Table 1 presents the composition of different OFMSW samples used to prepare the OFMSW hydrolysates. Non-biodegradable materials contained in OFMSW (glass, stones, plastics, sand, etc.) can cause serious technological problems in industrial facilities (clogging, erosion in equipment) and reduce the performance of biological processes. Unsorted biowaste obtained via mechanical sorting processing using mixed MSW from household bins contained higher contents of inert material (non-biodegradable) and ash as well as lower moisture content than sorted biowaste. The glucan content, representing both cellulose and starch, is slightly higher in sorted biowaste (ca. 40 %, db). Most of glucan originates from cellulose since the obtained starch content is 4 - 5.3% in all cases. The xylan content was lower than 5 % (db) in sorted biowaste, while it was higher than 5 % (db) in unsorted biowaste. The pectin content, originating mainly from fruit waste, is lower (10.1 – 12.19 %, db) in unsorted biowaste than sorted biowaste (15.87 – 18.25 %, db). The protein was higher (8.75 – 10.15 %, db) in sorted biowaste, while the fat content (4.59 – 5.86 %, db) was higher in unsorted biowaste. The lignin content varies (5.64 – 11.02 %, db) among all OFMSW samples. This low content of lignin in municipal biowaste is advantageous compared with other lignocellulosic wastes (typically >25% in woods).
The primary components of sorted municipal solid waste are cellulose (45%), hemicellulose (9%) and lignin (10%) . Garcia et al.  characterized different fractions of biodegradable municipal solid waste (meat, fish, fruit and vegetable, restaurant and household waste). Dry matter, ash content and crude protein in the different fractions varied between 11.9 - 59.0 %, 4.9 - 21.8 % and 11.6 - 57.0 % (on a dry basis, db), respectively .
OFMSW hydrolysate production
OFMSW mechanical pretreatment and enzymatic hydrolysis was performed by IMECAL S.A. with tailor-made enzymatic cocktails provided by Novozymes. The enzyme content and the corresponding enzyme activities are confidential and cannot be mentioned in this publication. Glucan content (including both cellulose and starch) varied in the range of 25 – 40% (db) and xylan content ranged from 0.2 – 8.7 % (db). Glucan (cellulose and starch) hydrolysis conversion yield was 75 % (w/w) and xylan conversion yield was around 12.5% (w/w). Table 2 presents the variation in the composition of different batches of OFMSW hydrolysates produced in this study. The total dry weight measured after hydrolysis was in the range of 114.17 – 118.81 g/L in all cases. In the liquid fraction, the total sugar concentration in OFMSW hydrolysate ranged between 31.2 – 107.3 g/L with 70.7 – 81.3% glucose (25.4 – 75.9 g/L), 7.1 – 12.6% xylose (3.95 – 7.6 g/L) and 0.3 to 14.4% fructose (0.1 – 15.5 g/L). Glycerol, sucrose, galactose, arabinose, mannose concentrations were less than 5% of the total sugar content. Free amino nitrogen (FAN) and inorganic phosphorus (IP) concentrations in the liquid fraction of OFMSW ranged between 203.6 - 638.7 mg/L and 100.6 - 553 mg/L, respectively.
Significant lactic acid concentrations (10.7 – 18.6 g/L) and lower acetic acid concentrations (1.5 – 3.7 g/L) were detected in all OFMSW hydrolysates. These organic acids were present since the beginning of the hydrolysis indicating contamination of OFMSW despite the origin of biowaste streams. No organic acid production or bacterial growth was observed during hydrolysis due to the aseptic conditions used. Furfural and 5-hydroxymethylfurfural were not detected in OFMSW hydrolysates. This was expected as these inhibitory compounds can be generated from the degradation of xylose and glucose under intensive chemical treatment.
In the solid fraction that remained after OFMSW hydrolysis, the ash (5.7 - 25 %, db) and protein (7 - 19.85 %, db) contents varied at a wide range depending on the origin of OFMSW. The lipid content was 6.8 - 7.6 % (db). Water and ethanol soluble extractives were ca. 33 % (db). Lignin, cellulose and hemicellulose ranged from 16.92 – 27.39 %, 9.07 – 9.46 % and 12.01 – 12.37 % (db), respectively. Hemicellulose consisted of xylan, galactan and mannan fractions.
Table 3 presents succinic acid production via fermentation using B. succiniciproducens and A. succinogenes at different initial total carbon source concentrations using either commercial carbon sources or OFMSW hydrolysate. Fermentations using the OFMSW hydrolysate enhanced the productivity of both microorganisms compared to the commercial medium, with 52% increase on average in the case of B. succiniciproducens and 32% in the case of A. succinogenes (Table 3). The highest succinic acid concentration that was observed in the case of B. succiniciproducens fermentations was 37.1 g/L in OFMSW hydrolysate and in the case of A. succinogenes was 37.9 g/L both in OFMSW hydrolysate and commercial substrate (Table 3).
The by-product to succinic acid ratio decreased with increasing initial total carbon source concentration for both A. succinogenes and B. succiniciproducens in both fermentation media. B. succiniciproducens resulted in a decrease of by-product to succinic acid ratio at around 50% in the case of glucose-based fermentations and up to 25% in the case of OFMSW hydrolysate. A. succinogenes by-product to succinic acid ratio decreased up to 84% in the case of glucose and up to 65% in the case of OFMSW hydrolysate (Table 3). The major difference between the two microorganisms lies on the fact that lactic acid production from B. succiniciproducens occurs throughout fermentation. When OFMSW hydrolysates were used, lactic acid and acetic acid were present at the beginning of fermentation and they have been excluded from the ratios presented in Table 3.
Figure 1 presents experimental results of batch fermentation carried out with A. succinogenes using OFMSW hydrolysate at initial total sugar concentration of 80 g/L. The final succinic acid concentration was 37.9 g/L with yield of 0.5 g/g and productivity of 0.57 g/L/h. The initial FAN concentration using both the synthetic medium and the OFMSW hydrolysate was in the range of 251 - 285 mg/L in all batch fermentations. FAN consumption occurred in the first 24 h and remained constant (at around 100 mg/L) until the end of fermentation (Figures 1a).
Babaei et al.  carried out batch fermentations with B. succiniciproducens cultivated in a hydrolysate from the organic fraction of household kitchen waste (the sugars contained 85% glucose and 15% xylose) with CO2 supply from either MgCO3 or raw biogas. Succinic acid production efficiency of around 5.5 g/L with yield 0.39 g/g and 3.8 g/L with yield 0.25 g/g, respectively . A. succinogenes has been employed for succinic acid production using deacetylated dilute acid pretreated corn stover hydrolysate leading to the production of 42.8 g/L succinic acid with yield of 0.74 g/g and maximum productivity of 1.27 g/L/h . Glucose rich food waste have also been used for the production of succinic acid in batch cultures by an engineered Yarrowia lipolytica strain resulting in 31.7 g/L succinic acid concentration with yield of 0.52 g/g and productivity of 0.60 g/L/h . Waste bread and bakery waste hydrolysates have also been used as raw materials for the production of succinic acid. Fermentation with bakery waste by A. succinogenes resulted in 47.3 g/L succinic acid with a yield of 1.16 g/g glucose and productivity of 1.12 g/L h . Cake and pastry hydrolysates resulted in 24.8 g/L (yield 0.8 g/g, productivity 0.79 g/L/h) and 31.7 g/L (yield 0.67 g/g, productivity 0.87 g/L/h) succinic acid concentration, respectively .
From a techno-economic viewpoint, it is crucial to identify the initial carbon source concentration in bioreactor cultures leading to the highest succinic acid concentration, yield and productivity. Results from this study clearly demonstrate that OFMSW hydrolysates at a 50 g/L initial carbon source concentration resulted in the highest productivity and yield for both microorganisms. Liu et al.  reported that growth inhibition of A. succinogenes was observed at 50 g/L initial glucose concentration. Salvachua et al.  carried out batch fermentations at different initial glucose concentrations (40-100 g/L) with the highest yield (0.72 g/g) achieved at initial glucose concentration of 60 g/L. A. succinogenes can tolerate up to 143 g/L glucose and cell growth was completely inhibited when glucose concentration was higher than 158 g/L . However, significant decrease of yield and prolonged lag phase were observed when glucose concentration was higher than 100 g/L . Using a xylose based medium, the initial inhibitory sugar concentration was around 50 g/L for both A. succinogenes and B. succiniciproducens .
Subsequent fed-batch fermentations on OFMSW hydrolysates were carried out with A. succinogenes at ca. 50 g/L initial carbon source concentration, where yield, productivity and by-product to succinic acid ratio were optimum. B. succiniciproducens was not selected due to significant lactic acid production during fermentation.
Fed-batch bioreactor fermentations
Table 4 presents the succinic acid production efficiency of A. succinogenes in fed-batch fermentations using OFMSW hydrolysate. Evaluation of the effect of different initial MgCO3 concentration (5, 10, 20 g/L) resulted in moderate improvement of succinic acid production efficiency at increasing concentrations. The highest succinic acid concentration (34.8 g/L), yield (0.6 g/g) and productivity (0.79 g/L/h) were achieved when 20 g/L MgCO3 concentration was used. The production of metabolic by-products was slightly reduced when MgCO3 concentration was increased to 10 g/L. Magnesium ions act as a cofactor for the key enzyme phosphoenolpyruvate (PEP) carboxykinase  and carbonate ions (HCO3−, CO3 2−) are a pool of additional CO2 . CO2 in the form of gas or carbonate salts have been previously investigated by McKinlay et. al.  resulting in increased succinic acid concentration in favor of by-product accumulation due to the suppression of OAAdec and Maldec towards pyruvate. According to Brink et al. , A. succinogenes is able to metabolize formate to CO2 and H2O towards the production of NADH. As a result, the generation of NADH contributes to enhanced succinic acid production. However, significant enhancement of succinic acid production efficiency was not observed in this study at high MgCO3 concentrations. For this reason, the lowest MgCO3 concentration (5 g/L) was used in subsequent fermentations in order to minimize raw material cost and environmental impact.
Figure 2 presents the metabolic product formation and lactic acid accumulation during fed-batch cultures carried out with different initial MgCO3 concentrations. Lactic acid (Figure 2) accumulation due to feeding of OFMSW hydrolysate reached concentrations close to 10 g/L when higher MgCO3 concentrations were used. The lactic acid concentration in batch cultures with 50 g/L initial carbon source concentration from OFMSW hydrolysates was constant (10 g/L) during fermentation resulting in higher productivity (0.89 g/L/h) and similar yield (0.56 g/g) (Table 3) as compared to fed-batch cultures carried out with 5 g/L and 10 g/L initial MgCO3 concentration.
Yeast extract (YE) and corn steep liquor (CSL) supplementation was also evaluated in fed-batch fermentations using OFMSW hydrolysate supplemented with 5 g/L MgCO3 (Figure 3). Addition of 5 g/L CSL resulted in 28.7 g/L of succinic acid with a yield of 0.5 g/g and a productivity of 0.41 g/L/h. Yeast extract supplementation (5 g/L) resulted in significantly higher succinic acid concentration (34.3 g/L) and productivity (0.75 g/L/h). However, the utilisation of yeast extract resulted in the highest by-product to succinic acid ratio (0.59) among all fed-batch fermentations presented in Table 4.
Yeast extract enhances cells growth and succinic acid production. Liu et al.  reported that yeast extract results in slightly higher succinic acid concentration than CSL in A. succinogenes CGMCC1593 cultures. CSL derived from corn refining has been widely employed as the sole nitrogen source resulting in high succinic acid concentrations (47.4 g/L) . Chen et al.  reported the production of 35.5 g/L succinic acid in A. succinogenes cultures carried out with 50 g/L glucose concentration and spent yeast cell hydrolysate, with a glucose utilization of 95.2%.
Figure 4 presents carbon source consumption and metabolic product accumulation during continuous fermentation of A. succinogenes. The continuous culture was carried out with glucose as carbon source until 900 h, since the major sugar fraction in OFMSW hydrolysate is glucose (73.2 %). OFMSW hydrolysate was used as feeding solution from 900 h until 2400 h. At 237 h, biofilm formation was developed and thus steady-state conditions were established. Three dilution rates (0.02, 0.04 and 0.08 h-1) were used with glucose and 6 dilution rates (0.02, 0.04, 0.05, 0.06, 0.08 and 0.1 h-1) were used with OFMSW hydrolysate. Figure 4 shows that steady-state was achieved within 2 – 4 days depending on the dilution rate used.
Using glucose and a dilution rate of 0.02 h-1, the average succinic acid production was 23.1 g/L with a yield of 0.51 g/g (Figure 5). Succinic acid concentration slightly decreased at increasing dilution rates. Specifically, it was 22.5 g/L (0.50 g/g yield) and 21.2 g/L (0.47 g/g yield) at dilution rates of 0.04 h-1 and 0.08 h-1, respectively. Increased dilution rates resulted in increased productivity, namely 0.46 g/L/h, 0.9 g/L/h and 1.71 g/L/h at dilution rates of 0.02 h-1, 0.04 h-1and 0.08 h-1, respectively.
OFMSW hydrolysate resulted in an average succinic acid production of 23.8 g/L with a yield of 0.53 g/g at dilution rate 0.02 h-1 (Figure 5). Succinic acid concentration (23.5 - 23.1g/L) and yield (0.52 - 0.51 g/g) were stable at 0.04 h-1 and 0.05 h-1. Higher dilution rates resulted in decreasing succinic acid concentration and yield. Specifically, succinic acid concentration was 21.2 g/L, 17.7 g/L and 10 g/L at dilution rates of 0.06 h-1, 0.08 h-1 and 0.1 h-1, respectively. The productivity increased at increasing dilution rates, up to 0.08 h-1, with an average productivity of 0.48 g/L/h, 0.94 g/L/h, 1.16 g/L/h, 1.27 g/L/h, and 1.41 g/L/h at dilution rates of 0.02 h-1, 0.04 h-1, 0.05 h-1, 0.06 h-1 and 0.08 h-1, respectively. Significant decrease in succinic acid production efficiency was observed at 0.1 h-1 due to cell wash-out.
Formic and acetic acid production was observed throughout continuous fermentation carried out either with glucose or OFMSW hydrolysate (Figure 4b). In the latter case, lactic acid accumulation was observed due to its presence in the OFMSW hydrolysate. Acetic acid concentrations produced by A. succinogenes were 7.1 g/L, 5.9 g/L and 5.4 g/L at dilution rates 0.02, 0.04 and 0.08 h−1, respectively, in glucose based medium. The respective formic acid concentrations were 5.7 g/L, 4.8 g/L and 4.4 g/L. The by-products to succinic acid ratio in glucose at dilution rate of 0.02 h-1 was 0.55 g/g, while at increasing dilution rates (0.04 and 0.08 h-1) by-products to succinic acid ratio was around 0.46 g/g.
When A. succinogenes was cultivated on OFMSW hydrolysate, the acetic acid concentration was always higher than formic acid concentrations at all dilution rates. The highest acetic acid concentrations were observed at dilution rates of 0.02 h-1 (9.9 g/L) and 0.04 h−1 (9.3 g/L). The highest concentration of total by-products for A. succinogenes was observed at dilution rate of 0.02 h−1 (12.7 g/L in glucose and 18 g/L in OFMSW hydrolysate). By-products to succinic acid ratio using OFMSW hydrolysate as feeding was 0.76 g/g, 0.73 g/g, 0.71 g/g, 0.62 g/g, 0.63 g/g and 0.6 g/g at dilution rates of 0.02 h-1, 0.04 h-1, 0.05 h-1, 0.06 h-1, 0.08 h-1 and 0.1 h-1, respectively.
Biofilm formation occurred on the wall and the mechanical parts of the bioreactor during continuous fermentation (Figure 6). Continuous A. succinogenes cultures under prolonged operation period result in biofilm formation [36, 37, 38]. Ladakis et al.  carried out continuous A. succinogenes fermentation using synthetic xylose as carbon source leading to succinic acid concentration of 24 g/L with yield 0.6 g/g at 0.02 h−1. Higher yield (0.77 g/g) and succinic acid concentration (26.4 g/L) was achieved in continuous cultures of immobilized A. succinogenes cells using synthetic xylose at dilution rate 0.1 h-1 . Continuous A. succinogenes cultures carried out on glucose at dilution rate 0.11 h−1 in a biofilm reactor packed with Poraver® beads led to 29.5 g/L succinic acid concentration with productivity of 3.24 g/L/h and yield of 0.9 g/g . Ferone et al.  reported continuous succinic acid fermentation of A succinogenes in a packed-bed biofilm reactor leading to 43 g/L succinic acid concentration at dilution rate 0.5 h-1 with glucose conversion of 88%. Continuous A. succinogenes cultures on glucose conducted in a fibrous-bed bioreactor led to 55.3 g/L succinic acid concentration with 0.8 g/g yield and 2.77 g/L/h production at dilution rate 0.05 h-1 .
Various crude hydrolysates from various biomass sources have been used for the production of bio-based succinic acid. Continuous fermentation on spent sulfite liquor resulted in 19.2 g/L succinic acid concentration with yield 0.48 g/g at dilution rate 0.02 h−1, while the highest productivity was 0.68 g/L/h at dilution rate 0.04 h−1 . Brandfield et al. achieved succinic acid concentration of 39.6 g/L via immobilized A. succinogenes continuous cultures carried out on corn stover hydrolysate .
Process feasibility evaluation
A preliminary techno-economic evaluation was carried out considering the optimum succinic acid production efficiency achieved in batch and continuous A. succinogenes cultures using OFMSW hydrolysate as feedstock. The batch fermentation resulted in 29.4 g/L succinic acid concentration with yield of 0.56 g/g and productivity of 0.89 g/L/h (Table 3). In continuous cultures, the succinic acid production efficiency (21.2 g/L, 0.47 g/g yield and 1.27 g/L/h productivity) achieved at dilution rate 0.06 h-1 was used in techno-economic evaluation. Fed-batch fermentations led to 34.3 g/L succinic acid concentration with a yield of 0.5 g/g and a productivity of 0.75 g/L/h. Fed-batch fermentations were not used in the technoeconomic evaluation due to the low productivity achieved.
The design and costing methodology were applied on the fermentation stage including media sterilization and the downstream separation and purification (DSP) stage that included centrifugation, activated carbon treatment, cation exchange resin treatment, evaporation, crystallization and drying unit operations. The estimation of the optimal design of the fermentation stage, the optimal scheduling of unit operations and the cost estimation of unit operations is based on the work presented by Dheskali et al. . The cost of manufacture for succinic acid did not consider the upstream stage of OFMSW pretreatment and hydrolysis. The production cost of OFMSW hydrolysate was subsequently estimated in order to achieve a minimum selling price (MSP) of bio-based succinic acid equal to its market price (2.94 $/kg according to ) or a potential market price of 2.5 $/kg at varying annual succinic acid production capacities (Figure 7). In this way, the profitability potential of succinic acid production could be assessed considering variable production cost for OFMSW derived carbon sources.
The cost of manufacture for bio-based succinic acid, considering only fermentation and DSP stages, is reduced at a lower rate for both continuous and batch cultures at annual capacity higher than 50,000 t (Figure 7a). This occurs because economies of scale have been reached. The implementation of continuous cultures leads to slightly lower cost of manufacture than batch cultures. Figure 7b shows the production cost of OFMSW-derived sugars that should be achieved at varying succinic acid production capacities in order to satisfy minimum selling prices of 2.94 $/kg and 2.5 $/kg. This means that if a lower OFMSW-derived sugar production cost than the one presented in Figure 7b at a specific plant capacity is achieved, then lower MSP than the targeted one could be reached. For instance, if an OFMSW hydrolysate production cost of 230 $/t total sugars is assumed, which is close to the market price of glucose syrup derived through enzymatic hydrolysis of corn starch produced by the wet milling process, then a MSP equal to the current market price of bio-based succinic acid (2.94 $/kg) could be achieved at annual succinic acid production capacity of 50,000 t if batch cultures are used and 40,000 t if continuous cultures are used. A MSP equal to 2.5 $/kg could be achieved at OFMSW hydrolysate production cost of 25 $/t total sugars, when the industrial plant produces annually 40,000 t succinic acid via continuous cultures or 60,000 t succinic acid via batch cultures.