and their extraction using two recovery techniques. ... Esterification allowed the recovery of VFA as ... favour of acetic acid from the fermentation broth.
Production and extraction of short chain carboxylic acids from the anaerobic mixed-culture fermentation of slaughterhouse blood
Jersson Plácido, Yue Zhang Water and Environmental Engineering Group, Faculty of Engineering and the Environment, University of Southampton, Southampton, United Kingdom
Keywords: Anaerobic mixed-culture fermentation; volatile fatty acids; esterification; pertraction
Aim of the research: to prove the concept that, by selection of reactor conditions and substrate type, anaerobic fermentation can be directed towards specified carboxylate products in concentrations suitable for extraction and further refining as intermediate bulk chemicals Short summary: This study evaluated the production of short chain carboxylic acids, in particular volatile fatty acids (VFA), from slaughterhouse blood using anaerobic fermentation and their extraction using two recovery techniques. The fermentation was operated under non-sterile conditions and used a mixed community of naturally occurring anaerobic microorganisms in biosolids digestate collected from a wastewater treatment plant (Millbrook, Southampton). To improve the VFA production different blood concentrations, iodoform (methanogens inhibitor), enzymatic hydrolysis pretreatment, acclimated inoculum, initial inoculum to substrate ratio, and different reactor configurations (batch, fed-batch and semi-continuous) were evaluated. All the treatments generated high VFA concentration up to 100000 mg L-1 with acetic, n-butyric and iso-valeric acids as the most predominant species. The different factors caused variations in the VFA concentration, profile, production rate and yield. Two routes, esterification and pertraction, were then tested for their efficiency to extract VFA from the fermentation broth. Esterification allowed the recovery of VFA as methyl esters which can be used industrially as scents and fragrances, and the production of ammonium sulphate, a mineral fertiliser, as a value-added byproduct of the esterification reaction. A pertraction system, comprised of a hydrophobic membrane and extractant using octanol/TOA (tri-n-octyl amine), was efficient to separate butyric and iso-valeric acids in favour of acetic acid from the fermentation broth. These results provided important information for the development of a carboxylate-platform bio-refinery using high-protein wastes as substrate.
Results The effective VFA production using mixed-culture anaerobic fermentation of slaughterhouse blood was demonstrated in this work. The flask batch experiments showed that blood concentration to a great degree controlled the VFA production extent and the resulting final VFA concentration, when the amount of inoculum was fixed. When the initial blood
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Figure 1. VFA, soluble protein, total ammoniacal-nitrogen and pH profile in batch reactor started with -1 a blood concentration of 143 g TS L and acclimated inoculum.
No apparent difference was identified between the IDF and No-IDF treatments at high initial blood concentrations mainly due to the inhibitory effect of high ammonium concentration to methanogens in both cases: as shown in Figure 1 the total ammoniacal-nitrogen centration increased rapidly from 500 to 7000 mg NH3-N L-1 in the first 5 days of the experiment. In addition, after the inoculum acclimation VFA production reached a concentration of 100000 mg L-1 when the initial blood dosing is 143 g dried blood L-1 (135 g VS L-1). This concentration is the highest VFA level reported by using protein wastes as substrate for carboxylic acid production (Kovács et al. 2013; Kovács et al. 2015).The enzymatic pretreatment improved the VFA production rate at the initial stage of fermentation due to the higher availability of free amino acids and small peptides for microorganisms to consume. In all tests, acetic, n-butyric and iso-valeric acids were the acids most produced, with isobutyric and propionic acids as minor components; those acids have been directly associated with at least one of the amino acids (Choi et al. 2013). 6
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Figure 2. VFA, soluble protein, total ammoniacal nitrogen, pH, CH4, CO2, and H2 profiles in semicontinuous reactor (inoculum: biosolids digestate, HRT: 7 days, no enzymatic pretreatment, no iodoform addition)
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In terms of fermenter configuration comparison, the batch fermenters with acclimated inoculum and the fed-batch fermenters achieved the largest concentration of VFA (100000 mg L-1) at an initial blood concentration of 143 g dried blood L-1, indicating a conversion rate of 0.66 g VFA g-1 VS. This level of VFA concentration was expected mainly because the duration of the experiments (45 days) allowed the conversion process to proceed to a greater extent than the semi-continuous configuration. The reduced amount of VFA produced in semi-continuous fermenters was caused by the much shorter hydraulic retention time (7 days). The best results of semi-continuous operation (Figure 2) during 45 days can produce in total 444000 mg of VFA L-1 (0.49 g VFA g-1 VS). The semi-continuous production is considerably larger than the batch and fed-batch reactors (100000 mg L-1). It is important to point it out that in batch processes the 80% of the total VFA production is achieved by day 10. If the batch fermentation is performed only during the initial 10 days a larger VFA production can be achieved. Batch processes can be improved by reducing the fermentation time, although this VFA yield increase is achieved at the expense of lower blood conversion rate.
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b) Figure 3. VFA recovery method a) esterification process, b) pertraction recovery system
Two recovery methodologies (esterification and pertraction) were evaluated in this study. The esterification method (Figure 3a) allowed the recovery of ammonium produced by the blood transformation into ammonium sulphate and the recovery of VFA as VFA-methyl esters. VFA-methyl esters production efficiency was dependent upon the VFA concentration and affected by the existence of other materials. The presence of untransformed blood proteins, for example, reduced the efficiency of the esterification reaction. This issue was more critical in semi-continuous fermentation where the blood degradation extent was lower than that of batch and fed-batch reactors. Therefore, batch processes were regarded to be more suitable for esterification reaction. In this process, the most produced VFA-methyl esters were methyl acetate, methyl butyrate, and methyl iso-valerate, and these esters have industrial importance as scents and fragrances. In addition, methyl-esters can also be converted back into VFA using an extra hydrolysis step. A pertraction system was also evaluated to recover VFA from the fermentation broth. Pertraction is the extraction of organic compounds from liquids with the aid of membranes. Figure 3b explains the possible recovery steps necessary for VFA recovery using pertraction methodology. In this study, the parameters of TOA/ octanol extraction were tested by a factorial design. The optimal pH of 3
was demonstrated and the relationship of 1:1 fermentation broth to TOA(25%)/octanol(75%) was identified. Under the optimal extraction conditions the pertraction system allowed the recovery of butyric and isovaleric acids (>80%) in favour of acetic acid (5 %) from fermentation froth. This pertractor system is a better fit for the continuous fermentation configuration because it reduces the product-induced inhibition effect on longer-chain VFA and allows the utilisation of acetic acid in recycled stream. Conclusions Anaerobic mixed-culture fermentation was proved to be an effective way of transforming slaughterhouse blood into VFA. In this process, the dominant acids were acetic, n-butyric and iso-valeric acids. The batch and semi-continuous reactors generated promising results in terms of total VFA concentration and yield. Integrated batch fermentation and esterification processes were proposed to be used for the recovery of both esters (scents and fragrances) and ammonium sulphate (fertiliser). For semi-continuous/continuous fermentation configuration, a pertractor system was regarded as a more suitable downstream process. The membrane extractor recovered butyric and iso-valeric acids from the fermenter effluent in favour of acetic acid, with the residual stream rich in acetic acid returned to mix up with dried substrate. These results highlighted some essential aspects for the development of a carboxylate-platform bio-refinery from high protein wastes.
References Choi K, Jeon BS, Kim B, Oh M, Um Y, Sang B (2013) In situ biphasic extractive fermentation for hexanoic acid production from sucrose by Megasphaera elsdenii NCIMB 702410. Appl. Biochem. Biotechnol. 171:1094-1107. Kovács E, Wirth R, Maróti G, Bagi Z, Rákhely G, Kovács KL (2013) Biogas production from protein-rich biomass: fed-batch anaerobic fermentation of casein and of pig blood and associated changes in microbial community composition. PloS one 8:e77265. Kovács E, Wirth R, Maróti G, Bagi Z, Nagy K, Minárovits J, Rákhely G, Kovács KL (2015) Augmented biogas production from protein-rich substrates and associated metagenomic changes. Bioresour. Technol. 178:254-261. http://dx.doi.org/10.1016/j.biortech.2014.08.111.
