Process Biochemistry 35 (2000) 923–929

The effect of microbial inoculation and pH on microbialcommunity 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 ofgrape pomace and rice straw. Composting studies were completed in 30 litre static-bed reactors with continuous temperature andoxygen monitoring. The effects of inoculation and pH adjustment on microbial community structure and level of decompositionduring composting were investigated. Principal components analysis (PCA) of the PLFA data showed that inoculation had littleeffect on the microbial community structure of the compost once temperature had peaked, while process temperature and theadjustment of initial pH had a significant effect. Adjustment of pH and inoculation did not significantly increase the level ofdecomposition as measured by oxygen consumption. © 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Microbial communities; Phospholipid fatty acid; Grape pomace

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1. Introduction

As legislation requires more food wastes to be di-verted from landfills, food industries must considerother options for the disposal of food processingresidues. One popular alternative to disposal is com-posting. Although composting is not a new waste treat-ment method, some of the characteristics of food wasteoffer unique challenges to processors. For example,grape pomace, a residue of wine processing, can havesignificant levels of phenolic compounds that mightinhibit the decomposition process. The low pH of theseresidues might also increase the time required for com-posting. Management of the process by the addition ofmicroorganisms capable of tolerating and/or decompos-ing inhibitory compounds, or the adjustment of pH,may offer a means of increasing the rate of decomposi-tion of these materials.

CO2 evolution and O2 consumption rates have beenused to measure decomposition rates for compostingprocesses performed under different management

schemes [1–5]. However, there have been very fewanalyses of the effect of different management alterna-tives on the microbial community dynamics duringcomposting. One method of studying microbial com-munity changes during composting is phospholipidfatty acid (PLFA) analysis. The rationale behind PLFAanalysis is that most microorganisms contain phospho-lipids in their membranes that are not stored but areturned over relatively rapidly during metabolism [6].Therefore, determining the total PLFA in an environ-mental sample can provide an estimate of viable micro-bial biomass contained within the sample [7]. Inaddition, certain PLFAs can be regarded as taxonomicor physiological biomarkers for microbial genera [8,9].

There have been several studies that have used PLFAanalysis in the monitoring of microbial communitychanges during composting processes. Hellman andco-workers [10] used lipid analysis to examine thechanges in community structure and biomass duringchanges in the composition of the effluent gas streamfrom a municipal solid waste windrow composting pro-cess. Samples taken during the early mesophilic stagesof composting contained high levels of lipids character-istic of eucaryotes, which decreased rapidly over time astemperature and CO2 evolution rate increased and pH

* Corresponding author. Tel.: +1-530-7520989; fax: +1-530-7522640.

E-mail address: jsvander@ucdavis.edu (J.S. VanderGheynst)

0032-9592/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.

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F. Lei, J.S. VanderGheynst / Process Biochemistry 35 (2000) 923–929924

Table 1Experimental variables in the rice straw and grape pomace composting studies

pH adjustmentTreatment (c ) Ratio of rice straw to grape pomaceInoculum Initial volatile solids Initial pH(% of total solids)(kg dry/kg dry)

–1 l:3.4+ 89 4.11–2 1:3.4– 89 4.10– 1:5.0– 883 4.08

–4 + 1:5.0 89 5.98

decreased. During the thermophilic phase of compost-ing, they observed high levels of lipids associated withactinomycetes and Gram-positive bacteria. In addition,simultaneous increases in methane emission and etherlipids, indicators of archaean methanogens, were ob-served. Herrmann and Shann [11] used PLFA profilesto characterize the microbial community changes dur-ing municipal solid waste composting managed by anaerated-mixed method. They found that samples ob-tained from a particular stage of composting (i.e.mesophilic, thermophilic and curing stages) had similarlipid contents. In the mesophilic stage they observedlipids characteristic of fungi, while in the thermophilicstages they observed lipids characteristic of ther-mophilic bacteria and actinomycetes. Lipid biomarkersassociated with actinomycetes and fungi were observedin cured samples. They found that very old samplestended to have common lipid profiles which suggeststhat PLFA analysis may provide a means of assessingcomposting maturity. In summary, analysis of PLFAdirectly extracted from compost samples shows greatpromise as a means of increasing our understanding ofthe composting process.

The objective of this study was to use PLFA analysisto study microbial community changes during the com-posting of rice straw and grape pomace. Additionallythe effects of compost inoculation and pH adjustmenton rates of decomposition and microbial communitystructure were investigated.

2. Materials and methods

Compost was produced from a mixture of grapepomace and rice straw. Experimental variables arelisted in Table 1. Fermented grape pomace (pressedskins, seeds and liquid) was obtained from a pilot-scalewine fermentation facility in the Department of Viticul-ture 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 byhammer milling through a 1 in. screen. The compostmixture was prepared by mixing thawed grape pomacewith straw using the ratios listed in Table 1. Reactorswere loaded with :5.4 kg of the wet compostmixtures.

The initial moisture content of the compost mixturesranged from 74 to 78% (g H2O/g wet solids). Inocula-tion of substrates was completed by mixing 5% (g dryinoculum/g total dry solids) several-week-old grape po-mace and rice straw compost with fresh grape pomaceand rice straw. Adjustment of pH was accomplished byuniformly adding a 5.0 N NaOH solution to the com-posting mixture.

All composts were produced using a 30 litre staticbed bioreactor with a 30 cm diameter (Fig. 1). Thebioreactor was aerated with humidified air at 5 litre/min to maintain effluent O2 concentrations above 10%.Temperature was measured at nine different locationsin the reactor and oxygen concentration was measuredin the effluent gas. Type T thermocouples were used tomeasure temperature and a zirconia oxide-based oxy-gen sensor was used to monitor O2 (NeuwGhent Tech-nology, LaGrangeville, NY). Temperature and oxygenreadings were recorded every 30 min using a datalogger (Campbell Scientific, Logan, UT).

Compost samples were collected for pH, moisturecontent, volatile solids and PLFA analysis at threepoints in the process; at the beginning, when the peaktemperature occurred and at the end of the processes.Sample analyses were completed in triplicate immedi-ately upon removal from the bioreactor. Moisture con-tent was measured by oven drying at 101°C for 24 h.Volatile solids content was determined by combustingsamples at 550°C for at least 6 h in a muffle furnace.Compost pH was measured on compost extracts diluted

Fig. 1. Static-bed composting reactor.

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Table 2Summary of experimental results

Final pHFinal moisture contentInitial PLFATreatment (c ) Final volatile solids content(g H2O/g wet weight* 100%)(ng/g dry compost) (% of total solids)

*10−4

70 881 3.784.1890.0371 875.2990.39 3.802

6.1391.433 74 88 3.8175 88 5.016.1190.334

2:1 (g H2O:g wet compost) with distilled deionizedwater.

PLFA analysis was conducted in a similar manner asdescribed by Bossio and Scow [12]. Compost samples of4 g dry weight were agitated in 33.25 ml of a single-phase mixture of chloroform, methanol and phosphatebuffer in an initial ratio of 1:2:0.8 [13]. Phosphatebuffer addition was adjusted to account for water in thecompost sample. After a 2 h extraction, the sampleswere centrifuged at 5000 rpm for 10 min and thesupernatant decanted into a separatory funnel. Com-post samples were re-extracted with 23 ml of extractantfor another 30 min. The supernatant from the secondextraction was added to the separatory funnel alongwith 15.14 ml of phosphate buffer and 14.75 ml ofCHCl3. The final CHCl3:buffer:methanol ratio was1:0.9:1. After shaking for 2 min, the samples wereallowed to stand overnight for phase separation. Thebottom layer from the separating funnel, which con-tained the lipids, was collected into a test tube anddried under N2 at 32°C in a water bath.

Phospholipids were separated from the neutral andglycolipids using solid phase extraction columns con-taining 500 mg of silicic acid (Phase Separations,Franklin, MA). The columns were conditioned with 3ml of CHCl3. Lipids were then transferred to thecolumn in 1 ml of CHC13. Neutral lipids, glycolipidsand polar lipids were fractionated with 5 ml CHCl3, 10ml acetone, and 5 ml methanol, respectively. Phospho-lipids (polar lipids) were dried under N2 at 32°C in awater bath. The phospholipids were subjected to mildalkaline methanolysis by exposure to 1 ml of 1:1methanol to toluene and 1 ml of 0.2 M KOH at 37°Cfor 10 min, which yielded fatty acid methyl esters(FAME). The FAME were extracted with 2 ml ofhexane, 2 ml of H2O and 0.3 ml of 1.0 M acetic acid,and then the hexane fraction was collected. Another 2ml of hexane was used for further extraction of thesample. The hexane samples containing the FAMEwere combined and washed with 4 ml of 0.03 N NaOHand then dried under N2 at room temperature. Thedried FAME were dissolved into 250 m1 hexane withC19:0 as an internal standard and analyzed using aHewlett Packard 6890 gas chromatograph with a 25-mUltra 2 (5% phenyl) methylpolysiloxane column (J&W

Scientific). Peaks were identified using fatty acid stan-dards and the microbial identification peak identifica-tion software (MIDI, Newark, DE).

The system of fatty acid nomenclature is as follows:the number before the colon gives the number of car-bons, the number after the colon gives the count ofdouble bonds, and the position of the double bondfrom the methyl end of the molecule is indicated last.The last number is followed by a ‘c’ for cis or a ‘t’ fortrans geometry. The prefixes ‘i’ and ‘a’ stand for iso oranteiso, respectively. The fatty acids with a hydroxygroup and a methyl group are given as OH and Me,respectively; the prefix number indicates position fromthe carboxyl end of the molecule. Cryclopropyl fattyacids are presented as ‘cy’.

Changes in the PLFA content of samples was ana-lyzed using principal components analysis (PCA).Analyses were done using the results for 27 individualPLFAs 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 pro-cesses are summarized in Table 2. A decrease in pH wasobserved for all of the treatments. The pH of treat-ments c1–3 dropped by :0.3 pH units during theprocess while the pH of treatment c4 dropped bynearly 1 pH unit. Moisture content and volatile solidscontent did not decrease appreciably during any of theprocesses. The final moisture and volatile solids con-tents of the samples ranged from 70–75 and 87–88%,respectively.

The total PLFAs in the initial samples for treatmentsc2–4 were not statistically different. Thus, increasingthe ratio of grape pomace to rice straw did not have asignificant effect on the total initial PLFAs in theprocesses. Treatment c1, which was inoculated withmatured grape pomace and rice straw compost, had alower initial PLFA content than the other treatments.The matured compost may have had a lower biomassconcentration than the raw compost substrates, therebyreducing the initial total PLFAs in treatment c1.

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Fig. 2. Temperature profiles during the composting experiments.Temperature measurements represent data taken from central ther-mocouples.

Table 3Variances of principal components from PCA for two groups oftreatments

Treatment c3 and 4Treatment c1 and 2cumulative percent variancecumulative percent variance

35 29PC145PC2 565767PC3

Fig. 4. PCA of PLFA compositions for samples from treatments c1and 2 (effect of inoculation).

Fig. 3. Cumulative oxygen depletion in the composting experiments.

isms, such as alcohol and phenolic compounds, whichmay have lowered the activity of the initial populationof micro-organisms in the composting materials. Inaddition, the temperature in treatment c4 peaked at ahigher level and remained above 40°C longer than theother three treatments. This may have been due to theadjustment of pH in this treatment.

Cumulative O2 depletion results for the four experi-ments are presented in Fig. 3. Early in the processtreatment c2 had the highest rate of O2 depletion, asindicated by higher cumulative O2 levels. However,total O2 depletion levels were very similar for all treat-ments by 150 h into the processes. These results suggestthat inoculation and pH adjustment did not improvethe overall level of decomposition for grape pomaceand rice straw compost.

Results from PCA of PLFA data from treatmentsc1 and 2 are presented in Table 3 and Figs. 4 and 5.This analysis emphasizes the effect of inoculation onthe microbial community structure. The first two princi-pal components (PCs) represent 56% of the variance.As illustrated in the principal component plots in Figs.4 and 5, there were differences observed between thetwo treatments and samples within treatments overtime. Differences in the initial samples were due toPLFAs 18:lw9c, 18:2w6,9c, and straight-chain saturatedPLFAs; levels of these PLFA were higher in treatmentc2 than in treatment c1. The PLFAs 18:1w9c and18:2w6,9c have been found to be enriched in samplescontaining fungi [14,15], while straight chain saturatedPLFA are common biomarkers for eubacteria [7,16].The higher levels of 18: lw9c and 18:2w6,9c in treat-

Temperature profiles for the four experiments areillustrated in Fig. 2. The peak temperatures for all fourtreatments fell within a range that could supportmesophilic and thermophilic microbial communities.The temperature in treatment c l started to increase at:50 h into the process and remained at a peak temper-ature for about 50 h. For treatment c2 the tempera-ture 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 rateof temperature rise, but did increase the duration oftime the composting temperature remained above 40°Cfor grape pomace and rice straw compost.

In treatments c3 and 4 the temperature started torise at about 75 h and peaked at about 150 h into theprocess. The difference in the rate of temperature risebetween treatments c l and 2 and treatments c3 and4 may have been due to the higher ratio of grapepomace to rice straw in treatments c3 and 4. Al-though the greater amount of grape pomace may haveincreased the level of biodegradable carbon sourcesavailable for microbial growth, it also may have in-creased some inhibiting compounds to micro-organ-

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Fig. 5. PCA of PLFAs from treatments c1 and 2 (effect of inocula-tion).

and rice straw compost that had been exposed tothermophilic temperatures, likely had low levels ofyeast, thus reducing the biomarkers 18: lw9c and18:2w6,9c in this treatment.

In both treatments c l and 2, differences in PLFAcontent were observed between the initial samples andsamples obtained when temperature peaked (Figs. 2,4 and 5). In general, the levels of straight chain satu-rated PLFA and branched-monounsaturated PLFAdecreased as temperature increased. Both types ofPLFA are common biomarkers for eubacteria [7,16].The decrease in the levels of these PLFA was likely aresult of the reduction in mesophilic bacteria as tem-perature increased during the processes. A similartrend in straight chain saturated PLFA was also ob-served by Hellmann and co-workers [10] as tempera-ture increased during windrow composting.

As the process temperatures peaked, the differencein PLFA content between treatments c1 and 2 de-creased as indicated by overlap of points representingsamples from 75 h in treatment c1 and samplesfrom 75 and 95 h from treatment c2 on the scoreplot (Fig. 4). These results suggest that inoculation ofthe composting process had little effect on the micro-bial community structure dominant in the high-mesophilic and low-thermophilic temperature regionfor this mixture.

The microbial community structure continued tochange over time as indicated by the difference inPC1 for samples taken at 75 and 170 h in treatmentc1 and 95 and 240 h in treatment c2 (Fig. 4). Thelevels of branched monounsaturated PLFA and termi-nally branched saturated PLFA increased over time.This change was greater in treatment c1 than intreatment c2. Terminally branched saturated PLFAhave been found in some thermophilic bacteria[15,17,18], and have been observed to be enriched inthermophilic composts [10,11]. Since the last samplefrom treatment c1 was taken shortly after the ther-mophilic phase, it is likely that thermophlic bacteriawere still present in the compost. However, the lastsample from treatment c2 was taken a few daysafter the thermophilic phase of composting hadended, which may explain the lower levels ofbiomarkers for thermophilic bacteria in this sample.

Results from PCA of PLFA data from treatmentsc3 and 4 are listed in Table 3 and illustrated inFigs. 6 and 7. This analysis represents the effect ofpH adjustment on the microbial community. The firsttwo PCs represent 45% of the variance. The PLFAcontent of initial samples from treatments c3 and 4were very similar as shown on the PC plot in Fig. 6.This suggests that pH adjustment did not affectthe initial microbial community structure of the com-post.

Fig. 6. PCA of PLFA compositions for samples from treatments c3and 4 (effect of initial pH adjustment).

Fig. 7. PCA of PLFAs from treatments c3 and 4 (effect of initialpH adjustment).

ment c2 may have been a result of yeast remainingin the grape pomace. The inoculum in treatment c1,which consisted of several-week-old grape pomace

F. Lei, J.S. VanderGheynst / Process Biochemistry 35 (2000) 923–929928

As the process temperature increased, differences inPLFA content in samples taken at 170 h in treatmentc3 and at 120 and 170 h in treatment c4 wereobserved. In both treatments an increase in straightchain saturated PLFA and branched monounsatu-rated PLFA was observed as temperature increased.In treatment c4 an increase in the levels of termi-nally branched saturated PLFA was also observed astemperature increased. These results indicate thatthere was an overall increase in eubacteria in bothtreatments. The higher temperature in treatment c4was likely responsible for the higher levels of ther-mophilic bacteria and thus terminally branched satu-rated PLFA in this treatment. The higher pH intreatment c4 may have made the compost substratemore favourable to colonization by thermophilic bac-teria.

The PLFA content of samples taken at the end ofthe processes was also quite different between treat-ments c3 and 4. In treatment c3 there was littledifference in the PLFA content of samples analyzedat 170 and 265 h. However, in treatment c4 levelsof terminally branched saturated PLFA and branchedmonounsaturated PLFA increased from 170 to 290 h.Treatment c3 had higher levels of branched unsatu-rated PLFAs than treatment c4, while treatmentc4 had higher levels of terminally branched satu-rated PLFAs. Treatment c4 experienced higher tem-peratures for longer times that treatment c3. Thislonger exposure to higher temperatures likely favoredthe growth of thermophilic micro-organisms in treat-ment c4, while the shorter exposure in treatmentc3 favored the growth of mesophilic micro-organ-isms.

Despite the large differences in microbial commu-nity structure observed in the PCA analysis, the cu-mulative O2 profiles from treatments c3 and 4followed very similar trends between 0 and 200 h(Fig. 2). Thus, PLFA analysis may provide a meansof identifying differences between composts with simi-lar trends in decomposition rate.

4. Conclusions

PLFA analysis was successfully used to study mi-crobial community differences in grape pomace andrice straw composting processes. PCA of PLFA datashowed that inoculation had little effect on the micro-bial community structure of the compost when thetemperature of the process peaked. Measurements ofcumulative O2 consumption also confirmed that inoc-ulation had little affect on the level of decomposition,however inoculated compost did remain above 40°Clonger than compost that was not inoculated.

Adjustment of pH had a significant effect on themicrobial community structure throughout the com-posting process. This was likely due to the effect pHadjustment had on increasing the time the processwas exposed to thermophilic temperatures. However,O2 consumption was the same regardless of pHadjustment.

Increasing the level of grape pomace in the com-post did increase the time to achieve thermophilictemperatures. This may have been due to the pres-ence of phenolic compounds that inhibited the initialmicrobial community. Higher levels of grape pomacehad only a small affect on the overall levels of de-composition as measured by cumulative O2 consump-tion.

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