Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6414-7
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Lactic acid fermentation within a cascading approach for biowaste treatment Maraike Probst & Janette Walde & Thomas Pümpel & Andreas Otto Wagner & Irene Schneider & Heribert Insam
Received: 12 November 2014 / Revised: 16 January 2015 / Accepted: 17 January 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Limited availability of resources and increased amounts of waste coupled with an ever-increasing demand for raw materials are typical characteristics of our times. As such, there is an urgent need to accordingly update waste treatment technology. The aim of this study was to determine whether a separate treatment of the liquid and the solid fraction of biowaste could enhance overall efficiency. Liquid fractions obtained from two different separation procedures were fermented at a pH of 5 and uncontrolled pH conditions for 72 h. The fermentation conditions leading to highest lactic acid productivity and yield were evaluated. The substrates gained by both separation procedures showed efficient lactic acid production up to 30 g LA L−1 were produced from biowaste by its indigenous microbiota with a productivity rate of 0.8±0.1 g L−1 h−1 (Probst et al. 2013, 2015). In a Fig. 1 Current technology of biowaste treatment applied on the sample collection plant and its proposed modification. Anaerobic digestion is only one possible treatment option. The collection sides and pretreatments of two substrates (Sliquid and Spressed) fermented within this study are indicated in grey
batch experiment, Lactobacilli were enriched to over 90 % of the total bacterial consortium within the first 24 h, and LA concentrations of around 30 g L−1 were obtained (Dreschke et al. 2015). Lactobacillus acidophilus and Lactobacillus acetotolerans were the microbial key players of fermentation. This idea of LA production with its indigenous microbiota profits from the adapted microflora and offers a more efficient and environmentally friendly waste treatment technology. However, the high potential of this technology remains to be further explored. The application of biowaste as an LA production substrate and its integration into the current waste treatment processes was elaborated following the idea of a biorefinery concept (Fig. 1). Collected biowaste was separated into a solid fraction, containing nutrients slowly and sparingly degradable, and a liquid fraction, containing nutrients fast and easily accessible. While the solid part could be used directly for anaerobic digestion, the liquid part could be added after fermentation and the extraction of LA (or serve as process water in the waste treatment facility, depending on dry matter) (Schneider et al., 2013). This separation is supposed to have less effect on the current treatment efficiency by still allowing efficient LA production. In order to further improve the current biowaste treatment technology, a biorefinery concept (Fig. 1) was applied in this study. The aims were to (i) separate the biowaste into a solid and a liquid fraction (by either a bar screen or pressing), (ii) utilise both biowaste substrates in a fermentation process under different conditions, and (iii) evaluate the substrate- and condition-dependent LA production process regarding
Appl Microbiol Biotechnol
microbial, physical, and chemical characteristics. The production of LA in addition to generating energy by anaerobic digestion would exploit more of the biowaste potential and contribute to environmental protection.
Bradford method (Bradford 1976), respectively. Concentrations of lactic, acetic, propionic and butyric acid and the optical ratio of LA were investigated as in Probst et al. (2013). DNA extraction
Material and methods Sampling, pre-treatment, and fermentation conditions Two substrates were fermented in this study. Both were taken from a waste treatment facility collecting urban biowaste in Tyrol, Austria (Fig. 1). The treatment of biowaste included the rough crushing of incoming biowaste and the initial solid–liquid separation by a bar screen. The liquid fraction (Sliquid) was collected and diluted 2:3 with water in order to achieve a dry matter content 97 % identity and >90 % coverage. Statistical analyses Univariate analysis of variance (ANOVA) was performed in order to investigate the influence of substrate, time, and treatment on the fermentation characteristics. According to residual diagnostics and the Levene’s test, the normal distribution and homoscedasticity of the residuals could be assumed. All tests were computed at a significance level of 5 %, and calculations were done using SPSS 21 (IMB). Tables can be found in the supplementary material. Principal component analysis (PCA) was performed on the correlation matrix (variables: dry matter, OM-LA, reducing sugar concentration, protein concentration, LA concentration, acetic acid concentration, copy numbers of Lactobacillus group and Shannon index of the Lactobacillus group) in SPSS 21 (IBM) in order to summarise the multidimensional character of the experiment into a two-dimensional graph. To obtain orthogonal components (eigenvalue>1) varimax rotation was applied. Communality matrices, rotated component matrices, and proportion of explained variances by the components can be found in the supplementary material. The different fermentations were clustered according to the most important chemical and physical parameters: LA, acetic acid, the Shannon index and the copy number of the Lactobacillus group, organic content, and protein and sugar concentration. In order to integrate the influence of characteristic OTUs, these variables were regressed onto the components using linear regression approach. Relationships are shown as arrows. In order to illustrate the relationship between the bacterial community and the ratio of lactic/acetic acid, principal components were extracted also from OTUs >5 % as described above. Finally, visualisation was performed using Microsoft Excel 2010.
Results Microbial community dynamics Next-generation sequencing has not only been shown to be a useful tool for identifying complex microbial communities, but it has also become a state-of-the-art tool in environmental
microbiology studies (Caporaso et al. 2011). In order to delve deeper into the complex bacterial communities driving the fermentations, microbial key players were identified using next generation sequencing of the bacterial 16S RNA gene (V1–V3 region). The initial quality trimming of sequencing data led to the separate analyses of the forward and reverse sequences. Since the reverse sequences showed similar results but did not provide as much detailed information as the forward sequences, we chose to focus only on the forward sequences. From nearly 2×106 sequences generated, 25 % passed the quality trimming. These 455,000 sequences were clustered into more than 4800 OTUs. The majority of these OTUs (23 %) were later identified as Lactobacillaceae. Of all OTUs, 3.3 % could not be classified at phylum level, and 54.4 % could not be classified at genus level. However, from all of the sequences, 87 % were identified as Lactobacillaceae. Only a very small number of 30 % variance) represented Lactobacillus OTUs predominantly present after 24 h rather than after 72 h, although these OTUs alone did not seem responsible for either LA or acetic acid production. The third component (10 % variance) represented OTUs associated with the influence of the pH control. Clearly, the enrichment of Acetobacter, L. buchneri, and L. reuteri observed at an uncontrolled pH correlated with increased production of acetic acid rather than LA. Whether the growth of these OTUs was caused by the higher amounts of acetic acid or the low pH in general remains unclear. Although bacterial OTUs correlated with the physicochemical characteristics of the fermentations no single OTUs accounted for specific fermentation features. Obviously, the complex interaction of microorganisms and their conditions determined fermentation behaviour and efficiency. Further experiments investigating these interactions are necessary. The dominant role of L. delbrueckii and its closest relatives in a fermentation process applying biowaste has never been described before. Prior studies reported Lactobacillus plantarum either alone (Sakai et al. 2004) or in combination with Lactobacillus brevis (Sakai 2000) or Lactobacillus fermentum (Zang 2006) as key players in the fermentations of kitchen and other organic wastes. In a preliminary study
Appl Microbiol Biotechnol Fig. 5 Correlation of lactic/acetic acid ratio and principal components extracted from OTUs>5 % at time points 0, 24, and 72 h of biowaste fermentation
offering a first insight into the microbiota of biowaste, L. plantarum was found to be the most frequently isolated organism. Furthermore, L. brevis and L. fermentum (Probst et al. 2013) and L. acidophilus and its closest relatives (Probst et al. 2015) were identified as bacterial key players. Whether these differences depend on seasonal changes, country- or region-dependent biowaste characteristics or methodology used for organism identification remains still a matter for future research. However, the domination of
Fig. 6 Biplot of principal coordinates extracted from physicochemical characteristics (cluster) and the bacterial consortium of biowaste (shown as arrows) observed after 0, 24, and 72 h of lactic acid fermentation. Samples were clustered according to an anabolic component extracted from the Lactobacillus group diversity (Shannon index) and 16S rRNA gene count and lactic and acetic acid produced and a catabolic component extracted from the nutrients, sugar, protein, and organic matter, metabolised over time
Lactobacilli within the bacterial biowaste community (Mayrhofer et al. 2006; Partanen et al. 2010; Probst et al. 2013; Probst et al. 2015) was shown once more. These findings and the establishment of a stable Lactobacilli community during the fermentations clearly indicate that biowaste, independent of the sampling season and region, can be applied for LA production. The fermentation at pH 5 seemed best for the fermentative production of LA. Despite the higher LA amounts per dry
Appl Microbiol Biotechnol
matter content in Spressed, Sliquid turns up to be preferable as a substrate. Reaching a productivity of >1 g L−1 h−1, fermentation of the liquid fractions of biowaste was able to compete with other biowaste fermentations (0.8±0.1 g L−1 h−1) (Probst et al. 2015) and alternate waste streams such as potato waste (0.7–1.0 g L−1 h−1) (Palaniraj and Nagarajan 2012) or molasses (1.0 g L−1 h−1) (Wang et al. 2010). Although the fermentation of Sliquid resulted in less acetic acid production compared to Spressed, the amount produced was relatively high compared to conventional LA production using highly productive and homofermentative strains. Silva et al. (2013), in contrast, reported a higher production of acetic acid from biowaste in batch fermentation at 37 °C but without strong LA accumulation. The initial pH of that study was higher (neutral pH in the beginning and pH decline to 5.5–6) probably allowing for higher acid production. However, fermentation of the liquid biowaste fraction needs further optimisation to prolong the high ratio of lactic/acetic acid and productivity beyond the first 24 h. In comparison to Yesil et al. (2014) who reported efficient production of LA for more than 7 days, the main production interval seemed rather short. However, pH values in his study never declined to a comparable level. Even so, the total amount produced in the current study was higher. A higher pH probably allows for prolonged LA production but does not necessarily increase total LA amount. However, this finding suggests the installation of a pH control. Future experiments will investigate whether the continuous extraction of LA can prolong the period of efficient LA production. Although recent studies have indicated that the selective enrichment of Bacillus species, mainly Bacillus coagulans (Akao et al. 2007; Hidaka et al. 2010), lead to the production of optically pure L-LA from organic residues, this genus was not identified during fermentation of biowaste. In accordance with the findings of Sakai et al. (2000) who produced LA from kitchen waste, fermentation of biowaste also resulted in a racemic mixture of LA. The production of poly-lactic acid demands optical pure LA. Despite this disadvantage, the implementation of a LA production step into current biowaste treatment technology would offer an environmentally friendly way of waste utilisation and better resourcing. Improved membrane technology for technical separation of the two isomers would alleviate the need for an optically pure product (Hadik et al. 2002). In conclusion, fermentative LA production was efficient from both liquid biowaste fractions. Sliquid, however, came with a higher nutrient content and pH. During fermentation, this substrate also produced more LA and reached higher productivity rates compared to Spressed. Therefore, the separation via a bar screen is the preferred choice of treatment prior to liquid biowaste fermentation. Despite their initially different bacterial community, all fermentations were dominated by L. delbrueckii and its closest relatives to a certain point. The increased process efficiency at a pH of 5 clearly suggested the installation of a pH control.
Acknowledgements We kindly thank Sebastian Waldhuber and Ljubica Begovic for their help in the lab. We also thank Paul Fraiz for improving the English language of the manuscript. This work was financed by the Klima- und Energiefonds (project KR11NE1F00380) in t h e f r a m e w o r k o f BN e u e E n e r g i e n 2 0 2 0 ^ a n d b y t h e BNachwuchsförderung^ of the Universität Innsbruck. Maraike Probst was partly funded by the doctoral fellowship BDoktoratsstipendium aus der Nachwuchsförderung^ and the BStipendium für österreichische Graduierte^ of the Universität Innsbruck.
References Abdel-Rahman MA, Tashiro Y, Sonomoto K (2011) Lactic acid production from lignocellulose-derived sugars using lactic acid bacteria: overview and limits. J Biotechnol 156:286–301. doi:10.1016/j. jbiotec.2011.06.017 Akao S, Tsuno H, Horie T, Mori S (2007) Effects of pH and temperature on products and bacterial community in-lactate batch fermentation of garbage under unsterile condition. Water Res 41:2636–2642. doi: 10.1016/j.watres.2007.02.032 Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254 Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, Fierer N, Knight R (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A 108:4516–4522. doi:10.1073/pnas. 1000080107 Dreschke G, Probst M, Walter A, Walde J, Pümpel T, Insam H (2015) Lactic acid and methane: Improved exploitation of biowaste potential. Bioresource Technol 176:47–55. doi:10.1016/j.biortech.2014.10.136 Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356 Dusselier M, Van Wouwe P, Dewaele A, Makshina E, Sels BF (2013) Lactic acid as a platform chemical in the biobased economy: the role of chemocatalysis. Energy Environ Sci 6:1415–1442. doi:10.1039/ C3ee00069a Endo A, Okada S (2005) Lactobacillus satsumensis sp nov., isolated from mashes of shochu, a traditional Japanese distilled spirit made from fermented rice and other starchy materials. Int J Syst Evol Microbiol 55:83–85. doi:10.1099/ijs.0.63248-0 Ge J, Chai Y, Chen L, Ping W (2012) The dynamics of bacteria community diversity during the fermentation process of traditional soybean paste Shengtai Xuebao. Acta Ecol Sin 32:2532–2538. doi:10.5846/ stxb201103110295 Hadik P, Szabo LP, Nagy E (2002) D,L-lactic acid and D,L-alanine enantio separation by membrane process. Desalination 148:193– 198. doi:10.1016/S0011-9164(02)00697-5 Hidaka T, Asahira T, Koshikawa H, Cheon J, Park Y, Tsuno H (2010) Effect of microbial composition on thermophilic acid fermentation. Enzym Microb Technol 47:127–133. doi:10.1016/j.enzmictec.2010. 06.005 Holzapfel W, Wood B (2014) Lactic acid bacteria—biodiversity and taxonomy. Blackwell, Wiley John RP, Nampoothiri KM, Pandey A (2007) Fermentative production of lactic acid from biomass: an overview on process developments and future perspectives. Appl Microbiol Biotechnol 74:524–534. doi:10. 1007/s00253-006-0779-6 Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD (2013) Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina
Appl Microbiol Biotechnol sequencing platform. Appl Environ Microbiol 79:5112–5120. doi: 10.1128/Aem.01043-13 Mayrhofer S, Mikoviny T, Waldhuber S, Wagner AO, Innerebner G, Franke-Whittle IH, Mark TD, Hansel A, Insam H (2006) Microbial community related to volatile organic compound (VOC) mission in household biowaste. Environ Microbiol 8:1960–1974. doi:10.1111/j.1462-2920.2006.01076.x Motte JC, Escudie R, Bernet N, Delgenes JP, Steyer JP, Dumas C (2013a) Dynamic effect of total solid content, low substrate/inoculum ratio and particle size on solid-state anaerobic digestion. Bioresource Technol 144:141–148. doi:10.1016/j.biortech.2013.06.057 Motte JC, Trably E, Escudie R, Hamelin J, Steyer JP, Bernet N, Delgenes JP, Dumas C (2013b) Total solids content: a key parameter of metabolic pathways in dry anaerobic digestion. Biotechnol Biofuels. doi:10.1186/1754-6834-6-164 Nguyen CM, Kim JS, Nguyen TN, Kim SK, Choi GJ, Choi YH, Jang KS, Kim JC (2013) Production of L- and D-lactic acid from waste Curcuma longa biomass through simultaneous saccharification and cofermentation. Bioresource Technol 146:35–43. doi:10.1016/ j.biortech.2013.07.035 Palaniraj R, Nagarajan P (2012) Kinetic studies in production of lactic acid from waste potato starch using Lactobacillus casei. Int J ChemTech Res 4:1601–1614 Partanen P, Hultman J, Paulin L, Auvinen P, Romantschuk M (2010) Bacterial diversity at different stages of the composting process. BMC Microbiol. doi:10.1186/1471-2180-10-94, 10 doi:Artn 94 Probst M, Fritschi A, Wagner A, Insam H (2013) Biowaste: a lactobacillus habitat and lactic acid fermentation substrate. Bioresource Technol 143:647–652. doi:10.1016/j.biortech.2013. 06.022 Probst M, Walde J, Pümpel T, Wagner AO, Insam H (2015) A closed loop for municipal organic solid waste by lactic acid fermentation. Bioresource Technol 175:142–151 Sakai K, Mori M, Fujii A, Iwami Y, Chukeatirote E, Shirai Y (2004) Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rRNA-targeted oligonucleotide probes. J Biosci Bioeng 98:48–56. doi:10.1263/Jbb.98.48 Sakai K, Murata Y, Yamazumi H, Tau Y, Mori M, Moriguchi M, Shirai Y (2000) Selective proliferation of lactic acid bacteria and accumulation of lactic acid during open fermentation of kitchen refuse with intermittent pH adjustment. Food Sci Technol Res 6:140–145
Schloss PD Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541. doi:10.1128/Aem.0154109 Schneider I, Wörle A, Schweitzer P, Probst M, Insam H, Bockreis A (2013) Cascading Approach of Biowaste Treatment to Obtain Value-added Organic By-Products and Biogas. ISWA World Congress Vienna ISBN 978-3-200-03229-3 Secchi N, Giunta D, Pretti L, Garcia MR, Roggio T, Mannazzu I, Catzeddu P (2012) Bioconversion of ovine scotta into lactic acid with pure and mixed cultures of lactic acid bacteria. J Ind Microbiol Biotechnol 39:175–181. doi:10.1007/s10295-011-1013-9 Silva FC, Serafim LS, Nadais H, Arroja L, Capela I (2013) Acidogenic fermentation towards valorisation of organic waste streams into volatile fatty acids. Chem Biochem Eng Q 27:467–476 Song DJ, Kang HY, Wang JQ, Peng H, Bu DP (2014) Effect of feeding bacillus subtilis natto on hindgut fermentation and microbiota of Holstein dairy cows. Asian Aust J Anim 27:495–502. doi:10. 5713/ajas.2013.13522 Walter J, Hertel C, Tannock GW, Lis CM, Munro K, Hammes WP (2001) Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis. Appl Environ Microbiol 67:2578–2585 Wang LM, Zhao B, Liu B, Yu B, Ma CQ, Su F, Hua DL, Li QG, Ma YH, Xu P (2010) Efficient production of L-lactic acid from corncob molasses, a waste by-product in xylitol production, by a newly isolated xylose utilizing Bacillus sp strain. Bioresource Technol 101: 7908–7915. doi:10.1016/j.biortech.2010.05.031 Yesil H, Tugtas AE, Bayrakdar A, Calli B (2014) Anaerobic fermentation of organic solid wastes: volatile fatty acid production and separation. Water Sci Technol 69:2132–2138. doi:10.2166/Wst.2014.132 Zang B, He P, Ye N, Shao L (2006) Enhanced isomer purity of lactic acid from the non-sterile fermentation of kitchen wastes. Bioresource Technol 99:855–862 Zhao WJ, Sun XH, Wang QH, Ma HZ, Teng Y (2009) Lactic acid recovery from fermentation broth of kitchen garbage by esterification and hydrolysis method. Biomass Bioenerg 33:21–25. doi:10.1016/j. biombioe.2008.04.010
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Lactic acid fermentation within a cascading approach for biowaste treatment Maraike Probst & Janette Walde & Thomas Pümpel & Andreas Otto Wagner & Irene Schneider & Heribert Insam
Received: 12 November 2014 / Revised: 16 January 2015 / Accepted: 17 January 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Limited availability of resources and increased amounts of waste coupled with an ever-increasing demand for raw materials are typical characteristics of our times. As such, there is an urgent need to accordingly update waste treatment technology. The aim of this study was to determine whether a separate treatment of the liquid and the solid fraction of biowaste could enhance overall efficiency. Liquid fractions obtained from two different separation procedures were fermented at a pH of 5 and uncontrolled pH conditions for 72 h. The fermentation conditions leading to highest lactic acid productivity and yield were evaluated. The substrates gained by both separation procedures showed efficient lactic acid production up to 30 g LA L−1 were produced from biowaste by its indigenous microbiota with a productivity rate of 0.8±0.1 g L−1 h−1 (Probst et al. 2013, 2015). In a Fig. 1 Current technology of biowaste treatment applied on the sample collection plant and its proposed modification. Anaerobic digestion is only one possible treatment option. The collection sides and pretreatments of two substrates (Sliquid and Spressed) fermented within this study are indicated in grey
batch experiment, Lactobacilli were enriched to over 90 % of the total bacterial consortium within the first 24 h, and LA concentrations of around 30 g L−1 were obtained (Dreschke et al. 2015). Lactobacillus acidophilus and Lactobacillus acetotolerans were the microbial key players of fermentation. This idea of LA production with its indigenous microbiota profits from the adapted microflora and offers a more efficient and environmentally friendly waste treatment technology. However, the high potential of this technology remains to be further explored. The application of biowaste as an LA production substrate and its integration into the current waste treatment processes was elaborated following the idea of a biorefinery concept (Fig. 1). Collected biowaste was separated into a solid fraction, containing nutrients slowly and sparingly degradable, and a liquid fraction, containing nutrients fast and easily accessible. While the solid part could be used directly for anaerobic digestion, the liquid part could be added after fermentation and the extraction of LA (or serve as process water in the waste treatment facility, depending on dry matter) (Schneider et al., 2013). This separation is supposed to have less effect on the current treatment efficiency by still allowing efficient LA production. In order to further improve the current biowaste treatment technology, a biorefinery concept (Fig. 1) was applied in this study. The aims were to (i) separate the biowaste into a solid and a liquid fraction (by either a bar screen or pressing), (ii) utilise both biowaste substrates in a fermentation process under different conditions, and (iii) evaluate the substrate- and condition-dependent LA production process regarding
Appl Microbiol Biotechnol
microbial, physical, and chemical characteristics. The production of LA in addition to generating energy by anaerobic digestion would exploit more of the biowaste potential and contribute to environmental protection.
Bradford method (Bradford 1976), respectively. Concentrations of lactic, acetic, propionic and butyric acid and the optical ratio of LA were investigated as in Probst et al. (2013). DNA extraction
Material and methods Sampling, pre-treatment, and fermentation conditions Two substrates were fermented in this study. Both were taken from a waste treatment facility collecting urban biowaste in Tyrol, Austria (Fig. 1). The treatment of biowaste included the rough crushing of incoming biowaste and the initial solid–liquid separation by a bar screen. The liquid fraction (Sliquid) was collected and diluted 2:3 with water in order to achieve a dry matter content 97 % identity and >90 % coverage. Statistical analyses Univariate analysis of variance (ANOVA) was performed in order to investigate the influence of substrate, time, and treatment on the fermentation characteristics. According to residual diagnostics and the Levene’s test, the normal distribution and homoscedasticity of the residuals could be assumed. All tests were computed at a significance level of 5 %, and calculations were done using SPSS 21 (IMB). Tables can be found in the supplementary material. Principal component analysis (PCA) was performed on the correlation matrix (variables: dry matter, OM-LA, reducing sugar concentration, protein concentration, LA concentration, acetic acid concentration, copy numbers of Lactobacillus group and Shannon index of the Lactobacillus group) in SPSS 21 (IBM) in order to summarise the multidimensional character of the experiment into a two-dimensional graph. To obtain orthogonal components (eigenvalue>1) varimax rotation was applied. Communality matrices, rotated component matrices, and proportion of explained variances by the components can be found in the supplementary material. The different fermentations were clustered according to the most important chemical and physical parameters: LA, acetic acid, the Shannon index and the copy number of the Lactobacillus group, organic content, and protein and sugar concentration. In order to integrate the influence of characteristic OTUs, these variables were regressed onto the components using linear regression approach. Relationships are shown as arrows. In order to illustrate the relationship between the bacterial community and the ratio of lactic/acetic acid, principal components were extracted also from OTUs >5 % as described above. Finally, visualisation was performed using Microsoft Excel 2010.
Results Microbial community dynamics Next-generation sequencing has not only been shown to be a useful tool for identifying complex microbial communities, but it has also become a state-of-the-art tool in environmental
microbiology studies (Caporaso et al. 2011). In order to delve deeper into the complex bacterial communities driving the fermentations, microbial key players were identified using next generation sequencing of the bacterial 16S RNA gene (V1–V3 region). The initial quality trimming of sequencing data led to the separate analyses of the forward and reverse sequences. Since the reverse sequences showed similar results but did not provide as much detailed information as the forward sequences, we chose to focus only on the forward sequences. From nearly 2×106 sequences generated, 25 % passed the quality trimming. These 455,000 sequences were clustered into more than 4800 OTUs. The majority of these OTUs (23 %) were later identified as Lactobacillaceae. Of all OTUs, 3.3 % could not be classified at phylum level, and 54.4 % could not be classified at genus level. However, from all of the sequences, 87 % were identified as Lactobacillaceae. Only a very small number of 30 % variance) represented Lactobacillus OTUs predominantly present after 24 h rather than after 72 h, although these OTUs alone did not seem responsible for either LA or acetic acid production. The third component (10 % variance) represented OTUs associated with the influence of the pH control. Clearly, the enrichment of Acetobacter, L. buchneri, and L. reuteri observed at an uncontrolled pH correlated with increased production of acetic acid rather than LA. Whether the growth of these OTUs was caused by the higher amounts of acetic acid or the low pH in general remains unclear. Although bacterial OTUs correlated with the physicochemical characteristics of the fermentations no single OTUs accounted for specific fermentation features. Obviously, the complex interaction of microorganisms and their conditions determined fermentation behaviour and efficiency. Further experiments investigating these interactions are necessary. The dominant role of L. delbrueckii and its closest relatives in a fermentation process applying biowaste has never been described before. Prior studies reported Lactobacillus plantarum either alone (Sakai et al. 2004) or in combination with Lactobacillus brevis (Sakai 2000) or Lactobacillus fermentum (Zang 2006) as key players in the fermentations of kitchen and other organic wastes. In a preliminary study
Appl Microbiol Biotechnol Fig. 5 Correlation of lactic/acetic acid ratio and principal components extracted from OTUs>5 % at time points 0, 24, and 72 h of biowaste fermentation
offering a first insight into the microbiota of biowaste, L. plantarum was found to be the most frequently isolated organism. Furthermore, L. brevis and L. fermentum (Probst et al. 2013) and L. acidophilus and its closest relatives (Probst et al. 2015) were identified as bacterial key players. Whether these differences depend on seasonal changes, country- or region-dependent biowaste characteristics or methodology used for organism identification remains still a matter for future research. However, the domination of
Fig. 6 Biplot of principal coordinates extracted from physicochemical characteristics (cluster) and the bacterial consortium of biowaste (shown as arrows) observed after 0, 24, and 72 h of lactic acid fermentation. Samples were clustered according to an anabolic component extracted from the Lactobacillus group diversity (Shannon index) and 16S rRNA gene count and lactic and acetic acid produced and a catabolic component extracted from the nutrients, sugar, protein, and organic matter, metabolised over time
Lactobacilli within the bacterial biowaste community (Mayrhofer et al. 2006; Partanen et al. 2010; Probst et al. 2013; Probst et al. 2015) was shown once more. These findings and the establishment of a stable Lactobacilli community during the fermentations clearly indicate that biowaste, independent of the sampling season and region, can be applied for LA production. The fermentation at pH 5 seemed best for the fermentative production of LA. Despite the higher LA amounts per dry
Appl Microbiol Biotechnol
matter content in Spressed, Sliquid turns up to be preferable as a substrate. Reaching a productivity of >1 g L−1 h−1, fermentation of the liquid fractions of biowaste was able to compete with other biowaste fermentations (0.8±0.1 g L−1 h−1) (Probst et al. 2015) and alternate waste streams such as potato waste (0.7–1.0 g L−1 h−1) (Palaniraj and Nagarajan 2012) or molasses (1.0 g L−1 h−1) (Wang et al. 2010). Although the fermentation of Sliquid resulted in less acetic acid production compared to Spressed, the amount produced was relatively high compared to conventional LA production using highly productive and homofermentative strains. Silva et al. (2013), in contrast, reported a higher production of acetic acid from biowaste in batch fermentation at 37 °C but without strong LA accumulation. The initial pH of that study was higher (neutral pH in the beginning and pH decline to 5.5–6) probably allowing for higher acid production. However, fermentation of the liquid biowaste fraction needs further optimisation to prolong the high ratio of lactic/acetic acid and productivity beyond the first 24 h. In comparison to Yesil et al. (2014) who reported efficient production of LA for more than 7 days, the main production interval seemed rather short. However, pH values in his study never declined to a comparable level. Even so, the total amount produced in the current study was higher. A higher pH probably allows for prolonged LA production but does not necessarily increase total LA amount. However, this finding suggests the installation of a pH control. Future experiments will investigate whether the continuous extraction of LA can prolong the period of efficient LA production. Although recent studies have indicated that the selective enrichment of Bacillus species, mainly Bacillus coagulans (Akao et al. 2007; Hidaka et al. 2010), lead to the production of optically pure L-LA from organic residues, this genus was not identified during fermentation of biowaste. In accordance with the findings of Sakai et al. (2000) who produced LA from kitchen waste, fermentation of biowaste also resulted in a racemic mixture of LA. The production of poly-lactic acid demands optical pure LA. Despite this disadvantage, the implementation of a LA production step into current biowaste treatment technology would offer an environmentally friendly way of waste utilisation and better resourcing. Improved membrane technology for technical separation of the two isomers would alleviate the need for an optically pure product (Hadik et al. 2002). In conclusion, fermentative LA production was efficient from both liquid biowaste fractions. Sliquid, however, came with a higher nutrient content and pH. During fermentation, this substrate also produced more LA and reached higher productivity rates compared to Spressed. Therefore, the separation via a bar screen is the preferred choice of treatment prior to liquid biowaste fermentation. Despite their initially different bacterial community, all fermentations were dominated by L. delbrueckii and its closest relatives to a certain point. The increased process efficiency at a pH of 5 clearly suggested the installation of a pH control.
Acknowledgements We kindly thank Sebastian Waldhuber and Ljubica Begovic for their help in the lab. We also thank Paul Fraiz for improving the English language of the manuscript. This work was financed by the Klima- und Energiefonds (project KR11NE1F00380) in t h e f r a m e w o r k o f BN e u e E n e r g i e n 2 0 2 0 ^ a n d b y t h e BNachwuchsförderung^ of the Universität Innsbruck. Maraike Probst was partly funded by the doctoral fellowship BDoktoratsstipendium aus der Nachwuchsförderung^ and the BStipendium für österreichische Graduierte^ of the Universität Innsbruck.
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