Results and discussion

microbial culture throughout all operating conditions was evaluated using Fluorescence in situ hybridization as previously described [17].

Calculations

In Condition 1, the feast to famine (FF) ratio was calculated as the ratio between the length of feast phase and the length of the famine phase. From Condition 2 to Condition 5, so that the carbon present in the Feed-N was taken into account, a global FF ratio was calculated as the sum of the length of the feast phase of Feed-C and length of the feast phase of Feed-N divided by the period with no exogenous carbon available (famine phase). The active biomass (XA) was calculated by subtracting PHA from the VSS. The specific PHA production rate (qPHA), specific substrate uptake rate (qFP), and specific nitrogen uptake rate (qN) were calculated from the slope of the linear regression of the concentrations of PHA, FP and N divided by XA, re-spectively. The storage yield (YPHA/FP) was calculated by dividing qPHA by -qFP and specific productivity (PPHA) was calculated by dividing the amount of PHA produced during the experi-ment by the length of the experiexperi-ment and by the amount of active biomass at the start of the experiment.

fermented waste [3,14]. Replacing the micronutrient and N&P solutions by the Feed-N (Con-dition 2) caused instability in the culture. This modification resulted in excess foam production, leading to significant biomass loss from the top of the reactor. For this reason, the working volume of the reactor was decreased from 53 L to 40 L (Condition 3) to allow a larger headspace for the foam.

Figure 5.1. FF ratio for the different operating conditions in the SBR.

In Condition 3, the global FF ratio increased 6-fold to 0.19 ± 0.06 h h^-1, though within the target value of 0.2 h h^-1, demonstrating the impact of using fermented food waste as a N&P and micronutrients source for the selection of a PHA-accumulating consortium. The variability in the FF ratio in the selection phase of this process also increased, which has often been re-ported when using complex feedstocks in a coupled feeding strategy [20]. Despite the fact that the working volume reduction helped with controlling the foam, settleability of the biomass was still an issue, which certainly contributed to the variability of the global FF ratio.

In an attempt to decrease the biomass loss, in Condition 4 a second settling phase before N-Feed feeding was added along with a decrease of the HRT from 1 d to 0.67 d. This process modification aimed at removing non-settling biomass along with potentially inhibiting and/or slowly biodegradable compounds from the broth. In spite of these modifications, the biomass

still presented poor settleability, which still resulted in extensive loss of biomass through the withdrawal of effluent. The main difference between Condition 1 and Conditions 2/3 was the replacement of the N&P solution by the Feed-N and the absence of a micronutrient media.

Thus, in order to assess the impact of any micronutrient limitation on the poor biomass settle-ability, a micronutrients solution was fed every cycle along with the feeds, similarly to what happened in the Condition 1 (Condition 5). The addition of the micronutrients solution led to improved settleability, and the FF ratio stabilized after 14 days of operation at around 0.18 ± 0.03 h h^-1. Due to the culture instability observed during Condition 2 and Condition 4, the culture was not characterized during these phases of the selection reactor and through accu-mulation tests.

Process performance under Conditions 1 and 3

As shown in the previous section, the replacement of the synthetic solution (Condition 1) by the N&P rich waste (Condition 3) resulted in higher and variable FF ratio and poor settling of the biomass. The latter caused biomass loss, observed by a decrease of active biomass from 5.71 to 2.43 gXA L^-1 (Table 5.3). Several factors may have contributed to this loss in performance:

substrate inhibition, the impossibility of establishing a proper famine phase or lack of micro-nutrients. Regarding substrate inhibition, it is known that some complex wastes may contain an inhibitory matrix, including a varied pollutants and heavy metals, among others [9,21]. De-spite the fact that Feed-N was fed only when Feed-C was exhausted, at the end of the cycle part of its content still remains in the bulk when the new cycle fed with Feed-C starts. The fact that the length of consumption of the Feed-C increased from Condition 1 to Condition 3 is actually consistent with substrate inhibition (Table 5.3). Although the active biomass decreased by 57%, the length of the consumption of Feed-C increased 2.6 fold. On the other hand, in Condition 1 all carbon was fed with the Feed-C, while in Condition 3, a meaningful fraction of the carbon source was fed with the Feed-N (approx. 20-33% of the OLR) and the difference was fed as Feed-C (approx. 66-80% of the OLR). As a consequence, the reduced active biomass cannot justify the increased FF ratio alone, suggesting a loss of performance, possibly by inhi-bition of the culture. Finally, the presence of solids in the feedstock and the content in non-VFA COD (up to 50%) can negatively impact on the performance as these compounds can promote the growth of non-PHA accumulating microorganisms. Hence, a proper famine phase, where no exogenous carbon source is available, would only occur after all these slowly biode-gradable sources of carbon were exhausted from the broth, which includes unfermented car-bohydrates, protein and lipids. As reported in the literature, a proper long famine phase is

critical to prevent a predominant growth response from the culture and thus to impose the selective pressure for the PHA accumulation in the following feast phase [19].

Table 5.3. Summary of the SBR performance

Condition 1 Condition 3 Condition 5 Feed-C Feed-C Feed-N Feed-C Feed-N Length of the feast phase

(h) 0.36 ± 0.07 0.93 ±

0.31

1.00 ± 0.23

1.12 ± 0.22

0.68 ± 0.22 Global FF ratio (h h^-1) 0.03 ± 0.01 0.19 ±

0.06

0.18 ± 0.03 Initial VSS (gVSS L^-1) 5.80 ± 0.97 2.60 ±

0.50

3.37 ± 0.60 Initial XA (gXA L^-1) 5.71 ± 1.10 2.43 ±

0.44

2.90 ± 0.24 -qFP (gCODFP gCODXA-1 h^-1) 0.67 ± 0.14 0.47 ±

0.0004

0.32 ± 0.08

0.50 ± 0.09

0.14 ± 0.001 -qN (mgN gCODXA-1 h^-1) 4.67 ± 1.7 15.7 ± 1.9 12.4 ± 0.6 PHA content at end of

C-Feed (gPHA gVSS-1) 0.22 0.16 ± 0.02

0.32 ± 0.01 YPHA/FP (gCODPHA gCODFP-1) 0.96 0.51 ±

0.09

0.85 ± 0.12 PHA composition

(gHB:gHV:gHHx) 58:23:19 76:24:0 78:22:0

The maximum PHA content was substantially lower in Condition 3 (0.16 gPHA gVSS-1) than the one obtained (0.22 gPHA gVSS-1) in Condition 1. This is possibly explained by a decrease of the fraction of PHA-accumulating microorganism in the SBR or of their efficiency in converting FP into PHA, or both. Indeed, the storage yield decreased (from 0.96 to 0.51 gCODPHA gCODFP -1) during this phase, which indicates a lower efficiency in converting carbon into PHA, and/or possibly the use of a fraction of the carbon to grow with residual ammonia of protein from the previous cycle.

FISH analysis showed that the culture changed with the feeding of Feed-N (Table 5.4).

While in Condition 1, genera Azoarcus, Paracoccus and phylum Firmicutes were dominant in the reactor, in Condition 3, genera Paracoccus and Amaricoccus were dominant. Along with the substantial increase in Amaricoccus, the predominance of Flavobacteria also increased

along with Thaurea and Zoogloea. Such changes in the microbial population might partially explain the performance changes that occurred.

Table 5.4. Semi-quantitative FISH characterization of the MMC enriched in the SBR during pseudo-steady state

Probe name Target group Inoculum Feed-N Condition 1

Condition 3

Condition 5

AMAR839 Amaricoccus - n.a. - +++ +++

Azo644 Azoarcus + n.a. +++ ++ ++

THA832 Thaurea - n.a. - + +

LAMP444 Lampropedia - n.a. - - -

Zra23a Zoogloea - n.a. - + +

UCB823

Plasticicumu-lans

acidi-vorans ^{- n.a. + + +}

Meg938/1028 Meganema - n.a. - - -

PAR651 Paracoccus +++ n.a. +++ +++ +++

LGC355 Firmicutes +++ +++ +++ + ++

CF319a Flavobacteria - ++ - ++ +

HGC69A Actinobacteria +++ + + + +

(-) Non-present (0%); (+) Almost non-existing (1-5%); (++) Present (5-25%); (+++) Abundant (25-50%); (++++) Dominant (> 50%).

The accumulation tests were carried out with the selected populations of Condition 1 and Condition 3 using the two feedstocks separately in a fed-batch mode and results summa-rized in Table 5.5. When the performance of both cultures fed with Feed-C are compared all relevant metrics decreased significantly for the culture selected on Condition 3: storage yield (0.59 to 0.38 gCODPHA gCODFP-1), final PHA content (0.64 to 0.32 gPHA gVSS-1), maximum specific PHA production rate (332 to 66.1 gCODPHA gCODXA-1 h^-1) and specific productivity (144 to 63.7 gPHA gXA-1 h^-1). This indicates that the culture from Condition 1 was better selected for PHA accumulators than the culture from Condition 3.

As expected, when the selected consortium (Condition 3) was fed with Feed-N in the fed-batch accumulation test, the overall performance was worse than when using Feed-C in the same metrics. The nitrogen-rich feed provided the consortium with plenty of non-VFA COD that might have been inhibited the culture, resulting in a final PHA content of only 0.15 gPHA

gVSS-1 and a specific productivity of 9.69 gPHA gXA-1 h^-1. Independently on the feed used, the

culture from Condition 3 shows to be less efficient in terms of PHA accumulation that the one selected in Condition 1.

Table 5.5. Summary of the fed-batch accumulation tests carried out after SBR was stabilized in each operation condition

Condition 1 Condition 3 Condition 5 Feed-C Feed-C Feed-N Feed-C Feed-N YPHA/FP (gCODPHA

gCODFP-1)

0.70 ± 0.05 0.38 ± 0.02

0.19 ± 0.001

0.80 ± 0.08

0.43 ± 0.03 YXA/FP (gCODXA

gCODFP-1)

0.18 ± 0.02

0.48 ± 0.03 Final PHA content

(gPHA gVSS-1)

0.64 ± 0.08 0.32 ± 0.03

0.15 ± 0.01

0.53 ± 0.001

0.27 ± 0.02 qPHA (mgCODPHA

gCODXA-1 h^-1)

332 ± 32 99.4 ± 5.2 66.1 ± 10.7

187 ± 0.01 210 ± 27 PPHA (mgPHA gXA-1 h^-1) 144 ± 1 63.7 ± 3.2 9.69 ±

0.39

89.9 ± 5.0 62.1 ± 2.9

Impact of feeding a micronutrients solution (Condition 3 vs Condi-tion 5)

Reasons for a less efficient selection of the culture in Condition 3 can also be related with the poor settleability of the biomass or the limiting availability of the micronutrients in the feedstocks (Fe, Co, Mn, etc). After adding a second step of settling following the end of con-sumption of the carbon of Feed-C with no improved settleability of the biomass (Condition 4), the impact of the lack of micronutrients was assessed in Condition 5, by supplementing a mi-cronutrients solution again in the SBR. With this change, the average length of the feast phase did not change (0.19 ± 0.06 h h^-1 in Condition 3 vs 0.18 ± 0.03 h h^-1 in Condition 5), but it varied considerably less (Figure 5.1). This was a consequence of the improved settling capacity of the biomass, which was observed, particularly in the newly introduced intermediate settling step.

With the operating conditions of Condition 3, the settling capability of the biomass had fluc-tuated between settling barely enough to no settling at all with some of the biomass actually even floating, resulting in significant biomass loss during effluent withdrawal. The poor settling behavior had possibly been caused by the adsorption of Feed-N compounds (e.g. fibers or

lipids) to the flocs of biomass that might have affected floc aggregation and/or chelation/lack of cations that promote aggregation and good settleability [22].

As a consequence of the addition of the micronutrients, and despite the OLR remaining unchanged, an increase (2.43 to 2.90 gXA L^-1) of active biomass was observed along with an increase of the maximum PHA content (from 0.16 to 0.32 gPHA gVSS-1) at the end of the Feed-C consumption. Additionally, the storage yield increased from 0.51 to 0.85 gCODPHA gCODFP-1, which is fairly close to the yield in Condition 1 (0.96 gCODPHA gCODFP-1). While the addition of a new fermented waste had resulted in more substantial changes in the composition microbial population selected in Condition 3 (Table 5.4), the addition of the second settling phase and micronutrients solutions did not modify the microbial community in a meaningful way, sug-gesting that the presence of the second fermented waste has more impact on microbial culture than the other operating conditions here studied.

The fed-batch tests also showed an increase in performance in Condition 5 with respect to Condition 3, corroborating the positive effects of the SBR performance. Considering the use of Feed-C as fermented feedstock in the fed-batch experiments: the storage yield increased 2-fold (from 0.38 to 0.80 gCODPHA gCODFP-1), the maximum specific PHA production rate in-creased nearly 2-fold (from 99.4 to 187 gCODPHA gCODXA-1 h^-1) and the PHA content at the final of the fed-batch increased by 69% (from 0.32 to 0.53 gPHA gVSS-1). Values are closer to those obtained in Condition 1 with the same feedstock in the fed-batch tests. Using the same se-lected culture (sese-lected in Condition 5) and Feed-N, the final PHA content was higher than that obtained with the culture selected in Condition 3 for the same feed (0.27 vs 0.15 gPHA gVSS-1), with the maximum specific storage rate and yields (YPHA/FP and YXA/FP) substantially increasing.

Similarly to what had happened in Condition 3, using biomass selected with Condition 5 and Feed-C yielded a higher PHA content (0.53 vs 0.27 gPHA gVSS-1) and productivity (89.9 ± 5.0 vs 62.1 mgPHA gXA-1 h^-1) than Feed-N.

The addition of the second settling phase and micronutrients solution resulted in a more efficient and active PHA-accumulating culture. While the sum of the storage and growth yields in Condition 3 added up to only 0.38 and 0.37 gCOD gCOD^-1, using Feed-C and Feed-N, re-spectively, in Condition 5, the sum of the yields added up to 0.80 and 0.91 gCOD gCOD^-1, using Feed-C and Feed-N, respectively. The yields in Condition 3 were fairly low and it shows how much FP were wasted into maintenance in far from optimal operating conditions. Since the new operating conditions did not affect the microbial culture significantly (Table 5.4), it can be assumed that the improved performance resulted from the addition of the micronutrients, which means that possibly one or more elements were missing before, leading to a more

efficient use of the substrate. It is unlikely that the addition of the second settling step was the major driver in recovering the performance of the culture, as this modification had been im-plemented before in Condition 4 with no clear improvements. Nevertheless, it is likely it helped removing slowly biodegradable substrates from the broth that can be used by a non-accumu-lating side population to grow [23,24]. The addition of the missing elements resulted in more carbon being metabolized towards PHA and growth, which means that before the culture was probably using alternative (and more costly) pathways for these processes or unable to assim-ilate a fraction of the carbon due to deficiency of certain elements, which reflected in increased maintenance costs and decreased overall productivity and performance.

Implications of the study

PHA production using MMC is usually carried out with the use of a single waste with additional nutritional supplements if necessary. The use of a second waste to support microbial growth with additional nutrients could reduce the costs of operation of the chemical additions while taking advantage of the benefits of uncoupling the carbon and nitrogen sources, even if only partially [4,6]. This study demonstrated that it is possible to replace a synthetic N&P feed by a nutrient-rich waste stream, thus simultaneously valorizing a second waste and reducing the costs of PHA production. However, the fermented food waste used in this study was not a sufficient supplement for the culture. The improvement in performance with the micronutrients addition suggests that one or more elements in this solution were missing from the fermented food waste stream, and that these were important requirements for the metabolic transfor-mations taking place in the PHA production process. Furthermore, performance did not fully recover to the level it was before when only one waste was used, even though the nutritional values (nitrogen, phosphorus and micronutrients) were returned to the same as in Condition 1. The nitrogen and phosphorus-rich fermented food waste contained also high solids, high non-VFA COD and even possibly inhibiting compounds that slowed down and hampered the metabolism of the PHA-accumulating culture. The solids and non-VFA COD may have func-tioned as a slowly biodegradable organic matter capable of supporting the growth of a flanking population and preventing a true famine phase to exist. Thus, the selective pressure, which favors the efficient selection of PHA-accumulating culture, was affected by these conditions.

This work unveiled some of the limitations of the dual feedstock to selecting a PHA-accumulating culture, which should be overcome prior to the use of this approach. Firstly, a characterization of the fermented waste should be carried out to identify any micronutrients potentially missing from it and the waste or process should be supplemented accordingly. Even

though complex wastes may have most of the nutrients, lacking one may be just enough to destabilize the process to the point that it is not feasible anymore. Secondly, complex wastes may contain plenty of solids and non-VFA organic matter that lower the efficiency of the se-lection of the PHA-accumulating culture. As carried out by Moretto et al., centrifugation and filtration of the fermented waste can remove most of the problematic fraction of the waste before use in the process, and even valorize it into biogas [25]. In this case, solids and a sub-stantial fraction of the non-VFA COD (e.g. proteins, lipids and carbohydrates) would have been removed and, possibly, some inhibiting compounds as well. A decrease in the availability of slowly biodegradable organic matter would have occurred, which would lead to an actual fam-ine phase and a better enrichment of PHA-accumulating microorganisms. These modifications together would have likely led to a better settleability of the biomass in the current study, which would have resulted in a more stable process. Thirdly, a process with a SRT equal to HRT may be taken into consideration, as it removes settling altogether from the process. In this study, the settleability of the culture was one of the main drivers of instability of the process, which could be avoided as it worsens the selection stage and consequently, the whole process.

Lastly, the presence of carbon in the second feeding could be limited with the use of a different waste. A carbon-deficient fermented waste with high N and P contents could simplify the issues encountered in this study by a allowing a fully uncoupled C and N feeding strategy.