Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 79:673–677 (online: 2004)DOI: 10.1002/jctb.1012

Volatile fatty acid production during anaerobicmesophilic digestion of solid potato wasteWilson Parawira,1,2 Marika Murto,1 John S Read3 and Bo Mattiasson1∗1Department of Biotechnology, Lund University, PO Box 124, SE-221 00 Lund, Sweden2Department of Biochemistry, University of Zimbabwe, PO Box MP167, Mt Pleasant, Harare, Zimbabwe3Department of Applied Biology and Biochemistry, NUST, PO Box 219 Ascot, Bulawayo, Zimbabwe

Abstract: The production of volatile fatty acids by anaerobic digestion of solid potato waste was investigatedusing a batch solid waste reactor with a working capacity of 2 dm−3 at 37 ◦C. Solid potato waste was packedinto the digester and the organic content of the waste was released by microbial activity by circulatingwater over the bed, using batch loads of 500 g or 1000 g potato waste. The sequence of appearance ofthe volatile fatty acids was (acetic, propionic); (n-butyric); (n-valeric, iso-valeric, caproic); (iso-butyric).After 300 h digestion of potato waste on a small scale, the fermentation products were chiefly (mg g−1 totalVFAs): acetic acid (420), butyric acid (310), propionic acid (140) and caproic acid (90), with insignificantamounts of iso-butyric acid, n-valeric and iso-valeric acids. When the load of potato solids was increased,the volatile fatty acid content was similar, but butyric acid constituted 110 mg g−1 and lactic acid 400 mg g−1

of the total volatile fatty acids. The maximum soluble chemical oxygen demand (COD) achieved underthe experimental conditions used was 27 and 37 g COD dm−3 at low and high loadings of potato solids,respectively. The total volatile fatty acids reached 19 g dm−3 of leachate at both loads of potato solid waste.Gas production was negligible, indicating that methanogenic activity was effectively inhibited. 2004 Society of Chemical Industry

Keywords: acidogenesis; anaerobic digestion; hydrolysis; leachate; potato; solid waste; volatile fatty acids

NOTATIONCA Caproic acidHAc Acetic acidHPr Propionic acidi-BA Iso-butyric acidi-VA Iso-valeric acidLA Lactic acidn-BA Butyric acidn-VA Valeric acidTVFAs Total volatile fatty acidsVFAs Volatile fatty acids

1 INTRODUCTIONIn recent years, research on the acidogenic phaseof anaerobic digestion has increased considerably.Hydrolysis and acidogenesis are the first steps in theanaerobic digestion of complex organic materials whenthey are degraded into methane and carbon dioxide.These basic steps involve conversion of the polymerspresent in organic matter into soluble monomers,which are quickly fermented into VFAs (HAc, HPr,n-BA, i-BA, n-VA and i-VA), hydrogen (H2) andcarbon dioxide (CO2) by the rapidly growing andpH-insensitive acidogenic bacteria.1,2 The HAc, H2

and CO2 are converted to methane by methanogens.2

Many kinds of bacteria are involved in hydrolysis andacidogenesis and, therefore, several kinds of organicacids and alcohols are usually produced.3

Hydrolysis is reported to be the rate-limiting stepduring anaerobic digestion of complex organic waste.4

The rate of hydrolysis depends on the extracellu-lar enzymes produced by fermentative acidogens,the biomass concentration, the substrate concentra-tion and the specific surface area of the particulatesubstrate.5 Accumulation of organic acids and low-ering of pH are known to lead to suppression ofmethanogenic activity and process failure in single-stage methanogenic, reactors.6 The concept of atwo-stage system was thus proposed to improve theprocess5 stability and efficiency.2 Using a two-stageanaerobic process, hydrolysis and acidification canbe conducted in the first digester at a pH, temper-ature and hydraulic retention time optimal for thefermentative, acidogenic bacteria. Acetogenesis andmethanogenesis can then be done in the second reactorat conditions optimal for those processes.

Detailed knowledge of the acid phase of anaerobicdigestion is useful in a number of situations. Microbialanaerobic acidogenesis could be a useful means of

∗ Correspondence to: Bo Mattiasson, Department of Biotechnology, Lund University, PO Box 124, SE-221 00 Lund, SwedenE-mail: bo.mattiasson@biotek.lu.seContract/grant sponsor: Swedish Agency for Research Cooperation (SAREC)(Received 19 September 2003; accepted 22 January 2004)Published online 6 May 2004

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producing organic acids from organic waste thatcould be used in denitrification, dephosphatationor methanisation.7,8 VFAs are essential as energyand carbon sources for the microorganisms involvedin the biological removal of nutrients (nitrogenand phosphorus) in wastewater treatment.9 Theorganic acids produced by the degradation of largequantities of residues from organic waste can alsobe used to make biodegradable plastic such aspolylactate polymers, an environmentally friendlyalternative to non-biodegradable plastics derived frompetrochemicals.10,11 Since the composition of organicacids in the medium influences the subsequentmethanogenesis reactions, it is not only interesting,but important, to study the product spectrum duringanaerobic acidogenesis. Initially, the liquefaction stageof each substrate could be performed in separatedigesters in order to obtain the best liquefactionyield and VFA conversion using a specialisedmicrobial ecosystem. The digestion pattern of eachindividual acid varies from one acid to anotherdepending on the substrate and physico-chemicalconditions.12–14 Optimum operational conditionsfor the acetogenic/methanogenic phase have beenextensively studied6,8 but little information is availableon the acidogenic phase. The characteristics of theleachate in terms of specific VFA concentrationshave important effects on the performance of theacidogenic digester as an excess of one type of acidfavours the predominance of specific kinds of microbialconsortia.2,12

This study focuses on investigating the VFAproduction profile and changes in the characteristicsof the leachate during the acid-phase digestion processof potato waste at two concentrations of solids. Theraw material can be seen as representative of manyother kinds of cheap starch-rich biomasses suitablefor conversion to VFAs and other biochemicalssuch as lactic acid. Throughout the world hundredsof millions of tonnes of potatoes are producedannually and when they are processed into consumableproducts, enormous amounts of starch-rich wastes,that can be detrimental to environmental quality, arealso produced.

2 MATERIALS AND METHODS2.1 Experimental set-upCylindro-conical anaerobic reactors with a 2 dm3

working capacity were used. Solid potato waste wascut into small pieces using a kitchen blender and 500 gor 1000 g portions were loaded into two separatereactors. The potato waste had a total solids (TS)content of 190 g kg−1 and a volatile solids (VS) contentof 950 g kg−1 TS. To both reactors 800 cm3 waterwas added, and 200 cm3 of anaerobic sludge fromthe Ellinge municipal wastewater treatment plant insouthern Sweden was used as inoculum. This plantreceives domestic waste and industrial effluent, thelatter mainly from a potato-processing plant. The

reactors, operated at 37 ◦C, were closed at the topwith butyl rubber stoppers to maintain anaerobicconditions. A wire sieve (3 mm gauge) was installed5 cm above the bottom of the cone in each reactor tosupport the solid waste substrate while still allowingthe liquid to pass through it. The leachate from thereactors containing microbes from the inoculum wasrecirculated at 9 cm3 min−1 and sprinkled over thepacked bed of potato waste. The leachate percolateddown through the bed and out of the reactor througha part at the bottom. Samples were collected everyhour for the first 24 h and thereafter every 12 h untilthe experiments were stopped after 300 h.

2.2 Analytical methodsThe samples from the reactors were centrifugedat 3000 × g for 3 min (WIFUG Lab CentrifugesSTUDIE-M, UK) and the supernatant was usedfor pH, COD and VFA analysis. Samples for VFAanalysis were acidified with concentrated sulfuricacid to stop microbial activity and stored at −20 ◦Cuntil analysis. Before analysis, the samples werethawed and filtered through a 0.45 µm filter (Minisart,Sartorius AG, Gottingen, Germany); the filtrates werecollected in HPLC sample vials. The individual VFAconcentrations were determined using HPLC, VarianStar 9000 (Varian, Walnut Creek, CA, USA), with aBiorad column, Cat 125-0115 (Hercules, CA, USA)for fermentation monitoring. The column temperaturewas 65 ◦C, sulfuric acid (1 mM) was used as mobilephase and the liquid flow was 0.8 cm3 min−1. Peakdetection was achieved with UV absorption at 208 nmand chromatograms were integrated and plotted usingStar chromatographic software. The TS, VS and CODwere determined according to standard methods.15

The COD equivalents of the five VFAs (derived fromthe complete oxidation reactions of the individualVFAs to CO2 and H2O), used to calculate the CODin the form of VFAs were: 1.066 (HAc), 1.512 (HPr),1.816 (BA), 2.036 (VA) and 2.204 mg dm−3 (CA).16

The following formula was used:

conversion of COD gg−1 = COD of VFA(mg dm−3)

Soluble COD(mg dm−3)

3 RESULTS AND DISCUSSIONVFAs are among the main products of the acidogenesisof organic matter. The appearance of VFAs in theleachate indicated the initiation of acidogenesis within1 h of digestion, as can be seen in Table 1. HAcand HPr were the first VFAs to appear at bothpotato loads. n-BA was detected after 6 h at bothloadings of digestion. i-BA was detected after 7 h and15 h of digestion of 500 g and 1000 g solid potatowaste, respectively. n-VA and i-VA appeared after7 h of digestion at both loading. CA was detectedafter 9 h and 7 h of digestion of 500 g and 1000 gpotato solids, respectively. The presence of VFAsin the reactors was indicative of bacterial activity.The appearance of HAc and HPr has also been

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Mesophilic digestion of solid potato waste

Table 1. Appearance of volatile fatty acids and lactic acid during

hydrolysis and acidification of solid potato waste at batch loads of

500 g and 1000 g

Time of appearance (h)

VFA 500 g 1000 g

HAc 1 1HPr 1 1n-BA 6 6n-VA 7 7i-VA 7 8CA 9 7LA 9 10i-BA 7 15

reported within 1 h in a study on anaerobic mesophilicdigestion of vegetable market waste.12 The anaerobicconditions and frequent contact of waste with leachatein the solid bed reactors provided conditions forenrichment, growth and development of acidogenicand hydrolytic bacteria.12 Their combined action onthe potato biomass resulted in the breakdown ofcomplex substrates to simpler materials, thus resultingin solubilisation of the organic content.

The time course for the production of VFAs andLA is shown in Fig 1. The fermentation productsafter 300 h digestion of potato were chiefly (mg g−1

total VFAs): HAc (420), n-BA (310), HPr (140)

Figure 1. Time course for the production of VFAs and lactic acidduring hydrolysis/acidification of potato solids at loads of 500 g (a)and 1000 g (b).

and CA (90) as well as low amounts of i-BA, n-VA and i-VA using 500 g potato solids. With 1000 gpotato solids, the fermentation products were mainly(mg g−1 total VFAs): HAc (410), LA (400), n-BA(110) and CA (40), low amounts of HPr and no n-VA or i-VA. The production of HAc, n-BA and i-BAincreased continuously with 500 g potato solids duringthe period of the study, while the production of CAand HPr was almost constant after 50 h and 150 h ofdigestion, respectively. The same pattern was observedfor HAc with 1000 g potato solids, but the productionof n-BA, i-BA and CA remained constant after 50 hof digestion. Identification of the individual VFAsformed is important, since it may provide valuableinformation on the metabolic pathways involved inthe process. HAc, HPr, n-BA and i-BA are formeddirectly from the fermentation of carbohydrates andproteins, as well as during the anaerobic oxidation oflipids.3,17 These results are similar to those found byHoriuchi et al3 when they studied the production oforganic acids from a complex medium in an anaerobicreactor with pH control. The high production of n-BAin this study is mainly attributed to the larger amountof carbohydrate (starch) present in the substrate.9,18

HAc is considered to be the major precursor ofmethane.12 The conversion of HAc and n-BA ishigher than that of HPr, n-VA and i-VA. The straight-chain (normal form) fatty acids were found to havea higher decomposition rate than their branched-chain (iso form) acids by Wang et al.1 In this study,straight-chain forms were the dominant products,therefore it would be expected that the amountof methanogenesis would be good when used as asecond step in the process of biogas production frompotato solid waste. The formation and consumptionof some VFAs, such as HPr, n-BA, i-BA, n-VAand i-VA, demonstrate the conversion between theseacids via β-oxidation followed by isomerisation andalso their conversion to acetic acid by acetogenicbacteria.18 The concentrations of VFAs increasedwith increasing concentrations of potato solids. Theseresults show that the concentration of the substratehad a considerable effect on the distribution of theacidification products.

The results also indicate that the occurrenceof the individual VFAs decreased as the chainlength increased. The higher molecular weightVFAs, including n-VA and i-VA, were presentat low concentrations at both loadings of potatosolids. These VFAs are mainly associated with thefermentation of proteins and they could be formedvia reductive deamination of single amino acids orby oxidation–reduction between pairs of amino acidsvia the Stickland reaction.12,18–20 The total Kjeldahlnitrogen in potato is very low (15 g kg−1 of the TS)hence the production of these acids was not assignificant as those of acetate and butyrate. It has beenreported that the production of the four acids, n-BA,i-BA, n-VA and i-VA, during the digestion of non-proteinaceous substrates is minimal. Neither of these

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VFAs is normally produced under the conditions oflow partial pressure of H2 encountered in acidogenicdigestion systems.18

The relationship between TVFAs concentrationand soluble COD at the different total solidsconcentrations is shown in Fig 2. The amount ofTVFAs reached approximately 19 g dm−3 at bothloadings after 300 h. However, the total organic acidcontent with 1000 g potato solids was 31 g dm−3 whenthe TVFA was combined with lactic acid. SolubleCOD is a parameter that represents the extent ofhydrolysis and solubilisation. Solubilisation of organicmatter, in terms of soluble COD, increased rapidlywithin the first 100 h and then gradually to reach amaximum of 27 g COD dm−3 with 500 g potato solids,and 37 g COD dm−3 with 1000 g potato solids after300 h of digestion. The recirculation of the percolatingculture through the solid bed promoted hydrolysisand acidification by virtue of repeated seeding of allregions of the bed. The COD of the leachate increasedslowly after 100 h and this could be interpreted asinhibition of hydrolysis possibly by the end-productsand low pH values (around 4.0), as opposed to 6.0–6.5which is optimal for hydrolysis.21,22 The maximumconcentration of organic acids achieved with 1000 gpotato solids waste was comparable to the 30 g dm−3

reported by de Baere et al.23

The conversion of soluble COD to VFAs for thetwo levels of potato solids is shown in Fig 3. The

Figure 2. Temporal variation of organic matter solubilisation in termsof TVFAs and soluble COD.

Figure 3. Conversion of COD to VFAs and changes in pH duringhydrolysis and acidification of solid potato waste.

soluble COD in the form of VFAs indicates the extentof acidogenesis in a bioreactor.4,8,9 The conversionof soluble COD to VFAs with 500 g potato solidsincreased with increasing soluble COD until all thesoluble organic compounds contributing to COD werepresent in the form of VFAs (1.06 g g−1) at 300 h,indicating that hydrolysis was the rate-limiting step.Inhibition of the hydrolysis of solid waste was alsoreported by Veeken et al22 when studying the effect ofpH and VFAs on the anaerobic hydrolysis of organicsolid waste. Maharaj and Elefsiniotis8 reported totalconversion of soluble COD to VFAs in their studyon the role of hydraulic retention time and lowtemperature on the acid-phase anaerobic digestion ofmunicipal and industrial wastewater. The proportionof COD in the form of VFAs with 1000 g potato solidsincreased rapidly during the first 60 h of incubationto 0.54 g g−1 and then only slowly (it appeared tohave reached a plateau) during the remainder of thestudy period to around 0.67 g g−1, indicating thatconversion of soluble organic compounds to VFAswas the rate-limiting step. Results from various acid-phase studies show a great deal of variation in theproportion of soluble COD in the form of VFAs,with mean values ranging from 0.4 to 0.9 g g−1.4,9,18

Complete conversion of soluble substrate to the majorend products of acidogenesis (ie VFAs) was favouredat the lower content of potato solids.

The remaining soluble COD can be attributed to themetabolic intermediates of the fermentation process.Some of the soluble compounds contributing to CODconsisted of LA, which was produced in large amounts,as shown in Fig 1(b). The total lactic acid productionwas 920 g kg−1 TS in this study, which was comparableto the 1166 g kg−1 TS reported by Huang et al.11

Formation and subsequent conversion of lactic acidoccur as normal processes in anaerobic digesters.17

Lactic acid (2-hydroxypropionic acid) is the mostwidely utilised multifunctional organic acid, of which85% is used in food and food-related applications.11 Inacid-phase digestion, a variety of other soluble C1 –C4

end products besides VFAs is formed, such as organicacids (formic), alcohols and ketones. The presence ofalcohols, which include methanol, ethanol, propanoland butanol, was not investigated in this study. All theabove compounds are fermentation end products ofcarbohydrate metabolism.

The pH in both reactors was reduced quickly fromapproximately 7.0 to around 4.0 (within 48 h afterstart-up) as the concentration of TVFAs increasedrapidly (Fig 3). The pH remained at around 4.0 forthe rest of the study at both feed concentrations,although it was slightly lower with the higher loading.Due to the low pH, methanogenesis was suppressedand acidogenic bacteria were enriched.

4 CONCLUSIONSAcidogenesis was feasible at both levels of potatosolids, as indicated by the low pH values, high VFA

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production and negligible gas production observed.The key acidification products were HAc, n-BAand HPr, with CA, i-BA, n-VA, and i-VA, in lowquantities. LA was also found in significant quantitiesat the higher loading of potato solids, contributingto the soluble COD in the leachate. The acidicconditions in the reactors appear to have inhibitedmethanogenesis, resulting in negligible gas productionthroughout the duration of the study. Our resultssuggest that a rational design for two-stage anaerobicdigestion can be achieved, and that biotechnologicaltreatment of starch wastes can produce valuable endproducts, such as VFAs and other organic acids.

ACKNOWLEDGEMENTFunding for this research was provided by the SwedishAgency for Research Cooperation (SAREC) andSwedish National Energy Administration (STEM).

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