodegradation results in what are essentially unavoidable CO₂ emissions to the atmosphere. A feasibility assessment was conducted to quantitatively consider reuse options for petroleum brownfields that would offset contaminant respiration emissions rates in the 2 to 10 micromoles CO₂ per meters squared per second (μmol CO₂ m^?2 s^?1) typically observed. Under a wide range of solar resource scenarios, placement of solar panels over only a fraction (no more than 35%) of the site footprint is estimated as necessary to achieve an emissions offset. Similarly, placement of one 30‐meter tall wind turbine of moderate rating (approximately 30 to 50 kW) is sufficient to provide an offset for a nominal 1,000 square meters site. For spreading of spent calcium‐rich construction materials, under even a high emissions scenario, the required footprint for the offset is less than the site footprint. While these approaches appear feasible, revegetation as forestland is estimated as sufficient only at contaminant respiration rates up to 2 μmol CO₂ m^?2 s^?1. Revegetation as rangeland and cropland, which sequesters CO₂ mainly in soil organic carbon, is estimated as requiring more than the site footprint under many contaminant respiration rates. Revegetation as a wetland fares slightly better from a carbon storage perspective, but it also has the potential for N₂O and CH₄ emissions that may largely undo the benefit from sequestration in soil organic matter. Overall, the results indicate several methods that are viable for achieving emissions offsets and a quantitation method that can be honed with site‐specific input parameters as appropriate.     

Assessing Aerobic Biodegradability of Plastics in Aqueous Environment by GC-Analyzing Composition of Equilibrium Gaseous Phase

Testing biodegradability of plastics under varied conditions of the environment as well as under laboratory conditions in accordance with valid international standards is very laborious, lengthy and often also economically demanding. For this reason, applicability was verified of gas chromatography to analyze gaseous phase when investigating the biodegradation course of plastics in an aqueous environment as an alternative to customary employed methods. A mathematical model of acid–basic CO₂ equilibrium in a gas–liquid system was worked out, enabling to determine quantity of produced CO₂ through chromatographic analysis of gaseous phase, in dependence on ratio of liquid and gas phase volumes (V _l/V _g) and on actual pH of liquid phase. Experimental conditions for organizing the tests were optimized. A ratio that proved suitable was V _l/V _g ≅ 0.1 at pH ≈ 7.1 of liquid phase. Under these test conditions, biodegradability of model samples, PHB, Gellan gum and Xanthan gum, was explored; course of biodegradation was studied through produced CO₂ (values ) determined by analyzing gaseous phase through gas chromatography on the one hand, and through customary “titration” procedure on the other. With water-soluble polymers, the decrement in dissolved organic carbon (values D _DOC) was also studied. Difference between values does not exceed 5%. The procedures in question are alternative “substituting” procedures for observing course of aerobic biodegradation of substances in an aqueous environment.     

Performance of Trickle-Bed Air Biofilter: A Comparative Study of a Hydrophilic and a Hydrophobic Voc

Two lab-scale trickle-bed air biofilters were operated for investigating the difference in performance between a hydrophilic and a hydrophobic volatile organic compound (VOC). Methyl isobutyl ketone (MIBK) and styrene were selected as a model hydrophilic and hydrophobic VOCs, respectively. Effects of loading rates, biofilter re-acclimation, removal profile along biofilter depth, nitrogen consumption, and CO₂ production were compared under three operating conditions, namely, backwashing and two non-use periods (starvation and stagnant). Consistent over 99% removal efficiency up to loading rates of 3.26 kg COD/m³-day was obtained for the MIBK biofilter at 0.76 min empty bed retention time (EBRT) and 1.5 L/d nutrient flow. A similar performance for the styrene biofilter was obtained for loading rates up to 1.9kg COD/m³-day at 2.02 min EBRT and 2.4 L/d nutrient flow. The MIBK biofilter required only an initial acclimation period of 16 days while styrene biofilter required 46 days. Non-use periods can be used as another means of biomass control for both biofilters when the employed loading rate did not exceed 1.27 and 2.17 kg COD/m³-day for styrene and MIBK biofilters, respectively. The re-acclimation of both biofilter was delayed with increase of loading rate. MIBK biofilter re-acclimated in 90 min, while styrene biofilter re-acclimated in more than 600 min. Under similar loading rates, MIBK biofilter utilized less biofilter depth than styrene biofilter. Nitrogen consumption behaviors were apparently different between the two biofilters. Styrene biofilter had higher CO₂ production than MIBK biofilter and its CO₂ production was closely related to the theoretical complete chemical oxidation.     

Use of kraft black liquors from a pulp mill for the production of soil conditioners

The possibility of utilizing kraft black liquors generated in a paper mill, using Eucaliptus globulus as raw material, for the production of a nitro-humic soil conditioner was studied. Two processes were considered: (1) oxidation of the kraft black liquors with nitric acid; and (2) precipitation of the lignin contained in kraft black liquors with CO₂ and further oxidation of this lignin by nitric acid. The second process was more efficient regarding product yield and safety of operation. Screening tests at different operating conditions of the latter process indicate that high quality nitro-humic materials can be obtained.     

Use of a novel biomarker,botryococcane, to monitor biodegradation of two lacustrine‐sourced crude oils

Bioremediation is a proven alternative for remediating petroleum‐impacted soils at exploration and production (E&P) sites. Monitoring remediation performance can involve detection and quantification of biodegradation resistant compounds such as C₃₀17α(H),21β(H)‐hopane, which requires the use of gas chromatography with mass spectrometry detection (GC/MS). Due to the remoteness of many E&P sites, this technology is not always available, and alternative methods are needed to provide reliable quantitative measurements of petroleum remediation efficiency. This study provides a detailed chemical characterization of lacustrine‐sourced crude oils and a technical basis for measuring the effectiveness of bioremediation efforts for soil impacted by those crudes. We show that the novel isoprenoid hydrocarbon botryococcane is relatively stable in lacustrine‐sourced crude oils compared with C₃₀17α(H),21β(H)‐hopane under moderate biodegradation conditions generally observed in field samples. We have also demonstrated that, due to the stability and relatively elevated concentration of botryococcane in lacustrine oils, it can be reliably measured using the more cost‐effective and available GC/FID methodology, and thereby be used to monitor the progress of ongoing soil bioremediation activities at remote sites.     

Carbon sequestration: Do N inputs and elevated atmospheric CO2 alter soil solution chemistry and respiratory C losses?

Soil respiration is a large C flux which is of primary importance in determining C sequestration. Here we ask how it is altered by atmospheric CO₂ concentration and N additions. Swards of Lolium perenne L. were grown in a Eutric cambisol under controlled conditions with and without the addition of 200 kg NO^? ₃ ?N ha^?1, at either 350 ppm or 700 ppm CO₂, for 3 months. Soil respiration and net canopy photosynthesis were both increased by added N and elevated CO₂, but soil respiration increased proportionately less than fixation by photosynthesis. Thus, both elevated CO₂ and N appeared to increase potential C sequestration, although adding N at elevated CO₂ reduced the C sequestered as a proportion of that fixed relative to elevated CO₂ alone. Across all treatments below-ground respiratory C losses were predicted by root biomass, but not by soil solution C and N concentrations. Specific root-dependent respiration was increased by elevated CO₂, such that below-ground respiration per unit biomass and per unit plant N was increased.     

Biodegradation of LDPE/Modified Starch Blends Sterilized with Gamma Radiation

Blends of LDPE/modified starch were prepared, sterilized by gamma radiation and investigated with respect to their microbial degradation by a mixture of fungal strains in liquid medium after 90 days, was analyzed by carbon dioxide (CO₂) production (Sturm test). Biodegradation of blends was evaluated by Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction; mechanical testing, scanning electron microscopy (SEM). The biodegradation of LDPE/modified starch blends was attributed to microbiological attack, with alterations in the chemical structure of the blend with an increase in the carbonyl and vinyl indices and the appearance of new crystalline symmetry generating a crystalline domain not existing before in the blend and decrease in the mechanical properties.     

Carbon Sequestration: Do N Inputs and Elevated Atmospheric CO2 Alter Soil Solution Chemistry and Respiratory C Losses?

Soil respiration is a large C flux which is of primary importance in determining C sequestration. Here we ask how it is altered by atmospheric CO₂ concentration and N additions. Swards of Lolium perenne L. were grown in a Eutric cambisol under controlled conditions with and without the addition of 200 kg NO ₃ ^– –N ha^–1, at either 350 ppm or 700 ppm CO₂, for 3 months. Soil respiration and net canopy photosynthesis were both increased by added N and elevated CO₂, but soil respiration increased proportionately less than fixation by photosynthesis. Thus, both elevated CO₂ and N appeared to increase potential C sequestration, although adding N at elevated CO₂ reduced the C sequestered as a proportion of that fixed relative to elevated CO₂ alone. Across all treatments below-ground respiratory C losses were predicted by root biomass, but not by soil solution C and N concentrations. Specific root-dependent respiration was increased by elevated CO₂, such that belowg-round respiration per unit biomass and per unit plant N was increased.     

n-Hexane Biodegradation in Trickle Bed Air Biofilters

While hydrophilic compounds are degraded easily in Trickling bed air biofilters (TBABs), hydrophobic compounds are retarded until biological cultures produce a sufficient RNA or enzyme/protein to utilize this compound. Hydrophobic compounds are not readily bio-available which makes them reluctant to biodegradation as mass transfer between the gas and liquid phases is a rate limiting step. To enhance the destruction of hydrophobic compounds in TBABs, the utilization of surfactant was introduced to increase the solubility which helps overcoming the rate limiting step. The surfactant was used as well to limit the growth of excess biomass ensuring smooth flow through the biofilter bed and preventing short circuits. Two different non-ionic non-toxic surfactants were used in this study: Triton X-100 and Tomadol® 25-7. Two lab-scale controlled TBABs were operated for investigating the performance difference for n-Hexane as an example of hydrophobic volatile organic compound (VOC) with and without the addition of surfactant. Operating conditions in both TBABs were as follows: nutrient feed rate (2L/day), air flowrate (1.4L/min), bed depth (60cm), empty bed retention time (120s), bed material (diatomaceous earth pellets) and room-temperature. The inlet concentration was changed from 50 to 100ppmv. Acclimation period, removal profile along biofilter depth, nitrogen consumption, and CO₂ production were compared under continuous loading operation condition. The optimum concentration of surfactant in the nutrient feed was determined by a batch experiment. The effect of different surfactant concentrations on VOC water solubility with time was studied by considering different VOC concentration sets within the TBAB loading rate range.     

Modelling Easily Biodegradability of Materials in Liquid Medium-Relationship Between Structure and Biodegradability

In the present project, twenty materials (e.g., polyhydroxybutyrate-hydroxyvalerate, polycaprolactone, cellulose acetate, polyacticacid, polyethylene), representing varied biodegradability levels were studied. An aerobic respirometric test, based on the CEN Draft, was setup. The biodegradability of each plastic film was evaluated by measuring the percentage of carbon converted into CO₂ during 35 days. The values of the CO₂ production were plotted versus days as a cumulative function. In order to reduce its number of points, the cumulative curve was modeled using a sigmoïd function (Hill sigmoïd). This model was compared to one found in the literature. A _i ² test showed that the biodegradation curve was more accurately fitted with the model than the previous one. Three kinetic parameters were determined by this Hill model: one represents the maximal percentage of carbon converted into CO₂, the second the half-life time in days of the degrading part of the material and the third one the curve radius.In addition, the following analyses were carried out on each sample: elemental analysis, thickness, hydrophobicity and surface free energy measurements. In order to compress the information and to keep only relevant pieces, these parameters were submitted to a Principal Component Analysis. PCA found linear combinations of variables that describe major trends in the data. The two principal components which separate groups of materials were closely related to a chemical and a physical axis respectively. Materials showing a high biodegradability were related to high oxygen (and nitrogen) contents and low hydrophobicity: Material thickness did not influence the likeliness to biodegradability described by the maximum biodegradation rate. Finally, this study established the correlation between the biodegradation and the structure of biopolymers.     

Anaerobic biodegradation of cellulosic material: Batch experiments and modelling based on isotopic data and focusing on aceticlastic and non-aceticlastic methanogenesis

Utilizing stable carbon isotope data to account for aceticlastic and non-aceticlastic pathways of methane generation, a model was created to describe laboratory batch anaerobic decomposition of cellulosic materials (office paper and cardboard). The total organic and inorganic carbon concentrations, methane production volume, and methane and CO₂ partial pressure values were used for the model calibration and validation. According to the fluorescent in situ hybridization observations, three groups of methanogens including strictly hydrogenotrophic methanogens, strictly aceticlastic methanogens (Methanosaeta sp.) and Methanosarcina sp., consuming both acetate and H₂/H₂CO₃ as well as acetate-oxidizing syntrophs, were considered. It was shown that temporary inhibition of aceticlastic methanogens by non-ionized volatile fatty acids or acidic pH was responsible for two-step methane production from office paper at 35 °C where during the first and second steps methane was generated mostly from H₂/H₂CO₃ and acetate, respectively. Water saturated and unsaturated cases were tested. According to the model, at the intermediate moisture (150%), much lower methane production occurred because of full-time inhibition of aceticlastic methanogens. At the lowest moisture, methane production was very low because most likely hydrolysis was seriously inhibited. Simulations showed that during cardboard and office paper biodegradation at 55 °C, non-aceticlastic syntrophic oxidation by acetate-oxidizing syntrophs and hydrogenotrophic methanogens were the dominant methanogenic pathways.     

How the origin of fresh household waste affects its ability to be biodegraded: An assessment using basic tools and its application to the city of Kara in Togo

Waste biodegradation has been largely investigated in the literature by using conventional tests like the BMP test and the respirometric test, whereas only few studies deal with the use of leaching tests in combination with biological activity measurements. Consequently, this study used an improved leaching test to evaluate the biodegradability of two deposits of fresh household waste from the city of Kara in Togo. The first deposit came from households in neighborhoods located in the outskirts of the city and the second consisted of fresh waste, mainly composed of business waste and household waste, collected in the urban center and aimed at being deposited in the landfill. A physicochemical characterization of the two deposits completed the leaching test. The biological activity was monitored by measuring O₂ consumption and CO₂ production. pH, DOC/OM, VFA/DOC ratios and the SUVA index was measured in the leaching juice to assess both the state of degradation of the waste in the deposits and the ability of the organic matter to be mobilized quickly and to be easily assimilated by microorganisms. The biodegradability of waste from the city of Kara correlated with their origin even though the physical characteristics of the two deposits studied differed greatly.     

Dialdehyde starch and zein plastic: Mechanical properties and biodegradability

Dialdehyde starch (DAS) and zein, a hydrophobic corn protein, were investigated to produce biodegradable plastics with improved water resistance and mechanical properties. In the study, dialdehyde starch and zein ratio, plasticizers, and degree of starch oxidation were examined. Increased molding temperature and level of starch oxidation decreased water absorption of the plastic. Tensile strength and Young's modulus increased with starch oxidation. The biodegradation of starting materials and ground plastic specimens was studied in aerobic soil reactors maintained at 25°C for 180 days. Biodegradation of corn starch, zein, and dialdehyde starch for 180 days produced CO₂ equivalent to 64, 63, and 10% of theoretical carbon, respectively. Specimens of molded DAS and zein (3 : 1) plastic showed accelerated CO₂ evolution compared to DAS and other raw materials alone. By 180 days, specimens made with starch of low oxidation (1 and 5% oxidized) demonstrated a 60% biodegradation, and specimens with highly oxidized starch (90% oxidized) achieved 37% biodegradation.Paper presented at the Bio/Environmentally Degradable Polymer Society—Third National Meeting, June 6–8, 1994, Boston, Massachusetts.Journal Paper J-15927 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Project No. 3258.