CHARACTERIZATION OF URBAN WASTES ACCORDING TO FERTILITY AND PHYTOTOXICITY PARAMETERS

Several urban wastes of different nature and level of organic matter stability (municipal solid wastes, sewage sludges and composts) have been characterized analysing fertility and phytotoxicity parameters. Sewage sludges showed the highest N and P values of all the wastes. The total K content of the wastes was low but almost all was available to plants. Mature composts had the lowest organic carbon and humic substances values as a consequence of organic matter mineralization during the composting process. The soluble C at pH 2/precipitated C ratio decreased as the stability of the organic matter increased. In spite of the heterogeneity of the wastes analysed, the organic matter/total organic C ratio was largely constant with a mean value of 2.05. In no case did the heavy metal levels exceed the maximum allowed by the EU disposition for sewage sludges use in agriculture. The highest levels of phytotoxic substances occurred in the fresh wastes. The germination rates and root length were highest with mature composts. Germination index and root length were negatively correlated with water soluble carbon (WSC) and WSC/N ratio. The principal component analysis showed that extractable C, soluble C at pH 2 and water soluble C were the C fractions which most contributed to the total variability.     

Techniques for the evaluation of maturity for composts of industrially contaminated lake sediments

Composting was applied as a bioremediation methodology for the reclamation of dredged sediments of Isnapur, Khazipally and Gandigudem lakes polluted with industrial wastes. The present study is an attempt to elaborate upon organic matter transformations and define the parameters for product maturity adapting chemical and spectroscopic methods during composting. The stability and maturity of sediments were evaluated by assessing parameters like C/N ratio, nitrification index (NH(4)-N/NO(3)-N), water-soluble organic carbon concentration, CO(2) evolution rate, cation exchange capacity and indices such as humification index, E4/E6 ratio, compost mineralization index (ash content/oxidizable carbon), germination index, dehydrogenase, polyphenoloxidase activities and FTIR spectroscopy. The results showed that the changes in the above chemical and biological parameters can be employed as reliable indicators of stability and maturity. The FTIR spectra revealed enrichment in the aromatic groups and a degradation of the aliphatic groups indicating stabilization of the final compost.     

Properties of slate mining wastes incubated with grape marc compost under laboratory conditions

The effect of the addition of spent grape marc compost (GMC) and vermicompost (GMV) as amendments to slate mining wastes was evaluated in a laboratory incubation experiment. Mixtures of slate processing fines (SPF), with three doses of each amendment (4%, 8% and 16% compost, dry weight), plus a control were incubated at 25 degrees C in the laboratory for 90 days. The changes in the chemical and biological properties of the mixtures (pH, total C, total N, inorganic N, available nutrients, microbial biomass carbon and dehydrogenase activity) were investigated during the incubation period, and once it was finished, the phytotoxicity of the mixtures was determined by the germination of Lolium multiflorum Lam. seeds. The addition of the amendments significantly increased the nutrient concentrations of the SPF and enhanced biological activity by increasing microbial biomass and enzymatic activity. Results improved with higher doses; within the composts, GMV showed a better performance than GMC. These results prove the suitability of grape marc-derived amendments for the biochemical amelioration of mining wastes, and highlight the benefits of organic amendment in restoration projects.     

Molecular composition of recycled organic wastes,as determined by solid-state 13C NMR and elemental analyses

Using solid state ¹³C NMR data and elemental composition in a molecular mixing model, we estimated the molecular components of the organic matter in 16 recycled organic (RO) wastes representative of the major materials generated in the Sydney basin area. Close correspondence was found between the measured NMR signal intensities and those predicted by the model for all RO wastes except for poultry manure char. Molecular nature of the organic matter differed widely between the RO wastes. As a proportion of organic C, carbohydrate C ranged from 0.07 to 0.63, protein C from <0.01 to 0.66, lignin C from <0.01 to 0.31, aliphatic C from 0.09 to 0.73, carbonyl C from 0.02 to 0.23, and char C from 0 to 0.45. This method is considered preferable to techniques involving imprecise extraction methods for RO wastes. Molecular composition data has great potential as a predictor of RO waste soil carbon and nutrient outcomes.     

Modelling C and N mineralization during decomposition of anaerobically digested and composted municipal solid waste.

Application of municipal solid waste (MSW) to arable land can be used to close the nutrient cycle between urban and rural areas. The aim of the current study was to quantify net N mineralization and respiration from composted MSW (CMSW) and anaerobically digested MSW (ADMSW) applied to soil, and to test whether a simple relationship between net N mineralization and respiration that was developed for plant materials, was applicable for these types of MSW. In a laboratory experiment, CMSW and ADMSW were incorporated into soil and incubated at 15 degrees C. During the 149-day experiment, netN mineralization and respiration were determined. Cumulative respiration derived from both MSW types was very steep during the first 30 days, after which it levelled off. However, calculated on the basis of applied C, the ADMSW was 10 times more degradable than the CMSW. Both MSW types caused initial net N immobilization followed by re-mineralization. A simple model based on the relationship between net N mineralization and respiration was only applicable for the MSW after significant modifications. If farmers are to recognize CMSW and ADMSW as valuable fertilizers, it is important that they can be produced with higher maturity, in order to avoid initial N immobilization.     

Use of thermal analysis techniques (TG-DSC) for the characterization of diverse organic municipal waste streams to predict biological stability prior to land application

The use of organic municipal wastes as soil amendments is an increasing practice that can divert significant amounts of waste from landfill, and provides a potential source of nutrients and organic matter to ameliorate degraded soils. Due to the high heterogeneity of organic municipal waste streams, it is difficult to rapidly and cost-effectively establish their suitability as soil amendments using a single method. Thermal analysis has been proposed as an evolving technique to assess the stability and composition of the organic matter present in these wastes. In this study, three different organic municipal waste streams (i.e., a municipal waste compost (MC), a composted sewage sludge (CS) and a thermally dried sewage sludge (TS)) were characterized using conventional and thermal methods. The conventional methods used to test organic matter stability included laboratory incubation with measurement of respired C, and spectroscopic methods to characterize chemical composition. Carbon mineralization was measured during a 90-day incubation, and samples before and after incubation were analyzed by chemical (elemental analysis) and spectroscopic (infrared and nuclear magnetic resonance) methods. Results were compared with those obtained by thermogravimetry (TG) and differential scanning calorimetry (DSC) techniques. Total amounts of CO₂ respired indicated that the organic matter in the TS was the least stable, while that in the CS was the most stable. This was confirmed by changes detected with the spectroscopic methods in the composition of the organic wastes due to C mineralization. Differences were especially pronounced for TS, which showed a remarkable loss of aliphatic and proteinaceous compounds during the incubation process. TG, and especially DSC analysis, clearly reflected these differences between the three organic wastes before and after the incubation. Furthermore, the calculated energy density, which represents the energy available per unit of organic matter, showed a strong correlation with cumulative respiration. Results obtained support the hypothesis of a potential link between the thermal and biological stability of the studied organic materials, and consequently the ability of thermal analysis to characterize the maturity of municipal organic wastes and composts.     

Revisiting the elemental composition and the calorific value of the organic fraction of municipal solid wastes

In this work, the elemental content (C, N, H, S, O), the organic matter content and the calorific value of various organic components that are commonly found in the municipal solid waste stream were measured. The objective of this work was to develop an empirical equation to describe the calorific value of the organic fraction of municipal solid waste as a function of its elemental composition. The MSW components were grouped into paper wastes, food wastes, yard wastes and plastics. Sample sizes ranged from 0.2 to 0.5 kg. In addition to the above individual components, commingled municipal solid wastes were sampled from a bio-drying facility located in Crete (sample sizes ranged from 8 to 15 kg) and were analyzed for the same parameters. Based on the results of this work, an improved empirical model was developed that revealed that carbon, hydrogen and oxygen were the only statistically significant predictors of calorific value. Total organic carbon was statistically similar to total carbon for most materials in this work. The carbon to organic matter ratio of 26 municipal solid waste substrates and of 18 organic composts varied from 0.40 to 0.99. An approximate chemical empirical formula calculated for the organic fraction of commingled municipal solid wastes was C₃₂NH₅₅O₁₆.     

Investigation of potentially toxic heavy metals in different organic wastes used to fertilize market garden crops

The benefits of using organic waste as fertilizer and soil amendment should be assessed together with the environmental impacts due to the possible presence of heavy metals (HMs). This study involved analysing major element and HM contents in raw and size-fractionated organic wastes (17 sewage sludges and composts) from developed and developing countries. The overall HM concentration pattern showed an asymmetric distribution due to the presence of some wastes with extremely high concentrations. HM concentrations were correlated with the size of cities or farms where the wastes had been produced, and HM were differentiated with respect to their origins (geogenic: Cr–Ni; anthropogenic agricultural and urban: Cu–Zn; anthropogenic urban: Cd–Pb). Size fractionation highlighted Cd, Cu, Zn and Pb accumulation in fine size fractions, while Cr and Ni were accumulated in the coarsest. HM associations with major elements revealed inorganic (Al, Fe, etc.) bearing phases for Cr and Ni, and sulfur or phosphorus species for Cd, Cu Pb and Zn.     

Nitrogen mineralization and nitrate leaching of a sandy soil amended with different organic wastes.

Organic wastes can be recycled as a source of plant nutrients, enhancing crop production by improving soil quality. However, the study of the dynamic of soil nutrient, especially the N dynamic, after soil application of any organic material is vital for assessing a correct and effective use of the material, minimizing the losses of nitrate in leachates and avoiding the negative environmental effects that it may cause in groundwater. To estimate the effect of three organic materials, a municipal solid waste compost (MWC), a non-composted paper mill sludge (PS), and an agroforest compost (AC) on the N dynamic of a sandy soil two experiments were carried out: an incubation experiment and a column experiment. The incubation experiment was conducted to estimate the N mineralization rate of the different soil-amendment mixtures. The soil was mixed with the organic amendments at a rate equivalent to 50,000 kg ha(-1) and incubated during 40 weeks at constant moisture content (70% of its water-holding capacity) and temperature (28 degrees C) under aerobic conditions. Organic amendment-soil samples showed an immobilization of N during the first weeks, which was more noticeable and longer in the case of PS-treated soil compared to the other two amendments due to its high C/N ratio. After this immobilization stage, a positive mineralization was observed for all treatment, especially in MWC treated soil. Contemporaneously a 1-year column (19 cm diameter and 60 cm height) experiment was carried out to estimate the nitrate losses from the soil amended with the same organic materials. Amendments were mixed with the top soil (0-15 cm) at a rate equivalent to 50,000 kg ha(-1). The columns were periodically irrigated simulating rainfall in the area of study, receiving in total 415 mm of water, and the water draining was collected during the experimental period and analysed for NO3-N. At the end of the experimental period NO3-N content in soil columns at three depths (0-20, 20-35 and 35-50 cm) was determined. The nitrate concentration in drainage water confirmed the results obtained in the incubation experiment: nitrate leaching was higher in soil treated with MWC due to its higher N-mineralization rate. Nevertheless, the nitrate losses represented a low amount compared with the total nitrogen added to soil. No clear signs of water-draining contamination were observed during the first year after the application of AC and PS; however, the nitrate leaching in soil treated with MWC slightly exceeded the limit allowed for the Drinking Water Directive 98/83/CE.     

Comparison of aerobic and anaerobic stability indices through a MSW biological treatment process

A complex mechanical-biological waste treatment plant designed for the processing of mixed municipal solid wastes (MSW) and source-selected organic fraction of municipal solid wastes (OFMSW) has been studied by using stability indices related to aerobic (respiration index, RI) and anaerobic conditions (biochemical methane potential, BMP). Several selected stages of the plant have been characterized: waste inputs, mechanically treated wastes, anaerobically digested materials and composted wastes, according to the treatment sequence used in the plant. Results obtained showed that the main stages responsible for waste stabilization were the two first stages: mechanical separation and anaerobic digestion with a diminution of both RI and BMP around 40% and 60%, respectively, whereas the third stage, composting of digested materials, produced lesser biological degradation (20-30%). The results related to waste stabilization were similar in both lines (MSW and OFMSW), although the indices obtained for MSW were significantly lower than those obtained for OFMSW, which demonstrated a high biodegradability of OFMSW. The methodology proposed can be used for the characterization of organic wastes and the determination of the efficiency of operation units used in mechanical-biological waste treatment plants.     

Microbial biomass in a soil amended with different types of organic wastes.

Application of different types of organic wastes may have a marked effect on soil microbial biomass and its activity. The objective of this study was to quantify the amount of microbial biomass in a loamy-clayey soil, amended with different types of organic waste residues (composts of municipal solid waste of different ages, sewage sludge and farmyard manure) and incubated for 8 weeks at 25 degrees C and two-thirds of field capacity, using the fumigation-extraction method. Both microbial biomass-C and -N (BC and BN, respectively) appeared to be dependent on the type of organic waste residues, on their degree of stability, and on their chemical characteristics. In general, organic wastes increased the microbial biomass-C content in the soil and the microbial BC was positively correlated with the organic C content, the C/N, neutral detergent fibre/N (NDF/N) and acid detergent fibre/N (ADF/ N) ratios. The microbial biomass content decreased according to the period of incubation, especially when the compost used was immature. The microbial biomass-N was positively correlated with the total N and percentage of hemicellulose. The microbial biomass-C was linearly related with the microbial biomass-N and the ratio BC/BN was exponentially related with the BC.     

Anaerobic co-digestion of dairy cow manure and high concentrated food processing waste

Anaerobic co-digestion of dairy manure (DM) and concentrated food processing wastes (FPW) under thermophilic (55 °C) and mesophilic (35 °C) temperatures, and fertilizer value of the effluent were investigated in this study. Two types of influent feedstock were utilized: 100 % DM and a 7:3 mixture (wet weight basis) of DM and FPW. The contents of the FPW, as feedstock were 3:3:3:1 mixture of cheese whey, animal blood, used cooking oil and residue of fried potato. Four continuous digestion experiments were carried out in 10 L digesters. Co-digestion under thermophilic temperature increased methane production per digester volume. However, co-digestion at 35 °C was inhibited. Total Kjeldahl nitrogen (N) recovered after digestion ranged from 73.1 to 91.9 %, while recoveries of ammonium nitrogen (NH₄-N) exceeded 100 %. The high recovery of NH₄-N was attributed to mineralization of influent organic N. The mixture of DM and FPW showed greater recoveries of NH₄-N after digestion compared to DM only, reflecting its greater organic N degradability. The ratios of extractable to total calcium, phosphorus and magnesium were slightly reduced after digestion. These results indicate that co-digestion of DM and FPW under thermophilic temperature enhances methane production and offers additional benefit of organic fertilizer creation.     

Carbon mineralization dynamics in soils amended with meat meals under laboratory conditions

Meat and bone meal (MBM) is obtained from the wastes produced during slaughtering operations. Its high concentration of N and P makes it interesting as an organic fertiliser but its use in soil has been barely studied previously. In this work four laboratory experiments were performed to study the influence of different variables (MBM composition, rate of application, temperature of incubation and the type of soil) on C mineralization dynamics of MBM in agricultural soils. The total CO2-C evolved (as % of added C) after 2 weeks ranged between 10% and 20%. The kinetics of mineralization were rapid, with C evolved as CO2 within the first 4 days representing more than 50% of total C mineralized. A linear correlation was found between the rate of application (added-C) and CO2-C evolved (r2: 0.997; P<0.001). A temperature coefficient (Q10) was used to assess the difference in biological activity at 5 degrees C intervals. Q10, which ranged from 1.0 to 2.7 (250h), was higher for the lower temperature range (Q10 (15-20 degrees C)>Q10 (20-25 degrees C)) and it was found to be related to the soil properties. Finally, the mineralization process was found to be highly dependent upon the different soil factors, although no simple linear correlation was found between mineralization and soil properties.     

Influences of chlorine content on emission of HCl and organic compounds in waste incineration using fluidized beds

HCl and some organic compounds are the precursors of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) in municipal solid waste incinerators. In this work, a lab-scale fluidized bed incinerator is adopted to study the relationship between the organic and the inorganic chlorine contents of artificial wastes and the emissions of HCl and organic compounds. The lower threshold limit (LTL) of chlorine content below which HCl and organic compounds are not generated is studied. Experimental results showed that organic chlorides had a greater potential to release chlorine than inorganic chlorides. The generation of organic pollutants fell, but the emissions of HCl increased with the temperature. The concentrations of chlorophenols (CPs)/chlorobenzenes (CBs) increased with chlorine contents. No LTL existed for HCl regardless of whether CaO was added. The LTL for CPs was between 0.1 and 0.3wt% of inorganic chloride, but there was none for organic sources. For CBs, the LTL was between 0.5 and 1.0wt% for inorganics at 700 and 800 degrees C, but 0.1-0.3 wt% at 700 degrees C and 0.3-0.5 wt% at 800 degrees C for organics. The production of PAHs and benzene, toluene, ethylbenzene and xylene (BTEX) was related to the surplus hydrogen ions that were not reacted with the chlorine. Adding CaO inhibited the production of HCl, CBs and CPs, but did not seriously affect PAHs and BTEX.     

New Numerical Model Study on a Tidal Flat System – Seasonal,Daily and Tidal Variations

We developed a new numerical model to investigate the dynamics of tidal flat ecosystems and their role in water quality in terms of the carbon cycle. This model was applied to Isshiki, a natural tidal flat area, which is the largest in Mikawa Bay, Japan.This model dealt with variations of biochemical or physical interaction among dissolved oxygen and C–N–P species (comprised of carbon, nitrogen, and phosphorus elements) both on a short-time scale (<24 h) as well as over a long-time scale (seasonal variation). The model indicated that the time dependence on phytoplankton, NH₄–N, NO_x–N, and PO₄–P are more sensitive to daily environmental variation than to seasonal environmental variation. This means that the rotation speed of these materials in the tidal flat area is fast. Here, we defined the rotation speed as the ratio of total fluxes of substance to the mass of the substance. Phytoplankton with a high rotation speed in the tidal flat area means that the tidal flat has the potential to recover from rapidly increasing phytoplankton: red tide. The model also indicated that the peculiar feature of the tidal flat is the mineralization of organic material. The effect on a long term base, is that it prevents the accumulation of sediment, which results in controlling the increase of oxygen consumption in benthic system, which is the cause of oxygen depleted water.     

Modelling of organic matter dynamics during the composting process

Composting urban organic wastes enables the recycling of their organic fraction in agriculture. The objective of this new composting model was to gain a clearer understanding of the dynamics of organic fractions during composting and to predict the final quality of composts. Organic matter was split into different compartments according to its degradability. The nature and size of these compartments were studied using a biochemical fractionation method. The evolution of each compartment and the microbial biomass were simulated, as was the total organic carbon loss corresponding to organic carbon mineralisation into CO₂. Twelve composting experiments from different feedstocks were used to calibrate and validate our model. We obtained a unique set of estimated parameters. Good agreement was achieved between the simulated and experimental results that described the evolution of different organic fractions, with the exception of some compost because of a poor simulation of the cellulosic and soluble pools. The degradation rate of the cellulosic fraction appeared to be highly variable and dependent on the origin of the feedstocks. The initial soluble fraction could contain some degradable and recalcitrant elements that are not easily accessible experimentally.     

Co-composting rice hulls and/or sawdust with poultry manure in NE Argentina

Rice hulls and sawdust are two common C-rich wastes derived from rice and timber agro-industries in subtropical NE Argentina. An alternative to the current management of these wastes (from bedding to uncontrolled burning) is composting. However, given their C-rich nature and high C/N ratio, adequate composting requires mixing with a N-rich waste, such as poultry manure. The effect of different proportions of poultry manure, rice hulls and/or sawdust on composting efficiency and final compost quality was studied. Five piles were prepared with a 2:1 and 1:1 ratio of sawdust or rice hulls to poultry manure, and 1:1:1 of all three materials (V/V). Different indicators of compost stability and quality were measured. Thermophilic phase was shorter for piles with rice hulls than for piles with sawdust (60 days vs. 105 days). Time required for stability was similar for both C-rich wastes (about 180 days). Characteristics of final composts were: pH 5.8–7.2, electrical conductivity 2.5–3.3 mS/cm, organic C 20–26%, total N 2.2–2.9%, lignin 19–22%, total Ca 18–24 g/kg, and extractable P 6–8 g/kg, the latter representing 60% of total P. Nitrogen conservation was high in all piles, especially in the one containing both C-rich wastes. Piles with sawdust were characterized by high total and available N, while piles with only rice hulls had higher Si, K and pH. Extractable P was higher in 1:1 piles, and organic C in 2:1 piles.     

Treatment of copper industry waste and production of sintered glass-ceramic.

Copper waste is iron-rich hazardous waste containing heavy metals such as Cu, Zn, Co, Pb. The results of leaching tests show that the concentration of these elements exceeds the Turkish and EPA regulatory limits. Consequently, this waste cannot be disposed of in its present form and therefore requires treatment to stabilize it or make it inert prior to disposal. Vitrification was selected as the technology for the treatment of the toxic waste under investigation. During the vitrification process significant amounts of the toxic organic and inorganic chemical compounds could be destroyed, and at the same time, the metal species are immobilized as they become an integral part of the glass matrix. The copper flotation waste samples used in this research were obtained from the Black Sea Copper Works of Samsun, Turkey. The samples were vitrified after being mixed with other inorganic waste and materials. The copper flotation waste and their glass-ceramic products were characterized by X-ray analysis (XRD), scanning electron microscopy and by the toxicity characteristic leaching procedure test. The products showed very good chemical durability. The glass-ceramics fabricated at 850 degrees C/2 h have a large application potential especially as construction and building materials.     

Sewage sludge composting: Influence of initial mixtures on organic matter evolution and N availability in the final composts

The influence of bulking agents on organic matter (OM) stability and nitrogen (N) availability in sewage sludge composts was investigated. The same sludge was composted on an industrial plant with different mixtures of bulking agents. The composting process included an active phase and a curing phase, both lasting 6 weeks, separated by the screening of composts. The OM evolution was characterised by carbon (C) and N mass balances in biochemical fractions. The OM stability and N potential availability of final composts were measured during soil incubations. During composting, the C and N losses reached more than 62% of the initial C and more than 45% of the initial N, respectively, due to C mineralisation or N volatilisation and screening. The bulking materials mostly influenced OM evolution during the active phase. They contributed to the mitigation of N losses during the active phase where N immobilisation through active microbial activity was favoured by bulking agents increasing the C:N ratio of the initial mixtures. However, the influence of bulking agents on OM evolution was removed by the screening; this induced the homogenisation of compost characteristics and led to the production of sludge composts with similar organic matter characteristics, C degradability and N availability.     

Assessment of chemical and biochemical stabilization of organic C in soils from the long-term experiments at Rothamsted (UK)

Biological and chemical stabilization of organic C was assessed in soils sampled from the long-term experiments at Rothamsted (UK), representing a wide range of carbon inputs and managements by extracting labile, non-humified organic matter (NH) and humic substances (HS). Four sequentially extracted humic substances fractions of soil organic matter (SOM) were extracted and characterized before and after a 215-day laboratory incubation at 25 degrees C from two arable soils, a woodland soil and an occasionally stubbed soil. The fractions corresponded to biochemically stabilised SOM extracted in 0.5M NaOH (free fulvic acids (FA) and humic acids (HA)) and chemically plus biochemically stabilised SOM extracted from the residue with 0.1M Na4P2O7 plus 0.1M NaOH (bound FA and HA). Our aim was to investigate the effects of chemical and biochemical stabilization on carbon sequestration. The non-humic to humic (NH/H) C ratio separated the soils into two distinct groups: arable soils (unless fertilised with farmyard manure) had an NH/H C ratio between 1.05 and 0.71, about twice that of the other soils (0.51-0.26). During incubation a slow, but detectable, decrease in the NH/H C ratio occurred in soils of C input equivalent or lower to 4Mgha(-1)y(-1), whereas the ratio remained practically constant in the other soils. Before incubation the free to bound humic C ratio increased linearly (R2=0.91) with C inputs in the soils from the Broadbalk experiment and decreased during incubation, showing that biochemical stabilization is less effective than chemical stabilization in preserving humic C. Changes in delta13C and delta15N after incubation were confined to the free FA fractions. The delta13C of free FA increased by 1.48 and 0.80 per thousand, respectively, in the stubbed and woodland soils, indicating a progressive biological transformation. On the contrary, a decrease was observed for the bound FA of both soils. Concomitantly, a Deltadelta15N of up to +3.52 per thousand was measured after incubation in the free FA fraction and a -2.58 Deltadelta15N in the bound FA. These changes, which occurred during soil incubation in the absence of C inputs, indicate that free FA fractions were utilised by soil microorganisms, and bound FA were decomposed and replaced, in part, by newly synthesized FA. The 13CPMAS-TOSS NMR spectra of free HA extracted before and after 215 days of incubation were mostly unchanged. In contrast, changes were evident in bound HA and showed an increase in aromatic C after incubation.