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The effect of microbial inoculation and pH on microbial community structure changes during composting
Process Biochemistry 35 (2000) 923 – 929 www.elsevier.com/locate/procbio
The effect of microbial inoculation and pH on microbial community structure changes during composting F. Lei, J.S. VanderGheynst * Department of Biological and Agricultural Engineering, Uni6ersity of California, One Shields A6enue, Da6is, CA 95616, USA Received 11 October 1999; received in revised form 26 October 1999; accepted 27 November 1999
Abstract Phospholipid fatty acid (PLFA) analysis was used to characterize microbial community structure during the composting of grape pomace and rice straw. Composting studies were completed in 30 litre static-bed reactors with continuous temperature and oxygen monitoring. The effects of inoculation and pH adjustment on microbial community structure and level of decomposition during composting were investigated. Principal components analysis (PCA) of the PLFA data showed that inoculation had little effect on the microbial community structure of the compost once temperature had peaked, while process temperature and the adjustment of initial pH had a significant effect. Adjustment of pH and inoculation did not significantly increase the level of decomposition as measured by oxygen consumption. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Microbial communities; Phospholipid fatty acid; Grape pomace
1. Introduction As legislation requires more food wastes to be diverted from landfills, food industries must consider other options for the disposal of food processing residues. One popular alternative to disposal is composting. Although composting is not a new waste treatment method, some of the characteristics of food waste offer unique challenges to processors. For example, grape pomace, a residue of wine processing, can have significant levels of phenolic compounds that might inhibit the decomposition process. The low pH of these residues might also increase the time required for composting. Management of the process by the addition of microorganisms capable of tolerating and/or decomposing inhibitory compounds, or the adjustment of pH, may offer a means of increasing the rate of decomposition of these materials. CO2 evolution and O2 consumption rates have been used to measure decomposition rates for composting processes performed under different management * Corresponding author. Tel.: +1-530-7520989; fax: + 1-5307522640. E-mail address: [email protected] (J.S. VanderGheynst)
schemes [1–5]. However, there have been very few analyses of the effect of different management alternatives on the microbial community dynamics during composting. One method of studying microbial community changes during composting is phospholipid fatty acid (PLFA) analysis. The rationale behind PLFA analysis is that most microorganisms contain phospholipids in their membranes that are not stored but are turned over relatively rapidly during metabolism [6]. Therefore, determining the total PLFA in an environmental sample can provide an estimate of viable microbial biomass contained within the sample [7]. In addition, certain PLFAs can be regarded as taxonomic or physiological biomarkers for microbial genera [8,9]. There have been several studies that have used PLFA analysis in the monitoring of microbial community changes during composting processes. Hellman and co-workers [10] used lipid analysis to examine the changes in community structure and biomass during changes in the composition of the effluent gas stream from a municipal solid waste windrow composting process. Samples taken during the early mesophilic stages of composting contained high levels of lipids characteristic of eucaryotes, which decreased rapidly over time as temperature and CO2 evolution rate increased and pH
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Table 1 Experimental variables in the rice straw and grape pomace composting studies Treatment ( c)
Inoculum
pH adjustment
Ratio of rice straw to grape pomace (kg dry/kg dry)
Initial volatile solids (% of total solids)
Initial pH
1 2 3 4
+ – – –
– – – +
l:3.4 1:3.4 1:5.0 1:5.0
89 89 88 89
4.11 4.10 4.08 5.98
decreased. During the thermophilic phase of composting, they observed high levels of lipids associated with actinomycetes and Gram-positive bacteria. In addition, simultaneous increases in methane emission and ether lipids, indicators of archaean methanogens, were observed. Herrmann and Shann [11] used PLFA profiles to characterize the microbial community changes during municipal solid waste composting managed by an aerated-mixed method. They found that samples obtained from a particular stage of composting (i.e. mesophilic, thermophilic and curing stages) had similar lipid contents. In the mesophilic stage they observed lipids characteristic of fungi, while in the thermophilic stages they observed lipids characteristic of thermophilic bacteria and actinomycetes. Lipid biomarkers associated with actinomycetes and fungi were observed in cured samples. They found that very old samples tended to have common lipid profiles which suggests that PLFA analysis may provide a means of assessing composting maturity. In summary, analysis of PLFA directly extracted from compost samples shows great promise as a means of increasing our understanding of the composting process. The objective of this study was to use PLFA analysis to study microbial community changes during the composting of rice straw and grape pomace. Additionally the effects of compost inoculation and pH adjustment on rates of decomposition and microbial community structure were investigated.
The initial moisture content of the compost mixtures ranged from 74 to 78% (g H2O/g wet solids). Inoculation of substrates was completed by mixing 5% (g dry inoculum/g total dry solids) several-week-old grape pomace and rice straw compost with fresh grape pomace and rice straw. Adjustment of pH was accomplished by uniformly adding a 5.0 N NaOH solution to the composting mixture. All composts were produced using a 30 litre static bed bioreactor with a 30 cm diameter (Fig. 1). The bioreactor was aerated with humidified air at 5 litre/ min to maintain effluent O2 concentrations above 10%. Temperature was measured at nine different locations in the reactor and oxygen concentration was measured in the effluent gas. Type T thermocouples were used to measure temperature and a zirconia oxide-based oxygen sensor was used to monitor O2 (NeuwGhent Technology, LaGrangeville, NY). Temperature and oxygen readings were recorded every 30 min using a data logger (Campbell Scientific, Logan, UT). Compost samples were collected for pH, moisture content, volatile solids and PLFA analysis at three points in the process; at the beginning, when the peak temperature occurred and at the end of the processes. Sample analyses were completed in triplicate immediately upon removal from the bioreactor. Moisture content was measured by oven drying at 101°C for 24 h. Volatile solids content was determined by combusting samples at 550°C for at least 6 h in a muffle furnace. Compost pH was measured on compost extracts diluted
2. Materials and methods Compost was produced from a mixture of grape pomace and rice straw. Experimental variables are listed in Table 1. Fermented grape pomace (pressed skins, seeds and liquid) was obtained from a pilot-scale wine fermentation facility in the Department of Viticulture and Enology at UC Davis and stored at − 20°C. Dry rice straw was obtained in northern California. The particle size of the rice straw was reduced by hammer milling through a 1 in. screen. The compost mixture was prepared by mixing thawed grape pomace with straw using the ratios listed in Table 1. Reactors were loaded with : 5.4 kg of the wet compost mixtures.
Fig. 1. Static-bed composting reactor.
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Table 2 Summary of experimental results Treatment ( c )
Initial PLFA (ng/g dry compost) *10−4
Final moisture content (g H2O/g wet weight* 100%)
Final volatile solids content (% of total solids)
Final pH
1 2 3 4
4.18 90.03 5.29 9 0.39 6.13 91.43 6.11 9 0.33
70 71 74 75
88 87 88 88
3.78 3.80 3.81 5.01
2:1 (g H2O:g wet compost) with distilled deionized water. PLFA analysis was conducted in a similar manner as described by Bossio and Scow [12]. Compost samples of 4 g dry weight were agitated in 33.25 ml of a singlephase mixture of chloroform, methanol and phosphate buffer in an initial ratio of 1:2:0.8 [13]. Phosphate buffer addition was adjusted to account for water in the compost sample. After a 2 h extraction, the samples were centrifuged at 5000 rpm for 10 min and the supernatant decanted into a separatory funnel. Compost samples were re-extracted with 23 ml of extractant for another 30 min. The supernatant from the second extraction was added to the separatory funnel along with 15.14 ml of phosphate buffer and 14.75 ml of CHCl3. The final CHCl3:buffer:methanol ratio was 1:0.9:1. After shaking for 2 min, the samples were allowed to stand overnight for phase separation. The bottom layer from the separating funnel, which contained the lipids, was collected into a test tube and dried under N2 at 32°C in a water bath. Phospholipids were separated from the neutral and glycolipids using solid phase extraction columns containing 500 mg of silicic acid (Phase Separations, Franklin, MA). The columns were conditioned with 3 ml of CHCl3. Lipids were then transferred to the column in 1 ml of CHC13. Neutral lipids, glycolipids and polar lipids were fractionated with 5 ml CHCl3, 10 ml acetone, and 5 ml methanol, respectively. Phospholipids (polar lipids) were dried under N2 at 32°C in a water bath. The phospholipids were subjected to mild alkaline methanolysis by exposure to 1 ml of 1:1 methanol to toluene and 1 ml of 0.2 M KOH at 37°C for 10 min, which yielded fatty acid methyl esters (FAME). The FAME were extracted with 2 ml of hexane, 2 ml of H2O and 0.3 ml of 1.0 M acetic acid, and then the hexane fraction was collected. Another 2 ml of hexane was used for further extraction of the sample. The hexane samples containing the FAME were combined and washed with 4 ml of 0.03 N NaOH and then dried under N2 at room temperature. The dried FAME were dissolved into 250 m1 hexane with C19:0 as an internal standard and analyzed using a Hewlett Packard 6890 gas chromatograph with a 25-m Ultra 2 (5% phenyl) methylpolysiloxane column (J&W
Scientific). Peaks were identified using fatty acid standards and the microbial identification peak identification software (MIDI, Newark, DE). The system of fatty acid nomenclature is as follows: the number before the colon gives the number of carbons, the number after the colon gives the count of double bonds, and the position of the double bond from the methyl end of the molecule is indicated last. The last number is followed by a ‘c’ for cis or a ‘t’ for trans geometry. The prefixes ‘i’ and ‘a’ stand for iso or anteiso, respectively. The fatty acids with a hydroxy group and a methyl group are given as OH and Me, respectively; the prefix number indicates position from the carboxyl end of the molecule. Cryclopropyl fatty acids are presented as ‘cy’. Changes in the PLFA content of samples was analyzed using principal components analysis (PCA). Analyses were done using the results for 27 individual PLFAs with carbon chain lengths between 15 and 20. PCA which was completed using the program SAS (SAS Institute, Cary, NC).
3. Results and discussion Experimental results from the four composting processes are summarized in Table 2. A decrease in pH was observed for all of the treatments. The pH of treatments c 1–3 dropped by : 0.3 pH units during the process while the pH of treatment c 4 dropped by nearly 1 pH unit. Moisture content and volatile solids content did not decrease appreciably during any of the processes. The final moisture and volatile solids contents of the samples ranged from 70–75 and 87–88%, respectively. The total PLFAs in the initial samples for treatments c2–4 were not statistically different. Thus, increasing the ratio of grape pomace to rice straw did not have a significant effect on the total initial PLFAs in the processes. Treatment c 1, which was inoculated with matured grape pomace and rice straw compost, had a lower initial PLFA content than the other treatments. The matured compost may have had a lower biomass concentration than the raw compost substrates, thereby reducing the initial total PLFAs in treatment c1.
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F. Lei, J.S. VanderGheynst / Process Biochemistry 35 (2000) 923–929 Table 3 Variances of principal components from PCA for two groups of treatments
PC1 PC2 PC3
Treatment c 1 and 2 cumulative percent variance
Treatment c3 and 4 cumulative percent variance
35 56 67
29 45 57
Fig. 2. Temperature profiles during the composting experiments. Temperature measurements represent data taken from central thermocouples.
Fig. 4. PCA of PLFA compositions for samples from treatments c 1 and 2 (effect of inoculation).
Fig. 3. Cumulative oxygen depletion in the composting experiments.
Temperature profiles for the four experiments are illustrated in Fig. 2. The peak temperatures for all four treatments fell within a range that could support mesophilic and thermophilic microbial communities. The temperature in treatment cl started to increase at : 50 h into the process and remained at a peak temperature for about 50 h. For treatment c 2 the temperature also started to increase at :50 h into the process, but peaked and began to drop between 75 and 80 h. Thus, inoculation did not significantly increase the rate of temperature rise, but did increase the duration of time the composting temperature remained above 40°C for grape pomace and rice straw compost. In treatments c 3 and 4 the temperature started to rise at about 75 h and peaked at about 150 h into the process. The difference in the rate of temperature rise between treatments c l and 2 and treatments c 3 and 4 may have been due to the higher ratio of grape pomace to rice straw in treatments c3 and 4. Although the greater amount of grape pomace may have increased the level of biodegradable carbon sources available for microbial growth, it also may have increased some inhibiting compounds to micro-organ-
isms, such as alcohol and phenolic compounds, which may have lowered the activity of the initial population of micro-organisms in the composting materials. In addition, the temperature in treatment c 4 peaked at a higher level and remained above 40°C longer than the other three treatments. This may have been due to the adjustment of pH in this treatment. Cumulative O2 depletion results for the four experiments are presented in Fig. 3. Early in the process treatment c2 had the highest rate of O2 depletion, as indicated by higher cumulative O2 levels. However, total O2 depletion levels were very similar for all treatments by 150 h into the processes. These results suggest that inoculation and pH adjustment did not improve the overall level of decomposition for grape pomace and rice straw compost. Results from PCA of PLFA data from treatments c 1 and 2 are presented in Table 3 and Figs. 4 and 5. This analysis emphasizes the effect of inoculation on the microbial community structure. The first two principal components (PCs) represent 56% of the variance. As illustrated in the principal component plots in Figs. 4 and 5, there were differences observed between the two treatments and samples within treatments over time. Differences in the initial samples were due to PLFAs 18:lw9c, 18:2w6,9c, and straight-chain saturated PLFAs; levels of these PLFA were higher in treatment c 2 than in treatment c 1. The PLFAs 18:1w9c and 18:2w6,9c have been found to be enriched in samples containing fungi [14,15], while straight chain saturated PLFA are common biomarkers for eubacteria [7,16]. The higher levels of 18: lw9c and 18:2w6,9c in treat-
F. Lei, J.S. VanderGheynst / Process Biochemistry 35 (2000) 923–929
Fig. 5. PCA of PLFAs from treatments c 1 and 2 (effect of inoculation).
Fig. 6. PCA of PLFA compositions for samples from treatments c 3 and 4 (effect of initial pH adjustment).
Fig. 7. PCA of PLFAs from treatments c 3 and 4 (effect of initial pH adjustment).
ment c2 may have been a result of yeast remaining in the grape pomace. The inoculum in treatment c 1, which consisted of several-week-old grape pomace
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and rice straw compost that had been exposed to thermophilic temperatures, likely had low levels of yeast, thus reducing the biomarkers 18: lw9c and 18:2w6,9c in this treatment. In both treatments cl and 2, differences in PLFA content were observed between the initial samples and samples obtained when temperature peaked (Figs. 2, 4 and 5). In general, the levels of straight chain saturated PLFA and branched-monounsaturated PLFA decreased as temperature increased. Both types of PLFA are common biomarkers for eubacteria [7,16]. The decrease in the levels of these PLFA was likely a result of the reduction in mesophilic bacteria as temperature increased during the processes. A similar trend in straight chain saturated PLFA was also observed by Hellmann and co-workers [10] as temperature increased during windrow composting. As the process temperatures peaked, the difference in PLFA content between treatments c 1 and 2 decreased as indicated by overlap of points representing samples from 75 h in treatment c 1 and samples from 75 and 95 h from treatment c 2 on the score plot (Fig. 4). These results suggest that inoculation of the composting process had little effect on the microbial community structure dominant in the highmesophilic and low-thermophilic temperature region for this mixture. The microbial community structure continued to change over time as indicated by the difference in PC1 for samples taken at 75 and 170 h in treatment c 1 and 95 and 240 h in treatment c 2 (Fig. 4). The levels of branched monounsaturated PLFA and terminally branched saturated PLFA increased over time. This change was greater in treatment c 1 than in treatment c 2. Terminally branched saturated PLFA have been found in some thermophilic bacteria [15,17,18], and have been observed to be enriched in thermophilic composts [10,11]. Since the last sample from treatment c 1 was taken shortly after the thermophilic phase, it is likely that thermophlic bacteria were still present in the compost. However, the last sample from treatment c 2 was taken a few days after the thermophilic phase of composting had ended, which may explain the lower levels of biomarkers for thermophilic bacteria in this sample. Results from PCA of PLFA data from treatments c 3 and 4 are listed in Table 3 and illustrated in Figs. 6 and 7. This analysis represents the effect of pH adjustment on the microbial community. The first two PCs represent 45% of the variance. The PLFA content of initial samples from treatments c 3 and 4 were very similar as shown on the PC plot in Fig. 6. This suggests that pH adjustment did not affect the initial microbial community structure of the compost.
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As the process temperature increased, differences in PLFA content in samples taken at 170 h in treatment c 3 and at 120 and 170 h in treatment c 4 were observed. In both treatments an increase in straight chain saturated PLFA and branched monounsaturated PLFA was observed as temperature increased. In treatment c 4 an increase in the levels of terminally branched saturated PLFA was also observed as temperature increased. These results indicate that there was an overall increase in eubacteria in both treatments. The higher temperature in treatment c 4 was likely responsible for the higher levels of thermophilic bacteria and thus terminally branched saturated PLFA in this treatment. The higher pH in treatment c 4 may have made the compost substrate more favourable to colonization by thermophilic bacteria. The PLFA content of samples taken at the end of the processes was also quite different between treatments c 3 and 4. In treatment c3 there was little difference in the PLFA content of samples analyzed at 170 and 265 h. However, in treatment c 4 levels of terminally branched saturated PLFA and branched monounsaturated PLFA increased from 170 to 290 h. Treatment c 3 had higher levels of branched unsaturated PLFAs than treatment c 4, while treatment c 4 had higher levels of terminally branched saturated PLFAs. Treatment c4 experienced higher temperatures for longer times that treatment c 3. This longer exposure to higher temperatures likely favored the growth of thermophilic micro-organisms in treatment c 4, while the shorter exposure in treatment c 3 favored the growth of mesophilic micro-organisms. Despite the large differences in microbial community structure observed in the PCA analysis, the cumulative O2 profiles from treatments c3 and 4 followed very similar trends between 0 and 200 h (Fig. 2). Thus, PLFA analysis may provide a means of identifying differences between composts with similar trends in decomposition rate.
4. Conclusions PLFA analysis was successfully used to study microbial community differences in grape pomace and rice straw composting processes. PCA of PLFA data showed that inoculation had little effect on the microbial community structure of the compost when the temperature of the process peaked. Measurements of cumulative O2 consumption also confirmed that inoculation had little affect on the level of decomposition, however inoculated compost did remain above 40°C longer than compost that was not inoculated.
Adjustment of pH had a significant effect on the microbial community structure throughout the composting process. This was likely due to the effect pH adjustment had on increasing the time the process was exposed to thermophilic temperatures. However, O2 consumption was the same regardless of pH adjustment. Increasing the level of grape pomace in the compost did increase the time to achieve thermophilic temperatures. This may have been due to the presence of phenolic compounds that inhibited the initial microbial community. Higher levels of grape pomace had only a small affect on the overall levels of decomposition as measured by cumulative O2 consumption. References [1] VanderGheynst JS, Cogan DJ, DeFelice PJ, Gossett JM, Walker LP. Effect of process management on the emission of organosulfur compounds and gaseous antecedents from composting processes. Environ Sci Technol 1998;32:3713 – 8. [2] Schulze KL. Rate of oxygen consumption and respiratory quotients during the aerobic decomposition of a synthetic garbage. Compost Sci 1960;1:36 – 40. [3] Walker LP, Nock TD, Gossett JM, VanderGheynst JS. The role of periodic water addition in managing moisture limitations during high solids aerobic decomposition. Process Biochem 1999;34(6-7):601– 12. [4] Bach PD, Shoda M, Kubota H. Composting reaction rate of sewage sludge in an autothermal packed bed reactor. J Ferment Technol 1985;63:271 – 8. [5] Elwell DL, Keener HM, Hansen RC. Controlled, high rate composting of mixtures of food wastes, yard waste and chicken manure. In: Seventh International Symposium on Agricultural and Food Processing Wastes. Chicago, IL: American Society of Agricultural Engineers, 1995. [6] White DC, Davis WM, Nickels JS, King JD, Bobbie RJ. Determination of sedimentary microbial biomass by extractable lipid phosphate. Oecologia 1979;40:51 – 62. [7] Vestal JR, White DC. Lipid analysis in microbial ecology. Bioscience 1989;39(8):535– 41. [8] White DC. Chemical ecology: possible linkage between macroand microbial ecology. Oikos 1995;74:177 – 84. [9] Zelles L, Bai QY, Rackwitz R, Chadwick D, Beese F. Determination of phospholipid- and lipopolysaccharide-derived fatty acids as an estimate of microbial biomass and community structures in soils. Biol Fertil Soils 1995;19:115 – 23. [10] Hellmann B, Zelles L, PalojN0 rvi A, Bai Q. Emission of climate-relevant trace gases and succession of microbial communities during open-windrow composting. Appl Environ Microbiol 1997;63(3):1011– 8. [11] Herrmann RF, Shann JF. Microbial community changes during the composting of municipal solid waste. Microb Ecol 1997;33:78 – 85. [12] Bossio DA, Scow KM. Impacts of carbon and flooding on soil microbial communities: phospholipid fatty acid profiles and substrate utilization patterns. Microb Ecol 1998;35:265–78. [13] Thigh EG, Dyer WJ. A rapid method of total lipid extraction and purlfication. Can J Biochem Physiol 1959;37:911 –5. [14] Frostega¨rd A, , Ba¨a¨th E. The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil. Biol Fertil Soils 1996;22:59 – 65.
F. Lei, J.S. VanderGheynst / Process Biochemistry 35 (2000) 923–929 [15] Federle TW. Microbial distribution in soil. In: Megusar F, Gantar M, editors. Perspectives in Microbial Ecology. Ljubljana, Yugoslavia: Slovene Society for Microbiology, 1986:493– 8. [16] Lechevalier H, Lechevalier MP. In: Ratledge C, Wilkinson SG, editors. Chemotaxonomic Use of Lipids and Overview. London: Academic Press, 1988:869–902.
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[17] Kroppenstedt RM, Goodfellow M. The family Thermomonosporaceae. In: Balows A, editor. The Prokaryotes, 2nd Edition. New York: Springer Verlag, 1992:1085 – 114. [18] O’Leary WM, Wilkinson SG. G-positive bacteria. In: Ratledge C, Wilkinson SG, editors. Microbial Lipids. New York: Academic Press, 1988:117 – 202.
The effect of microbial inoculation and pH on microbial community structure changes during composting F. Lei, J.S. VanderGheynst * Department of Biological and Agricultural Engineering, Uni6ersity of California, One Shields A6enue, Da6is, CA 95616, USA Received 11 October 1999; received in revised form 26 October 1999; accepted 27 November 1999
Abstract Phospholipid fatty acid (PLFA) analysis was used to characterize microbial community structure during the composting of grape pomace and rice straw. Composting studies were completed in 30 litre static-bed reactors with continuous temperature and oxygen monitoring. The effects of inoculation and pH adjustment on microbial community structure and level of decomposition during composting were investigated. Principal components analysis (PCA) of the PLFA data showed that inoculation had little effect on the microbial community structure of the compost once temperature had peaked, while process temperature and the adjustment of initial pH had a significant effect. Adjustment of pH and inoculation did not significantly increase the level of decomposition as measured by oxygen consumption. © 2000 Elsevier Science Ltd. All rights reserved. Keywords: Microbial communities; Phospholipid fatty acid; Grape pomace
1. Introduction As legislation requires more food wastes to be diverted from landfills, food industries must consider other options for the disposal of food processing residues. One popular alternative to disposal is composting. Although composting is not a new waste treatment method, some of the characteristics of food waste offer unique challenges to processors. For example, grape pomace, a residue of wine processing, can have significant levels of phenolic compounds that might inhibit the decomposition process. The low pH of these residues might also increase the time required for composting. Management of the process by the addition of microorganisms capable of tolerating and/or decomposing inhibitory compounds, or the adjustment of pH, may offer a means of increasing the rate of decomposition of these materials. CO2 evolution and O2 consumption rates have been used to measure decomposition rates for composting processes performed under different management * Corresponding author. Tel.: +1-530-7520989; fax: + 1-5307522640. E-mail address: [email protected] (J.S. VanderGheynst)
schemes [1–5]. However, there have been very few analyses of the effect of different management alternatives on the microbial community dynamics during composting. One method of studying microbial community changes during composting is phospholipid fatty acid (PLFA) analysis. The rationale behind PLFA analysis is that most microorganisms contain phospholipids in their membranes that are not stored but are turned over relatively rapidly during metabolism [6]. Therefore, determining the total PLFA in an environmental sample can provide an estimate of viable microbial biomass contained within the sample [7]. In addition, certain PLFAs can be regarded as taxonomic or physiological biomarkers for microbial genera [8,9]. There have been several studies that have used PLFA analysis in the monitoring of microbial community changes during composting processes. Hellman and co-workers [10] used lipid analysis to examine the changes in community structure and biomass during changes in the composition of the effluent gas stream from a municipal solid waste windrow composting process. Samples taken during the early mesophilic stages of composting contained high levels of lipids characteristic of eucaryotes, which decreased rapidly over time as temperature and CO2 evolution rate increased and pH
0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 9 ) 0 0 1 5 5 - 7
F. Lei, J.S. VanderGheynst / Process Biochemistry 35 (2000) 923–929
924
Table 1 Experimental variables in the rice straw and grape pomace composting studies Treatment ( c)
Inoculum
pH adjustment
Ratio of rice straw to grape pomace (kg dry/kg dry)
Initial volatile solids (% of total solids)
Initial pH
1 2 3 4
+ – – –
– – – +
l:3.4 1:3.4 1:5.0 1:5.0
89 89 88 89
4.11 4.10 4.08 5.98
decreased. During the thermophilic phase of composting, they observed high levels of lipids associated with actinomycetes and Gram-positive bacteria. In addition, simultaneous increases in methane emission and ether lipids, indicators of archaean methanogens, were observed. Herrmann and Shann [11] used PLFA profiles to characterize the microbial community changes during municipal solid waste composting managed by an aerated-mixed method. They found that samples obtained from a particular stage of composting (i.e. mesophilic, thermophilic and curing stages) had similar lipid contents. In the mesophilic stage they observed lipids characteristic of fungi, while in the thermophilic stages they observed lipids characteristic of thermophilic bacteria and actinomycetes. Lipid biomarkers associated with actinomycetes and fungi were observed in cured samples. They found that very old samples tended to have common lipid profiles which suggests that PLFA analysis may provide a means of assessing composting maturity. In summary, analysis of PLFA directly extracted from compost samples shows great promise as a means of increasing our understanding of the composting process. The objective of this study was to use PLFA analysis to study microbial community changes during the composting of rice straw and grape pomace. Additionally the effects of compost inoculation and pH adjustment on rates of decomposition and microbial community structure were investigated.
The initial moisture content of the compost mixtures ranged from 74 to 78% (g H2O/g wet solids). Inoculation of substrates was completed by mixing 5% (g dry inoculum/g total dry solids) several-week-old grape pomace and rice straw compost with fresh grape pomace and rice straw. Adjustment of pH was accomplished by uniformly adding a 5.0 N NaOH solution to the composting mixture. All composts were produced using a 30 litre static bed bioreactor with a 30 cm diameter (Fig. 1). The bioreactor was aerated with humidified air at 5 litre/ min to maintain effluent O2 concentrations above 10%. Temperature was measured at nine different locations in the reactor and oxygen concentration was measured in the effluent gas. Type T thermocouples were used to measure temperature and a zirconia oxide-based oxygen sensor was used to monitor O2 (NeuwGhent Technology, LaGrangeville, NY). Temperature and oxygen readings were recorded every 30 min using a data logger (Campbell Scientific, Logan, UT). Compost samples were collected for pH, moisture content, volatile solids and PLFA analysis at three points in the process; at the beginning, when the peak temperature occurred and at the end of the processes. Sample analyses were completed in triplicate immediately upon removal from the bioreactor. Moisture content was measured by oven drying at 101°C for 24 h. Volatile solids content was determined by combusting samples at 550°C for at least 6 h in a muffle furnace. Compost pH was measured on compost extracts diluted
2. Materials and methods Compost was produced from a mixture of grape pomace and rice straw. Experimental variables are listed in Table 1. Fermented grape pomace (pressed skins, seeds and liquid) was obtained from a pilot-scale wine fermentation facility in the Department of Viticulture and Enology at UC Davis and stored at − 20°C. Dry rice straw was obtained in northern California. The particle size of the rice straw was reduced by hammer milling through a 1 in. screen. The compost mixture was prepared by mixing thawed grape pomace with straw using the ratios listed in Table 1. Reactors were loaded with : 5.4 kg of the wet compost mixtures.
Fig. 1. Static-bed composting reactor.
F. Lei, J.S. VanderGheynst / Process Biochemistry 35 (2000) 923–929
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Table 2 Summary of experimental results Treatment ( c )
Initial PLFA (ng/g dry compost) *10−4
Final moisture content (g H2O/g wet weight* 100%)
Final volatile solids content (% of total solids)
Final pH
1 2 3 4
4.18 90.03 5.29 9 0.39 6.13 91.43 6.11 9 0.33
70 71 74 75
88 87 88 88
3.78 3.80 3.81 5.01
2:1 (g H2O:g wet compost) with distilled deionized water. PLFA analysis was conducted in a similar manner as described by Bossio and Scow [12]. Compost samples of 4 g dry weight were agitated in 33.25 ml of a singlephase mixture of chloroform, methanol and phosphate buffer in an initial ratio of 1:2:0.8 [13]. Phosphate buffer addition was adjusted to account for water in the compost sample. After a 2 h extraction, the samples were centrifuged at 5000 rpm for 10 min and the supernatant decanted into a separatory funnel. Compost samples were re-extracted with 23 ml of extractant for another 30 min. The supernatant from the second extraction was added to the separatory funnel along with 15.14 ml of phosphate buffer and 14.75 ml of CHCl3. The final CHCl3:buffer:methanol ratio was 1:0.9:1. After shaking for 2 min, the samples were allowed to stand overnight for phase separation. The bottom layer from the separating funnel, which contained the lipids, was collected into a test tube and dried under N2 at 32°C in a water bath. Phospholipids were separated from the neutral and glycolipids using solid phase extraction columns containing 500 mg of silicic acid (Phase Separations, Franklin, MA). The columns were conditioned with 3 ml of CHCl3. Lipids were then transferred to the column in 1 ml of CHC13. Neutral lipids, glycolipids and polar lipids were fractionated with 5 ml CHCl3, 10 ml acetone, and 5 ml methanol, respectively. Phospholipids (polar lipids) were dried under N2 at 32°C in a water bath. The phospholipids were subjected to mild alkaline methanolysis by exposure to 1 ml of 1:1 methanol to toluene and 1 ml of 0.2 M KOH at 37°C for 10 min, which yielded fatty acid methyl esters (FAME). The FAME were extracted with 2 ml of hexane, 2 ml of H2O and 0.3 ml of 1.0 M acetic acid, and then the hexane fraction was collected. Another 2 ml of hexane was used for further extraction of the sample. The hexane samples containing the FAME were combined and washed with 4 ml of 0.03 N NaOH and then dried under N2 at room temperature. The dried FAME were dissolved into 250 m1 hexane with C19:0 as an internal standard and analyzed using a Hewlett Packard 6890 gas chromatograph with a 25-m Ultra 2 (5% phenyl) methylpolysiloxane column (J&W
Scientific). Peaks were identified using fatty acid standards and the microbial identification peak identification software (MIDI, Newark, DE). The system of fatty acid nomenclature is as follows: the number before the colon gives the number of carbons, the number after the colon gives the count of double bonds, and the position of the double bond from the methyl end of the molecule is indicated last. The last number is followed by a ‘c’ for cis or a ‘t’ for trans geometry. The prefixes ‘i’ and ‘a’ stand for iso or anteiso, respectively. The fatty acids with a hydroxy group and a methyl group are given as OH and Me, respectively; the prefix number indicates position from the carboxyl end of the molecule. Cryclopropyl fatty acids are presented as ‘cy’. Changes in the PLFA content of samples was analyzed using principal components analysis (PCA). Analyses were done using the results for 27 individual PLFAs with carbon chain lengths between 15 and 20. PCA which was completed using the program SAS (SAS Institute, Cary, NC).
3. Results and discussion Experimental results from the four composting processes are summarized in Table 2. A decrease in pH was observed for all of the treatments. The pH of treatments c 1–3 dropped by : 0.3 pH units during the process while the pH of treatment c 4 dropped by nearly 1 pH unit. Moisture content and volatile solids content did not decrease appreciably during any of the processes. The final moisture and volatile solids contents of the samples ranged from 70–75 and 87–88%, respectively. The total PLFAs in the initial samples for treatments c2–4 were not statistically different. Thus, increasing the ratio of grape pomace to rice straw did not have a significant effect on the total initial PLFAs in the processes. Treatment c 1, which was inoculated with matured grape pomace and rice straw compost, had a lower initial PLFA content than the other treatments. The matured compost may have had a lower biomass concentration than the raw compost substrates, thereby reducing the initial total PLFAs in treatment c1.
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F. Lei, J.S. VanderGheynst / Process Biochemistry 35 (2000) 923–929 Table 3 Variances of principal components from PCA for two groups of treatments
PC1 PC2 PC3
Treatment c 1 and 2 cumulative percent variance
Treatment c3 and 4 cumulative percent variance
35 56 67
29 45 57
Fig. 2. Temperature profiles during the composting experiments. Temperature measurements represent data taken from central thermocouples.
Fig. 4. PCA of PLFA compositions for samples from treatments c 1 and 2 (effect of inoculation).
Fig. 3. Cumulative oxygen depletion in the composting experiments.
Temperature profiles for the four experiments are illustrated in Fig. 2. The peak temperatures for all four treatments fell within a range that could support mesophilic and thermophilic microbial communities. The temperature in treatment cl started to increase at : 50 h into the process and remained at a peak temperature for about 50 h. For treatment c 2 the temperature also started to increase at :50 h into the process, but peaked and began to drop between 75 and 80 h. Thus, inoculation did not significantly increase the rate of temperature rise, but did increase the duration of time the composting temperature remained above 40°C for grape pomace and rice straw compost. In treatments c 3 and 4 the temperature started to rise at about 75 h and peaked at about 150 h into the process. The difference in the rate of temperature rise between treatments c l and 2 and treatments c 3 and 4 may have been due to the higher ratio of grape pomace to rice straw in treatments c3 and 4. Although the greater amount of grape pomace may have increased the level of biodegradable carbon sources available for microbial growth, it also may have increased some inhibiting compounds to micro-organ-
isms, such as alcohol and phenolic compounds, which may have lowered the activity of the initial population of micro-organisms in the composting materials. In addition, the temperature in treatment c 4 peaked at a higher level and remained above 40°C longer than the other three treatments. This may have been due to the adjustment of pH in this treatment. Cumulative O2 depletion results for the four experiments are presented in Fig. 3. Early in the process treatment c2 had the highest rate of O2 depletion, as indicated by higher cumulative O2 levels. However, total O2 depletion levels were very similar for all treatments by 150 h into the processes. These results suggest that inoculation and pH adjustment did not improve the overall level of decomposition for grape pomace and rice straw compost. Results from PCA of PLFA data from treatments c 1 and 2 are presented in Table 3 and Figs. 4 and 5. This analysis emphasizes the effect of inoculation on the microbial community structure. The first two principal components (PCs) represent 56% of the variance. As illustrated in the principal component plots in Figs. 4 and 5, there were differences observed between the two treatments and samples within treatments over time. Differences in the initial samples were due to PLFAs 18:lw9c, 18:2w6,9c, and straight-chain saturated PLFAs; levels of these PLFA were higher in treatment c 2 than in treatment c 1. The PLFAs 18:1w9c and 18:2w6,9c have been found to be enriched in samples containing fungi [14,15], while straight chain saturated PLFA are common biomarkers for eubacteria [7,16]. The higher levels of 18: lw9c and 18:2w6,9c in treat-
F. Lei, J.S. VanderGheynst / Process Biochemistry 35 (2000) 923–929
Fig. 5. PCA of PLFAs from treatments c 1 and 2 (effect of inoculation).
Fig. 6. PCA of PLFA compositions for samples from treatments c 3 and 4 (effect of initial pH adjustment).
Fig. 7. PCA of PLFAs from treatments c 3 and 4 (effect of initial pH adjustment).
ment c2 may have been a result of yeast remaining in the grape pomace. The inoculum in treatment c 1, which consisted of several-week-old grape pomace
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and rice straw compost that had been exposed to thermophilic temperatures, likely had low levels of yeast, thus reducing the biomarkers 18: lw9c and 18:2w6,9c in this treatment. In both treatments cl and 2, differences in PLFA content were observed between the initial samples and samples obtained when temperature peaked (Figs. 2, 4 and 5). In general, the levels of straight chain saturated PLFA and branched-monounsaturated PLFA decreased as temperature increased. Both types of PLFA are common biomarkers for eubacteria [7,16]. The decrease in the levels of these PLFA was likely a result of the reduction in mesophilic bacteria as temperature increased during the processes. A similar trend in straight chain saturated PLFA was also observed by Hellmann and co-workers [10] as temperature increased during windrow composting. As the process temperatures peaked, the difference in PLFA content between treatments c 1 and 2 decreased as indicated by overlap of points representing samples from 75 h in treatment c 1 and samples from 75 and 95 h from treatment c 2 on the score plot (Fig. 4). These results suggest that inoculation of the composting process had little effect on the microbial community structure dominant in the highmesophilic and low-thermophilic temperature region for this mixture. The microbial community structure continued to change over time as indicated by the difference in PC1 for samples taken at 75 and 170 h in treatment c 1 and 95 and 240 h in treatment c 2 (Fig. 4). The levels of branched monounsaturated PLFA and terminally branched saturated PLFA increased over time. This change was greater in treatment c 1 than in treatment c 2. Terminally branched saturated PLFA have been found in some thermophilic bacteria [15,17,18], and have been observed to be enriched in thermophilic composts [10,11]. Since the last sample from treatment c 1 was taken shortly after the thermophilic phase, it is likely that thermophlic bacteria were still present in the compost. However, the last sample from treatment c 2 was taken a few days after the thermophilic phase of composting had ended, which may explain the lower levels of biomarkers for thermophilic bacteria in this sample. Results from PCA of PLFA data from treatments c 3 and 4 are listed in Table 3 and illustrated in Figs. 6 and 7. This analysis represents the effect of pH adjustment on the microbial community. The first two PCs represent 45% of the variance. The PLFA content of initial samples from treatments c 3 and 4 were very similar as shown on the PC plot in Fig. 6. This suggests that pH adjustment did not affect the initial microbial community structure of the compost.
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As the process temperature increased, differences in PLFA content in samples taken at 170 h in treatment c 3 and at 120 and 170 h in treatment c 4 were observed. In both treatments an increase in straight chain saturated PLFA and branched monounsaturated PLFA was observed as temperature increased. In treatment c 4 an increase in the levels of terminally branched saturated PLFA was also observed as temperature increased. These results indicate that there was an overall increase in eubacteria in both treatments. The higher temperature in treatment c 4 was likely responsible for the higher levels of thermophilic bacteria and thus terminally branched saturated PLFA in this treatment. The higher pH in treatment c 4 may have made the compost substrate more favourable to colonization by thermophilic bacteria. The PLFA content of samples taken at the end of the processes was also quite different between treatments c 3 and 4. In treatment c3 there was little difference in the PLFA content of samples analyzed at 170 and 265 h. However, in treatment c 4 levels of terminally branched saturated PLFA and branched monounsaturated PLFA increased from 170 to 290 h. Treatment c 3 had higher levels of branched unsaturated PLFAs than treatment c 4, while treatment c 4 had higher levels of terminally branched saturated PLFAs. Treatment c4 experienced higher temperatures for longer times that treatment c 3. This longer exposure to higher temperatures likely favored the growth of thermophilic micro-organisms in treatment c 4, while the shorter exposure in treatment c 3 favored the growth of mesophilic micro-organisms. Despite the large differences in microbial community structure observed in the PCA analysis, the cumulative O2 profiles from treatments c3 and 4 followed very similar trends between 0 and 200 h (Fig. 2). Thus, PLFA analysis may provide a means of identifying differences between composts with similar trends in decomposition rate.
4. Conclusions PLFA analysis was successfully used to study microbial community differences in grape pomace and rice straw composting processes. PCA of PLFA data showed that inoculation had little effect on the microbial community structure of the compost when the temperature of the process peaked. Measurements of cumulative O2 consumption also confirmed that inoculation had little affect on the level of decomposition, however inoculated compost did remain above 40°C longer than compost that was not inoculated.
Adjustment of pH had a significant effect on the microbial community structure throughout the composting process. This was likely due to the effect pH adjustment had on increasing the time the process was exposed to thermophilic temperatures. However, O2 consumption was the same regardless of pH adjustment. Increasing the level of grape pomace in the compost did increase the time to achieve thermophilic temperatures. This may have been due to the presence of phenolic compounds that inhibited the initial microbial community. Higher levels of grape pomace had only a small affect on the overall levels of decomposition as measured by cumulative O2 consumption. References [1] VanderGheynst JS, Cogan DJ, DeFelice PJ, Gossett JM, Walker LP. Effect of process management on the emission of organosulfur compounds and gaseous antecedents from composting processes. Environ Sci Technol 1998;32:3713 – 8. [2] Schulze KL. Rate of oxygen consumption and respiratory quotients during the aerobic decomposition of a synthetic garbage. Compost Sci 1960;1:36 – 40. [3] Walker LP, Nock TD, Gossett JM, VanderGheynst JS. The role of periodic water addition in managing moisture limitations during high solids aerobic decomposition. Process Biochem 1999;34(6-7):601– 12. [4] Bach PD, Shoda M, Kubota H. Composting reaction rate of sewage sludge in an autothermal packed bed reactor. J Ferment Technol 1985;63:271 – 8. [5] Elwell DL, Keener HM, Hansen RC. Controlled, high rate composting of mixtures of food wastes, yard waste and chicken manure. In: Seventh International Symposium on Agricultural and Food Processing Wastes. Chicago, IL: American Society of Agricultural Engineers, 1995. [6] White DC, Davis WM, Nickels JS, King JD, Bobbie RJ. Determination of sedimentary microbial biomass by extractable lipid phosphate. Oecologia 1979;40:51 – 62. [7] Vestal JR, White DC. Lipid analysis in microbial ecology. Bioscience 1989;39(8):535– 41. [8] White DC. Chemical ecology: possible linkage between macroand microbial ecology. Oikos 1995;74:177 – 84. [9] Zelles L, Bai QY, Rackwitz R, Chadwick D, Beese F. Determination of phospholipid- and lipopolysaccharide-derived fatty acids as an estimate of microbial biomass and community structures in soils. Biol Fertil Soils 1995;19:115 – 23. [10] Hellmann B, Zelles L, PalojN0 rvi A, Bai Q. Emission of climate-relevant trace gases and succession of microbial communities during open-windrow composting. Appl Environ Microbiol 1997;63(3):1011– 8. [11] Herrmann RF, Shann JF. Microbial community changes during the composting of municipal solid waste. Microb Ecol 1997;33:78 – 85. [12] Bossio DA, Scow KM. Impacts of carbon and flooding on soil microbial communities: phospholipid fatty acid profiles and substrate utilization patterns. Microb Ecol 1998;35:265–78. [13] Thigh EG, Dyer WJ. A rapid method of total lipid extraction and purlfication. Can J Biochem Physiol 1959;37:911 –5. [14] Frostega¨rd A, , Ba¨a¨th E. The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil. Biol Fertil Soils 1996;22:59 – 65.
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