Protocol of the strategies to develop a sustainable long-term SSF at a bench scale

This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 689229.

2017.11.13 Version 3 689229

Ref. Ares(2017)5845148 - 29/11/2017

Deliverable 4.6 2

A Decentralised Management Scheme for Innovative Valorisation of Urban Biowaste

D4.6 - Protocol of the strategies to develop a sustainable long-term SSF at bench scale

Grant Agreement N° 6689229

Acronym DECISIVE

Full Title A Decentralised Management Scheme for Innovative Valorisation of Urban Biowaste

Work Package (WP) WP4.2

Authors A. Cerda (UAB) A. Sánchez (UAB)

Document Type Report

Document Title Protocol of the strategies to develop a sustainable long-term SSF at bench scale

Dissemination Level (mark with an«X» in the column to the far right)

CO Confidential, only for partners of the Consortium (including the Commission’s Services)

PU Public X

PP Restricted to other programme participants (including the Commission Services)

RE Restricted to a group specified by the Consortium (including the Commission Services)

ABSTRACT

This document aims to provide a complete methodology for the development of a solid state fermentation process using a batch configuration for biopesticide production. Extensive details on the biopesticide production using digestate as the substrate and inoculum preparation are described.

DOCUMENT HISTORY

version date editor modification

Draft 01 06/11/2017 A.Cerda First draft

Draft 02 13/11/2017 A.Cerda Second draft after approval of the Coordinator

Draft 03 20/11/2017 A.Cerda Third draft after approval by reviewers

This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 689229.

Deliverable 4.6 3

Release 01 28/11/17 A. Tremier Approval by the Coordinator

Distributed Distributed to the Team Leaders after final approval by the Coordinator

CONTRIBUTORS

name company contributions include

ALEJANDRA CERDA LLANOS

UAB AUTHOR

ANTONI SÁNCHEZ FERRER

UAB CO-AUTHOR

Deliverable 4.6 4

Table of contents

1. INTRODUCTION .................................................................................................................................................. 6

2. STATE OF THE ART ............................................................................................................................................ 7

3. PROTOCOL FOR A BATCH OPERATION FOR BIOPESTICIDE PRODUCTION AT A BENCH

SCALE ......................................................................................................................................................................... 8

3.1 Materials and reagents .................................................................................................................. 8

3.2 Inoculum preparation ................................................................................................................... 8

3.2.1 Reconstitution of the lyophilized strain of Bt ........................................................................ 8

3.2.2 Bt storage ........................................................................................................................... 9

3.2.3 Bt inoculum preparation ...................................................................................................... 9

3.3 Digestate conditioning ................................................................................................................. 10

3.4 Batch operation at bench scale.................................................................................................... 10

3.5 Conservation ................................................................................................................................ 11

3.6 Considerations for the demonstration plant ............................................................................... 11

4. FINAL REMARKS .............................................................................................................................................. 12

Deliverable 4.6 5

Glossary

Bt stands for "Bacillus thuringiensis"

C stands for “Cold Chamber”

CC, SC stands for "cell count" and "spore count”, respectively

CFU stands for "Colony Forming Units"

RT stands for "Room Temperature"

sOUR stands for “specific Oxygen Uptake Rate”

SB stands for “Sequential Batch”

SmF stands for “Submerged Fermentation”

SSF stands for "Solid State Fermentation"

Deliverable 4.6 6

1. Introduction

DECISIVE project aims to demonstrate the ability to decrease the generation of urban waste (from households and assimilated) and increase recycling and recovery by focusing efforts on decentralised management and valorisation of the organic fraction of waste (i.e. biowaste), in a short cycle. To achieve this objective, the DECISIVE project will develop and demonstrate eco-innovative solutions that include, among other tools, the use of Solid State Fermentation (SSF) technology.

The potential transformation of digestate into value-added products through SSF is of high interest for all the communities, because it provides a solution for the management of these materials and an economic benefit by the use or commercialization of the product obtained. A small scale multi-feedstock biorefinery will allow for the management of wastes in-situ, avoiding economic and environmental costs related to waste transportation, and providing new business models for the circular economy, also very necessary in many areas.

As reported in Deliverable 4.5, Bt-derived biopesticide was selected as the most suitable bioproduct to be obtained from digestate. In this document digestate was used as a sole substrate for Bt-biopesticide production under non sterile conditions with promising results when compared with related bibliography. Deliverable 4.6 from WP 4.2.1 aims at providing a detailed protocol for the production of biopesticide using digestate as the sole substrate by means of SSF. To ensure a clear reading and understanding of this document, D4.6 is divided into four parts, being this introduction the first of them:

Chapter 2 proposes a brief state of the art of SSF developments and the biopesticide production using this technology.

Chapter 3 describes the complete materials and methodology for the development of the productive process to obtain biopesticides. Special attention is focused on the inoculum preparation. This chapter is sorted into six categories: materials, inoculum preparation, digestate conditioning, batch operation, conservation and considerations for the demonstration SSF pilot.

Chapter 4 is the section of final remarks of this report.

Deliverable 4.6 7

2. State of the art Biopesticides have arisen as an alternative to conventional pesticides because of different reasons, such as the physiological resistance to the vectors, environmental pollution resulting in bio-amplification of food chain contamination and harmful effects on beneficial insects (Poopathi et al., 2016). In this context, there has been an increased interest in developing more sustainable strategies to produce biopesticides. Bacteria like Bacillus sphaericus (Bs) or Bacillus thuringiensis subsp. israelensis (Bt) have been reported as good biopesticide producers (Copping and Menn, 2000, Ballardo et al., 2016, Poopathi et al., 2016) because of their high toxicity to dipteran larvae and possibility of their use in mosquito eradication operations. Among these species, Bt is the most relevant: it has more than 26 Bt-based products registered by the US-EPA and marketed world-wide. Of these, 15 are derived from naturally occurring strains, e.g. Able (Thermo Trilogy), Bactospeine (Valent BioSciences), Dipel (Valent BioSciences), Javelin (Thermo Trilogy), Thuricide (Thermo Trilogy) and Xentari (Valent BioSciences) (Baum et al., 1999).

Conventionally, Bt-based biopesticide production is performed using pure substrates in a highly controlled submerged fermentation process (SmF). These fermentations are normally carried out using expensive and defined growth media, with temperature and pH control and under sterile conditions. Hence, these fermentations are commonly reported as expensive due to the high operational costs associated (Poopathi et al., 2016). These costs are greatly attributed to the use of pure substrates, sterilisation process and extensive use of water (Thomas et al., 2016). In this sense, solid-state fermentation (SSF) appears as an emerging technology to partially overcome some of these issues, in particular, by using organic wastes instead of pure substrates. There is a great interest in the study of these processes that is reflected on the high rate of production of related scientific literature. For example, some studies explore the use of the SSF to produce enzymes (Vassilev et al., 2009; Abraham et al. 2013, Dhillon et al., 2012, Cerda et al., 2016, Cerda et al., 2017), aromas (Martinez et al., 2017), biosurfactants (Jimenez-Peñalver et al., 2017) and other compounds of pharmaceutical and industrial interest (El Bakry et al., 2015). However, most of the research carried out in SSF is performed using few grams of substrate under initial sterile conditions and using systems with controlled temperature. Taking into account these conditions, the scaling up of the process appears as a challenging issue, considering the widely known constraints of working with large amounts of solid substrates and the costs associated to keeping the conventional operational conditions.

For an efficient SSF scale-up it is necessary to have a deep knowledge of the process engineering: mass transfer phenomena and energy flow models, etc., and a wide experience on methodologies used to analyse physical properties affecting these phenomena (Ruggieri et al., 2009a; Ruggieri et al., 2009b). Additionally, there is the necessity to develop a long term SSF operation in continuous or semi-continuous regime in order to reduce costs associated with inoculum requirements. Some attempts have been reported on the production of enzymes (Cheirsilp and Kitcha, 2015; Astolfi et al., 2011), however, these studies have been performed using only a few grams of sterile substrate which is not representative for a future scale-up. Lately, a novel and more suitable approach has been assessed for enzymatic production by SSF using a sequential batch operation with non-sterile wastes, showing promising applications (Cerda et al., 2016).

The optimization of the biopesticide production systems can lead to a new business niche of high value markets such as ‘organic’ farming. This could provide the possibility of a reduction in the production costs and better formulations.

http://www.sciencedirect.com/science/article/pii/S0048969717304412?via%3Dihub#bb0040

Deliverable 4.6 8

3. Protocol for a batch operation for biopesticide production

at a bench scale

3.1 Materials and reagents

Reagents:

- Nutrient Broth

- Nutrient Agar

- Ringer

Materials - 0.5 L Erlenmeyer flasks

- Air filters

- Mechanical stirrer

- Centrifuge vessels 15 mL, 50mL and 250mL

- Spatula

- Funnel

- Beaker of 500 mL

- Drigalski spatula - Micropipettes of 100 µL, 1mL and 10 mL

- Microscope slide and cover slip.

- Glass fiber filter paper Whatman™

- Cuvettes for spectrophotometer

- Petri dishes

Equipment: - Centrifuge

- Microscope

- Laminar flux chamber

- Oven

- Incinerator

- Incubator

- Reactors of 0.5-2L fully equipped with different sensors for on-line process monitoring.

Additionally, these reactors must be implemented with a data acquisition system.

3.2 Inoculum preparation

B. thuringiensis var. Kurstaky NRRL HD-73 (CECT 4497) (Bt) was acquired from the “Colección Española de cultivos tipo“CECT.(www.cect.org) (Valencia, Spain). The strain is available in a lyophilized form, therefore, it has to be reconstituted prior to its use.

3.2.1 Reconstitution of the lyophilized strain of Bt

The strain has to be reconstituted according to the following instructions:

- Properly store ampoules containing the inoculum. Do not open immediately, protect them from light and keep them at a temperature between 4 and 24 °C, preferably 18 °C. Do not freeze. In

http://www.cect.org/

Deliverable 4.6 9

this state, the viability can be maintained during very long periods of time. It is advisable to plan the recovery of lyophiles as soon as possible after receipt and not later than one year.

- Check that you have the recommended culture medium for each strain (Table 1). In addition to the liquid medium necessary for the reconstitution, it is advisable in most cases to count with solid medium (agar plates).

- The culturing media to be used must be freshly prepared or preserved in good conditions (not dry or with excessive humidity, without contaminants or precipitates and for a maximum period depending on its composition).

Resuspend the pellet with 5-10 mL of sterile liquid medium (Table1, Nutrient Broth). Then use the entire suspension to inoculate in a solid medium as recommended by the provider (Table 1, Nutrient Agar). Use Petri dishes to perform the solid culture and incubate at 30ºC for 20h on an incubator.

TABLE 1.COMPOSITION OF OXOID® COMMERCIAL MEDIA.

Nutrient Agar Nutrient Broth nº2

Typical Formula 28 g L-1 25 g L-1

Dust “Lab-Lemco” 1.0 10

Peptone 5.0 10

Sodium chloride 5.0 5

pH 7.4±0.2 7.5±0.2

Yeast extract 2.0 -

Agar 15.0 -

3.2.2 Bt storage

To store the original Bt strain, it is required to:

- Take a loop of one properly identified colony obtained from the previous section (3.2.1).

- Place the loop inside a cryovial (with skirt) containing protective liquid (normally, glycerol) and homogenize. Inside the cryovial there are 25 cryopearls (CRYOINSTANT®) covered with cryoprotectants. These pearls will be covered with the Bt suspension.

- Remove the cryopearls and then place them into a vial with a saline solution.

- The vials must be stored at -80 °C for posterior utilization.

This storage procedure allows the perfect conservation of the strain and eases the propagation of the microorganism into a liquid medium. Additionally, this procedure prevents the formation of ice crystals in the vials, minimizing the risk of cross contamination.

3.2.3 Bt inoculum preparation

a) SSF at a laboratory scale in 0.5L reactors

All media used for inoculum preparation have to be adjusted to pH 7.5±0.2 before autoclaving for 30 min at 121ºC. Table 1 presents the complete composition of these commercial growing media.

One Bt-cryopearl has to be inoculated into a 0.5L Erlenmeyer flask containing 100 mL of previously sterilized culture media. After that, the flask has to be incubated in an orbital shaker at 180 rpm and 30ºC for 20 h. The optimum incubation conditions were determined by Ballardo (2016) taking into consideration Bt kinetics. After that time, it is necessary to check the optical density of the culture at 600nm, which must

Deliverable 4.6 10

be around 3.5±0.2 ( considering the proper dilution, normally 1:5 using culture media) in order to ensure the proper amount of biomass to inoculate in the SSF process (Annex I).

After that time, the culture broth has to be centrifuged at 3500 rpm for 10 minutes at room temperature. The prepared inoculum can be used for two purposes:

1) to inoculate a larger SmF as described in the following section.

2) to inoculate a small SSF reactor (0.5 L), according to the specifications described in Section 3.4.

b) SSF at a larger scale

If the SSF is going to be carried out at bench or pilot scale, the amount of inoculum required is higher, then a propagation fermentation has to be performed. The main goal of this additional fermentation is to obtain a highly active biomass and in great quantities.

The propagation consists in two steps:

1) Repeat the complete procedure indicated in section 3.2.3a.

2) Once the first stage carried out in 0.5 L reactors is completed, the liquid culture (0.1 L) obtained is transferred to a 2 L reactor containing 1.1 L of nutrient media (composition in Table 1). This propagation has to be performed during 24 h at 30ºC under static conditions with an airflow of 20 mL min

-1 (Annex II).

At the end of the fermentation, check the optical density, which must be 3.5±0.2, as explained in Annex I. Finally, culture broth has to be centrifuged at 3500 rpm for 10 minutes.

3.3 Digestate conditioning

The digestate has to be used as soon as it arrives at the facilities. If not, the material should be stored in a cold chamber (4 ºC) until its use for a maximum of ten days.

Prior to its use, the material has to be conditioned. If a high presence of impurities is observed, the digestate has to be sieved using a 0.5 mm net, in order to remove them. After this process, the digestate will undergo a hygienisation process. This process consists in holding the residue for 1 h into a previously heated oven at 70ºC. Then, the residue is cooled down to room temperature.

3.4 Batch operation at bench scale

The batch operation of a SSF process at bench scale, is carried out using a thermally isolated reactor with a continuous air supply, as the described in Deliverable 4.5 (Cerda and Sánchez, 2017).

The mixture of the batch has to be prepared considering a ratio of 0.035-0.05 g Bt g-1

digestate and using the solid inoculum obtained from the 2L reactors after centrifugation (Section 3.2.3b). Wood chips are added as bulking agent in a ratio of 1:1 (v/v) in order to provide enough porosity to promote proper oxygen transfer (Ruggieri et al., 2009a). During the fermentation the airflow must be adjusted in values between 0.1-0.2 L kg

-1 digestate min

-1 in order to ensure full aerobic conditions inside the reactor. One

complete batch for biopesticide production has to be performed for 72h in order to obtain the highest Bt concentration.

From the obtained product, measurements of viable cell count, spore count and other routine parameters have to be carried out. The measurement methods are described in Deliverable 4.5 (Cerda and Sánchez, 2017).

Deliverable 4.6 11

Note that if a SSF is going to be carried out using small reactors (0.5 L), then the same operational conditions must be considered with exception of the inoculum use. In this particular case, fermentation should be inoculated with the inoculum obtained in a 0.5 L SmF, as described in Section 3.2.3a. After the fermentation, the product obtained is a fermented solid enriched with Bt. This fermented solid can be submitted to a downstream process in order to have the biopesticide. The biopesticide can be presented in a solid form or in a liquid form.

3.5 Conservation

There are two ways for the storage of the biopesticide depending on the application form (solid or liquid). If the product is presented in a solid form, then it must be stored at room temperature or cold chamber for a maximum of 30 days. If the product is presented in a liquid form, it must be extracted in a 1:10 solid: liquid ratio using a Ringer solution and stored in a cold chamber for a maximum of 30 days (Annex III).

3.6 Considerations for the demonstration plant

In order to fully optimize the biopesticide productive process, there are some considerations to take into account for the demonstration pilot plant. One of these considerations is the configuration of the operation of the SSF process. In this report, a batch process is proposed but, there is the possibility to develop a sequential batch operation as reported by Cerda el al., (2017). Some preliminary experiments have been carried out with very interesting and promising results (Annex IV). In these experiments, it was possible to obtain a continuous production of fermented solid enriched with Bt and therefore it would be an alternative for the operation of the demonstration pilot plant. This continuous operation would avoid the periodical demand for new inoculum needed in batch operation.

Another aspect to take into consideration is the temperature regime for the SSF. Bt is a strict aerobic bacterium with an optimum growth temperature of 30±2ºC and, hence, any increment on the temperature will affect its growth and consequently the biopesticide production. As reported in Deliverable 4.5, there is a substantial increase in the temperature when the SSF is carried out at a large scale, affecting the process productivity. For these reasons, several strategies need to be studied to overcome this constrain, among them: reactor configuration, inoculation strategies or even the setting up of a pretreatment unit.

One of the most attractive strategies to take into consideration for temperature control is the time of inoculation. The proposed SSF process has a clear thermophilic stage during 10-48 h of fermentation as reported in Deliverable 4.5. This temperature increase is caused by the metabolic heat generated due the biological activity during the fermentation. It is possible that, if the inoculation of Bt is performed after the thermophilic stage, the microorganism would be able to thrive in the solid matrix. Additionally, these first 48h of fermentation would perform as an aerobic pretreatment which would hydrolyse the substrate and make it more available for the targeted microorganisms.

Deliverable 4.6 12

4. Final remarks

This report describes the protocol for biopesticide production at a bench scale using digestate as a sole substrate. Also, considering the results obtained so far for biopesticide production, some considerations for the operation of the pilot demonstration plant were given. The most attractive alternative to avoid the productivity drop due to heat surplus during the fermentation is the development of inoculation strategies in order to avoid the presence of Bt during the thermophilic stage. Additionally, preliminary experiments for the sequential batch operation of a biopesticide production SSF were performed with promising results.

Deliverable 4.6 13

Deliverable 4.6 14

Annex I

Standardization of the inoculum monitoring

In order to monitor the cell growth during the preparation of the inoculum two measures were taken and utterly correlated. With this purpose, 7 fermentations were performed measuring optical density and dry matter at the moment of maximum biological activity (48h). The results of these analysis are presented in Figure 1.

y= 1.6619·x+0.7275

R2

=0.9156

Dry Matter [g L-1

]

0.0 0.5 1.0 1.5 2.0

Opti

cal D

ensi

ty

0

1

2

3

4

y= 1.6619 x + 0.7276

R2= 0.9156

FIGURE 1. CORRELATION OBTAINED FROM OPTICAL DENSITY AND DRY MATTER

CONTENT FROM SAMPLES OBTAINED FROM 7 INDEPENDENT FERMENTATIONS FOR

INOCULUM PREPARATION.

Results showed that after 20h of fermentation, the average content of dry matter of the fermentations was 1.6±0.2 g L

-1 which corresponds to a measured optical density of 3.5±0.3. The measurement of the

optical density in a liquid culture is easy and reliable and therefore it contributes to a better monitoring of the biomass growth during the inoculum preparation process.

Deliverable 4.6 15

Annex II

Stirring requirements

An experiment was carried out in order to reduce costs associated with the agitation of the reactor for the inoculum preparation. This experiment consisted in the development of two inoculum preparation processes at the conditions described in section 3.2.3b using i) mechanical stirring and aeration (150 rpm and 20 mL min

-1) and ii) only aeration (20 mL min

-1). The results are presented in Table 2.

TABLE 2. AVERAGE OPTICAL DENSITY, DRY MATTER AND CELL COUNT OBTAINED IN 7

FERMENTATIONS FOR INOCULUM PREPARATION CARRIED OUT USING DIFFERENT

AGITATION MECHANISMS.

Stirring requirements OD Dry matter

(%d.b) Cell count

(1011

CFU g-1

DM)

Mechanical stirring + Aeration

3.32±0.01 1.91±0.01 3.37±0.09

Aeration 3.42±0.03 2.11±0.04 4.35±0.09

As it is observed, the optical density and dry matter of the fermentation reported similar values and are similar to the reported in the 0.5L reactors (Annex I). The difference between the two fermentations relies on the viable cell count obtained. Using only aeration as the source of homogenization of the culture promoted the proper cell growth, which is observed in the increase of nearly 30% in the viable cell count.

In light of the obtained results, the inoculum preparation carried out in 2L reactors will be performed using only aeration as a technique to homogenize the culture media.

Deliverable 4.6 16

Annex III

Conservation of the biopesticide For the conservation of the obtained material two presentations were considered: solid and liquid form. The scheme presented in Figure 4 shows the two alternatives of biopesticide storage. Liquid biopesticide was obtained after a liquid extraction of the fermented solid using Ringer solution at different ratios (1:10, 1:5 and 1:2). On the other hand, solid biopesticide was stored as obtained from the fermentations. Both types of biopesticides were stored in cold chamber (C,4ºC) and room temperature (RT, 20ºC) for a maximum of 30 days. Additionally, samples were taken at the beginning, at day 7 and at 30 days of storage. In all sampling cell and spore count, pH and conductivity were measured.

FIGURE 4. EXPERIMENTAL DESIGN OF THE CONSERVATION CONFIGURATION FOR

BIOPESTICIDE IN A LIQUID AND SOLID PRESENTATION.

Table 4 presents the results of the storage conditions for the biopesticide in liquid and solid forms. The results are expressed as the percentage of variation in the viable cell and spore count in comparison with the initial sample. In this sense, if the percentage resulted in positive values implied that CC and SC presented an increase when compared with the initial values. On the other hand, if the percentages were negative implied that CC and SC values decreased in comparison with the initial content.

The initial sample was obtained from a SSF carried out in a 0.5L reactor, according to the specifications indicated in Section 3.4. The initial sample contained a value of CC and SC of 4.92±0.19 and 6.35±0.15 (∙10

8) CFU g

-1DM, respectively.

Deliverable 4.6 17

TABLE 3. PERCENTAGE OF SURVIVAL OF THE VIABLE CELLS AND SPORE COUNT AFTER

STORAGE CONDITIONS FOR BIOPESTICIDE IN A LIQUID AND SOLID PRESENTATION.

7 days 30 days

Viable Cells

(%) Spores

(%) Viable Cells

(%) Spores

(%)

SOLID

C 20.40 -9.06 21.19 -1.62

RT 14.22 3.78 8.13 -0.07

LIQUID

RT 1:10 -10.33 -6.8 11.91 -25.80

RT 1:5 -8.03 0.08 -0.50 -5.26

RT 1:2 -22.06 -25.15 6.23 -29.75

C1:10 -4,23 -21,93 57.65 18.80

C1:5 -3.42 -17.67 19.15 -33.64

C1:2 5.78 -21.46 20.34 -39.61

From these experiments, it can be stated that for the biopesticide in solid form, both assessed storage strategies are suitable for the conservation of the viable cells for a maximum of 30 days. Conservation in a cold chamber and room temperature at 30 days of storage reported an increase of viable cell content of 21 and 8% respectively, when compared with the initial sample. Spores content showed no significant differences at 30 days of storage when compared with the initial spore content of the fermented solid.

Biopesticide in the liquid form presented a different behaviour. The best results were obtained using the extraction ratio of 1:10 and stored in the cold chamber for a maximum period of 30 days. Using this strategy, the viable cell and spore count increased in 58 and 19% when compared with the original sample. Despite this storage system is adequate for 30 days of storage, there is a slight decrease in viable cell count in most of the measurements at 7 days of storage of the liquid form of biopesticide. This decrease might be attributed to the presence of hydrolytic enzymes (such as proteases) that could negatively affect the viable cell counting.

Deliverable 4.6 18

Annex IV

Sequential batch operation

In order to provide information about the possibility of establishing a sequential batch (SB) operation in the demonstration pilot, a series of experiments were performed at lab scale. Two 0.5L reactors were set using a ratio of 0.035 g Bt g

-1 digestate and using the inoculum obtained as described in section 3.2.1a.

Wood chips were added as bulking agent in a 1:1 (v/v) ratio. The fermentations were carried out in duplicates, using a fixed airflow of 20mL min

-1 and a temperature of 37ºC.

The operation was initially performed as a conventional batch fermentation until the system reached the maximum biological activity. As reported in Deliverable 4.5 (Cerda and Sánchez, 2017) the system achieves the maximum biological activity at 72h of fermentation, therefore for the SB operation the solid retention time (SRT) was set at this time. SB operation starts after the first SRT is completed. At that moment 50% of the wet fermented solids is removed from the reactor. The removed material can be used as product for further analysis. The remaining 50% of the fermented solid is used as inoculum to start a new batch, with the addition of 50% of fresh digestate. Note that these operational conditions have not been yet optimized, however, this exchange ratio has been proven to be useful in similar SB operations for other bioproducts generation (Cerda et al., 2017)

During the sequential batch operation, a continuous on-line monitoring of temperature and sOUR were carried out in order to monitor the biological activity. Sampling was performed after a complete manual homogenization of the fermented solids to obtain a full representative sample and prior to feeding the reactor with fresh substrate. From these solid samples, measurements of viable cell count and spore count and other routine methods as detailed in Deliverable 4.5 were determined. The sOUR profiles of these experiments are presented in Figure 2.

Deliverable 4.6 19

Time [h]

0 50 100 150 200 250

sOU

R [

mg O

2 g

-1D

M h

-1 ]

0.0

0.5

1.0

1.5

2.0

Cel

l C

ount

and S

po

re C

ount

[CF

U g

-1 D

M]

0

2e+8

4e+8

6e+8

8e+8

R1

R2

CC

SC

FIGURE 2. DUPLICATES OF THE SEQUENTIAL BATCH OPERATION FOR BIOPESTICIDE

PRODUCTION. SOUR PROFILES OF THE SOLID STATE FERMENTATION PROCESS USING BT

AS INOCULUM AND DIGESTATE AS THE SUBSTRATE

As observed in Figure 2, the SB operation was carried out for nearly 12 days. In the first cycle of the SB there is a quick increase of the sOUR, reaching a maximum average of 1.7±0.1 mg O2 g

-1 DM h

-1. The

following two batches showed a decrease in the maximum sOUR values, with average values of 1.5±0.1 mg O2 g

-1 DM h

-1. 1.4±0.2 mg O2 g

-1 DM h

-1 in the second and third batch, respectively. It is likely that

reduction in the sOUR value is associated to an adaptation of the biomass to a fresh substrate. Additionally, the 50% removal of the content of the reactor affects the substrate content in the solid matrix, but also affects the biomass content of the fermentation. In this sense, these two facts could have generated the sOUR reduction. In the final batch, there is an increase in the sOUR value, reaching a maximum value of 1.8±0.1 mg O2 g

-1 DM h

-1. Even though the

maximum sOUR value changed during the fermentation, the cumulative oxygen consumption (COC) was stable in an average value of 45.5±0.5 mg O2 g

-1 DM.

Regarding the growth of Bt the results are presented in Figure 3, the fermentations started with a number of viable cells of 5.49 ± 0.02 (∙10

8) CFU g

-1DM. During the complete SB operation, the viable cells and

spore count showed a decrease in the second batch followed by a sustained recovery in CC and SC. Bt was able to colonize and survive at the provided conditions, presenting a final viable cell count of 5.11 ± 0.3 (∙10

8) CFU g

-1DM. These results showed that it is possible to establish a sequential batch operation

for biopesticide production using digestate as a sole substrate. This positive outcome is of great relevance when the pilot demonstration is taken into consideration. This operational configuration allows

Deliverable 4.6 20

not only the continuous production of a fermented solid enriched with Bt, but to avoid the continuous production of inoculum by SmF. This optimization of the process can provide economic benefits that need to be taken into account when developing the pilot SSF reactor. Further research is needed to determine the solid exchange ratio for each batch.

Time [h]

Initial 1º Batch 2º Batch 3º Batch 4º Batch

Cel

l C

ou

nt

and

Sp

ore

Co

unt

[CF

U g

-1 D

M]

0

1e+8

2e+8

3e+8

4e+8

5e+8

6e+8

7e+8

CC

SC

FIGURE 3. AVERAGE CELL AND SPORE COUNT OBSERVED DURING A SEQUENTIAL BATCH

OPERATION FOR BIOPESTICIDE PRODUCTION BY MEANS OF SOLID STATE

FERMENTATION PROCESS USING BT AS INOCULUM AND DIGESTATE AS THE SUBSTRATE

Deliverable 4.6 21

Documentary references

Abraham, J., T. Gea, and A. Sánchez. 2013. Potential of the solid-state fermentation of soy fibre residues by native microbial populations for bench-scale alkaline protease production. Biochemical Engineering Journal.74, 15-19.

Astolfi, V., Joris, J., Verlindo, R., Oliveira, J., Maugeri, F., Mazutti, M., de Oliveira, D., Treichel, H. 2011.Operation of a fixed-bed bioreactor in batch and fed-batch modes for production of inulinase by solid-state fermentation. Biochemical Engineering Journal, 58–59, 39-49.

Ballardo, C., Abraham, J., Barrena, R., Artola, A., Gea, T., Sánchez, A. 2016a.Valorization of soy waste through SSF for the production of compost enriched with Bacillus thuringiensis with biopesticide properties. Journal of Environmental Management, 169, 126-131.

Ballardo, C. 2016b. Valoración de residuos sólidos orgánicos como sustrato para el crecimiento de Bacillus Thuringensis mediante fermentación en estado sólido. in: Chemical, Biological and Environmental Engineering Department, Vol. PhD Universitat Autònoma de Barcelona. Barcelona, Spain. Available in: https://ddd.uab.cat/record/175011.

Baum J., Johnson T., Carlton B. 1999. Bacillus thuringiensis. In: Hall F.R., Menn J.J. (eds) Biopesticides: Use and Delivery. Methods in Biotechnology, vol 5. Humana Press.

Cerda, A., El-Bakry, M., Gea, T., Sánchez, A. 2017. Long term enhanced solid state fermentation: Inoculation strategies for amylase production from soy and bread wastes by Thermomyces sp. in a sequential batch operation. Journal of Environmental Chemical Engineering.4(2), 2394-2401.

Cerda, A., Gea, T., Vargas-García, M., Sánchez, A. 2017. Towards a competitive solid state fermentation: Cellulases production from coffee husk by sequential batch operation and role of microbial diversity. Science of the Total Environment. 589, 56-65.

Cerda, A., Sánchez, A. 2017. Report of the possibilities of digestate (and other wastes) as raw materials for SSF processes to obtain certain bioproducts. Deliverable 4.5 from the H2020 project DECISIVE. Cheirsilp, B., Kitcha, S. 2015. Solid state fermentation by cellulolytic oleaginous fungi for direct conversion of lignocellulosic biomass into lipids: Fed-batch and repeated-batch fermentations. Industrial Crops and Products, 66, 73-80. Copping, L. Menn, J. 2000. Biopesticides: a review of their action, applications and efficacy. Pest Management Science.,56: 651–676.

Dhillon, G.S., Kaur, S., Brar, S.K., Verma, M. 2012b. Potential of apple pomace as a solid substrate for fungal cellulase and hemicellulase bioproduction through solid-state fermentation. Industrial Crops and Products, 38(1), 6-13.

El-Bakry, M., Abraham, J., Cerda, A., Barrena, R., Ponsá, S., Gea, T., Sánchez, A. 2015. From Wastes to High Value Added Products: Novel Aspects of SSF in the Production of Enzymes. Critical Reviews in Environmental Science and Technology, 45(18), 1999-2042.

Jiménez-Peñalver, P., Gea, T., Sánchez, A., Font, X. 2016. Production of sophorolipids from winterization oil cake by solid-state fermentation: Optimization, monitoring and effect of mixing. Biochemical Engineering Journal, 115, 93-100.

Mahanta, N., Gupta, A., Khar, S. 2008. Production of protease and lipase by solvent tolerant Pseudomonas aeruginosa PseA in solid-state fermentation using Jatropha curcas seed cake as substrate. Bioresource Technology. 99, 1729-1735.

Martínez, O., Sánchez, A., Font, X., Barrena, R. 2017. Valorization of sugarcane bagasse and sugar beet molasses using Kluyveromyces marxianus for producing value-added aroma compounds via solid-state fermentation. Journal of Cleaner Production, 158(Supplement C), 8-17.

Mitchell, D., Berovič, M., Krieger, N. 2006. Solid-State Fermentation Bioreactor Fundamentals: Introduction and Overview. in: Solid-State Fermentation Bioreactors: Fundamentals of Design and Operation, (Eds.) D.A. Mitchell, M. Berovič, N. Krieger, Springer Berlin Heidelberg. Berlin, Heidelberg, pp. 1-12.

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Poopathi S., Muruga, K., , Selvakumari, J., Mani C., Bala, P, de Britto R. , Pattnaik S. , Santhosh, G., Prabakaran K. 2016. Biopesticide production from environmental wastes. JSM Tropical Medicine and Research, 1(2): 1008.

Ruggieri, L., Gea, T., Artola, A., Sanchez, A. 2009a.Air filled porosity measurements by air pycnometry in the composting process: a review and a correlation analysis. Bioresource Technology, 100(10), 2655-66.

Ruggieri, L., Cadena, E., Martínez-Blanco, J., Gasol, C.M., Rieradevall, J., Gabarrell, X., Gea, T., Sort, X., Sánchez, A. 2009b.Recovery of organic wastes in the Spanish wine industry.Technical, economic and environmental analyses of the composting process. Journal of Cleaner Production, 17(9), 830-838.

Thomas, L., Larroche, C., Pandey, A. 2013. Current developments in solid-state fermentation. Biochemical Engineering Journal, 81, 146-161.

Vassilev, N., Requena, A., Nieto, L., Nikolaeva, I., Vassileva, M. 2009. Production of manganese peroxidase by Phanerochaete chrisosporium grown on medium containing agro-wastes/rock phosphate and biocontrol properties of the final product. Industrial Crops and Products,30, 28-32.

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Contact

Antoni Sánchez Ferrer antoni.sanchez@uab.cat UAB Carrer de Sitges, Edifici Q 08197Bellaterra

DISCLAIMER The content of this report does not reflect the official opinion of the European Union. Responsibility for the information and views expressed in the report lies entirely with the authors.