Protein extraction using Tris-EDTA saturated phenol and precipitation using 0.1 M ammonium acetate prepared in 80% methanol was done as per the protocol suggested by Wang et al. (2006). Microalgal biomass (wet) was homogenized in a mortar and pestle using liquid nitrogen and out of that 200 mg was transferred to a 2 mL tube. The tube was filled with 2 mL of 10% acetone and mixed with the microalgal biomass using a cyclomixture. After 30 min of incubation at room temperature, the mixture was centrifuged at 6000 rpm for 10 min. After centrifugation, the supernatant was discarded and the tube was filled with 2 mL of 0.1 M ammonium acetate prepared in 80% methanol. After proper mixing by vortexing and incubation at room temperature for 15 min, the mixture was centrifuged at 6000 rpm for 10 min. The supernatant was again discarded and the pellet was mixed with 2 mL of 80% acetone. After keeping the mixture undisturbed for 15 min, the acetone was removed after centrifugation under the above mentioned conditions. The pellet was then air dried to remove the residual acetone. To this microalgal pellet, 1 mL of phenol was added, mixed thoroughly and incubated for 5 min at room temperature. The mixture was then centrifuged at 6000 rpm for 5 min and the supernatant containing the proteins dissolved in phenol, was transferred to a fresh 2 mL tube. To this separated supernatant, 1 mL of 0.1 M ammonium acetate prepared in 80% methanol was added, mixed properly and incubated overnight at −20◦C for proper protein precipitation. The other day, the mixture was centrifuged at 6000 rpm for 5 min, the supernatant was carefully discarded and the pellet obtained was washed once with 80% methanol and once with 80% acetone. The protein pellet was then processed for quantification using Bradford method (Bradford, 1976).

### Protocol 3

The protocol recommended by Cilia et al. (2009) was followed for extraction and precipitation of protein from wet S. obliquus biomass using 10% TCA prepared in 80% acetone containing 2% β-mercaptoethanol. The wet microalgal biomass (200 mg) was dissolved in 10 mL 10% TCA prepared in 80% acetone containing 2% β-mercaptoethanol. The mixture was mixed thoroughly by vortexing. It was then incubated at −20◦C for at least 12 h for protein precipitation. The precipitated protein was separated from the supernatant by centrifugation at 5000 rpm for 15 min and then washed three times in 5 mL of cold acetone. The protein pellet was then processed for quantification using Bradford method (Bradford, 1976).

### Protocol 4

The protocol suggested by Bhardwaj and Yadav (2013) was followed for extraction of proteins using Tris-HCl saturated phenol and precipitation using 0.1 M ammonium acetate prepared in 80% cold methanol. After homogenization of the wet microalgal biomass using liquid nitrogen in a mortar pestle, 200 mg of homogenized microalgal biomass was suspended in 3 mL of extraction buffer in a 15 mL centrifuge tube, vortexed and incubated for 10 min on ice. Afterward, equal volume, i.e., 3 mL of Tris-HCl saturated phenol was added and the solution was incubated with intermittent shaking at room temperature for 10 min for phase separation to occur. Following this, the solution was centrifuged for 10 min at 5000 rpm. The phenolic phase on the top of the tube, was collected carefully to avoid contact with the interphase, and was transferred to a fresh tube. This phenol phase was then re-extracted with 3 mL of extraction buffer. The sample was shaken and incubated at room temperature for 5 min, after which, it was centrifuged for 10 min at 5000 rpm. The phenol phase, still on the top of the tube, was recovered carefully and transferred to a new tube. To this 10 mL of precipitation solution was added. The contents were mixed by a cyclomixture and then incubated overnight at −20◦C. The other day, the protein was finally pelleted after centrifugation at 5000 rpm for 10 min. The pellet was washed once with 80% cold methanol and once with 80% cold acetone. The protein pellet was then processed for quantification using Bradford method (Bradford, 1976).

### Protocol 5

As proposed by Gerde et al. (2013), protein extraction was carried out by alkali hydrolysis method and ultrapure millipore water followed by precipitation using 2M HCl. 200 mg of wet microalgal biomass was dissolved in 20 mL of 2 M NaOH prepared in ultrapure millipore water containing 0.05% (v/v) of β-mercaptoethanol thus raising the pH of the biomass suspension to pH 11. The mixture was incubated for 5 h at 60◦C. The biomass suspension was then centrifuged at 5000 rpm for 15 min and the supernatant collected was acidified using 2M HCl (added drop wise) till the pH was brought down to pH 3.2. After incubation of the acidified mixture at room temperature for 30 min, it was centrifuged at 5000 rpm for 15 min. The supernatant was discarded and the protein pellet was then processed for quantification using Bradford method (Bradford, 1976).

### Ethics Statement

This study was carried out in accordance with the principles of the Basel Declaration and recommendations of Institutional Animal Ethics Committee (IAEC), IIT Kharagpur. The protocol was approved by the IAEC, IIT Kharagpur.

# Experimental Set-Up for Fish Feeding Experiments

Freshwater fish, namely, rohu (Labeo rohita), mrigal (Cirrhinus mrigala) and catla (Catla catla), were obtained from natural ponds at Agricultural and Food Engineering Department, IIT Kharagpur. Before distributing the fish in different tanks, fish fingerlings (no = 90) were treated with potassium permanganate solution (1 mgL−1 ) for 1 h to remove any external parasites. Then they were randomly divided over six 50 L glass tanks fitted with lights on top of the tanks (15 fish fingerlings of mixed species in each tank). The fingerlings were then allowed to acclimatize to the new growth conditions for 2 weeks. The weight of the fish fingerlings ranged from 3.7 to 4.0 g at the beginning of the acclimatization period. During this period of acclimatization, the fish were fed with their standard diet at 2% of their body weight, only once during the daylight period between 9:00 – 10:30 am. The fecal matter and uneaten feed were removed once every 7 days from each tank by draining approximately 80% of the tank water volume using a siphon pipe and replacing it with an equal volume of clean ground water. The rectangular glass tanks/aquariums were properly aerated with the use of air stones.

### Experimental Set-Up 1

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90 days feeding experiment was conducted with fish fed with three different test diets in three different tanks. The three test diets were standard diet (control), whole S. obliquus biomass and standard (control) + whole microalgal biomass (50:50) diets. The control diet consisted of a standard fish meal-based pelleted feed containing fishmeal, groundnut-oil cake, rice bran, wheat flour, and vitamins and minerals mixture in appropriate proportions. Biochemically the control feed had 30% crude protein, 3.5% lipid, and 40% carbohydrate, with the rest of the composition being crude fiber, minerals, ash, and moisture. Contrarily, the whole S. obliquus biomass was the harvested microalgal cells without any pre-treatment consisting of 53.2% protein, 12.5% lipid, and 22% carbohydrate. These diets were fed to the fish fingerlings at 2% of their body weight per day. At the start of the experimental set-up 1, the weight of the fish fingerlings ranged from 4.5 to 4.9 g. The amount of feed was calculated and readjusted every 15 days according to change in the body weight. The treatments were executed in duplicate. During this experiment, seven water quality parameters (analyzed as explained below in Section "Analysis of Water Quality Parameters," daily for pH, temperature and DO, and once every 3 days for the rest of the parameters) and eight growth parameters (analyzed as explained below in Section "Analysis of Fish Growth Performance and Nutrient Utilization Parameters," once every 15 days) were tested for ensuring proper fish health and management through the progress of the experiment.

### Experimental Set-Up 2

Another 90 days feeding experiment was conducted with fish fed with three different test diets, standard diet (control), standard (control) + whole microalgal biomass (50:50) diets and standard (control) + extracted microalgal protein (50:50) diets at 2% body weight per day. The treatments were done in duplicate and all the experimental analyses were done in a similar manner as explained above.

### Experimental Set-Up 3

The third feeding experiment for 90 days was conducted with fish fed with three different test diets, standard diet (control), standard (control) + whole microalgal biomass (50:50) diets and standard (control) + whole microalgal + extracted microalgal protein (25:25:50) diets at 2% body weight per day. The treatments were done in duplicate and all the experimental analyses were done in a similar manner as explained above.

### Analysis of Water Quality Parameters

Water quality parameters such as temperature, pH, dissolved oxygen (DO) and turbidity were measured using standard instruments. Nitrate, nitrite and total ammonia nitrogen (TAN), contents in the water samples were measured according to the protocols suggested by Nicholas and Nason (1957), Lowe and Evans (1964), and Herbert et al. (1971), respectively.

# Analysis of Fish Growth Performance and Nutrient Utilization Parameters

### **Body weight gain**

The body weight gain (BWG) was calculated as per the formula suggested by Badwy et al. (2008) and was expressed as g fish−1 . The weight of the fish was measured using a weighing balance (Pioneer Scale Industries, Kolkata, India). Fish were caught using a fish net and were then placed on a weighing balance. Weight of the fish was measured after the fish became stable on the balance. An average value of the wet weight was taken after noting down three independent readings. The fish fingerlings were not anesthetized during their weight measurement.

Body weight gain (g fish−1 ) = Weight (g) at the end of the experimental period – weight (g) at the beginning of the experimental period.

### **Specific growth rate**

The specific growth rate (SGR) of the fish was calculated as per the formula suggested by Badwy et al. (2008).

Specific growth rate (SGR) = (ln W1 − ln W0)/(t2 − t1).

where W1 was the final body weight of the fish after time t2 and, W0 was the initial body weight of the fish at time t1.

### **Feed conversion ratio (FCR)**

The FCR was calculated as per the formula suggested by Siddhuraju and Becker (2003). Total feed fed to the fishes for every 15 days period was calculated and expressed as (g fish−1 ). Total weight gained by the fishes (in each tank) after every 15 days of feeding was measured as per the method detailed for measurement of BWG.

Feed conversion ratio (FCR) = Total feed fed (g fish−1 )

/Total wet weight gain (g).

### **Protein efficiency ratio (PER)**

The PER was calculated as per the method suggested by Siddhuraju and Becker (2003). Fish were caught in a fish net and euthanized by rapid chilling. Fish flesh were then collected and grounded using liquid nitrogen. Protein extraction using ultrapure water, NaOH and TCA was done following Barbarino and Lourenco (2005). Protein estimation was done following Bradford (1976).

Protein efficiency ratio (PER)

= Wet weight gain (g fish−1 )/Amount of protein fed (g fish−1 ).

### **Apparent net lipid utilization (ANLU)**

The ANLU was calculated as per the formula suggested by Becker et al. (1999). Fish were first caught in a fish net and euthanized by rapid chilling. Fish flesh was then collected and grounded using liquid nitrogen. Lipid extraction using methanol and chloroform was done following Bligh and Dyer (1959). The amount of lipid obtained/lipid yield was calculated by subtracting W1 (weight of the empty vial) from W2 (weight of the vial containing the extracted lipid) and was expressed as gL−1 .

fmicb-10-02114 September 14, 2019 Time: 12:26 # 5

ANLU = [final fish body lipid (g) − initial fish body lipid (g)

/crude lipid fed (g)] × 100.

### **Protein productive value (PPV)**

The PPV was calculated using the formula suggested by Siddhuraju and Becker (2003).

Protein productive value (PPV)

= [gain in fish body protein (g)/crude protein fed (g)] × 100.

### **Metabolic growth rate (MGR)**

The MGR was calculated as per the formula suggested by Becker et al. (1999).

Metabolic growth rate (MGR)

= live body weight gain (g)/[(initial body weight (g)/1000)0.8

.

+ (final body weight (g)/1000)0.8 /2]day−1

### **Omega-3 Fatty acid analysis**

Fish were first caught in a fish net and euthanised by rapid chilling. Fish flesh was collected and grounded using liquid nitrogen. Lipid extraction was done following Bligh and Dyer (1959) following which the amount of lipid obtained was expressed as gL−1 . Transesterification of the extracted lipids was done following Mandal et al. (2013) and analyzed using GC-MS as per the method detailed in Patnaik and Mallick (2015).

### Proposition for an Algal Refinery

This manuscript being a sequel to the previously published paper by Patnaik and Mallick (2015), the test microalga for this part of the study has been grown under the optimized condition for designing the algal refinery. The optimized condition comprised of S. obliquus grown in N 11 medium supplemented with 0.17% acetate, 0.17% citrate and 0.4 g L−1 nitrate and incubated for a period of 9 days in a temperature controlled culture room. The sequential extraction of all the components from the test microalga other than protein was done as per the methods detailed in Patnaik and Mallick (2015) whereas the protein part was extracted following the method standardized in this manuscript.

To find out the correct sequence of extraction of different components (so as to avoid any negative impact on the extraction yields of different components) four different sequences were tried as shown in **Table 1**.

### Statistical Analysis

Microsoft Excel (Microsoft Corporation, United States) was used for graphical representations and determination of the standard error values (obtained from experiments carried out by using three independent cultures to confirm their reproducibility) for all experiments related to standardization of protein extraction protocols and microalgal refinery design. Fish feeding experiments were done in duplicate. Statistical analysis and graphical representations of the fish growth and nutrient utilization studies were also done using Microsoft Excel (Microsoft Corporation, United States).

### RESULTS AND DISCUSSION

### Selection of Protocol for Protein Extraction From S. obliquus Biomass

For extraction of protein from S. obliquus biomass, five different protocols were tested by following the methods described in Section "Selection of Protocol for Protein Extraction From S. obliquus Biomass." The extracted proteins were then analyzed using Bradford method (Bradford, 1976). The protein yield from the whole S. obliquus biomass grown under control condition for a period of 21 days was found to be 0.65 gL−1 (53.2% dcw), as measured before the initiation of the extracting protocols. Of all the protocols tested, Protocol 1 proved to be the most efficient. The selected protocol produced maximum extraction of protein up to 0.58 gL−1 (47.5% dcw) after 21 days of incubation. So, this protocol was able to extract 89% of the protein present in the whole S. obliquus biomass under control condition. As compared to the other protocols, Protocol 1 was found to extract maximum amount of proteins, probably due to the presence of more watersoluble proteins in the test microalgal biomass (Algae PARC, 2016), the denaturation of the disulfide bonds in the protein molecules by β-mercaptoethanol (Nelson and Cox, 2000), and the disruption of the solvation layers of the proteins and their partial denaturation, thus exposing even more hydrophobic surface to the solvent and enhancing the hydrophobic aggregation of the protein molecules by using TCA (trichloroacetic acid) (Barbarino and Lourenco, 2005; Cilia et al., 2009).

Hence, this experiment suggested the significance of a thorough knowledge of the type of proteins present inside the test organism along with their solubility and structure for selecting/designing an efficient protein extraction protocol. Additionally, the selected method should be compliable for a broad range of microalgal species, in both, their wet and lyophilized forms. Furthermore, the extraction protocol should not be extremely harsh so as to jeopardize the complete protein functionality ultimately compromising with quantification of the extracted protein using dye-based reactions. Another important point worth keeping in mind is that, the extraction protocol

alone, ultimately decides the use to which the extracted protein can be put to in a refinery design. The results of different protein extraction protocols used have been given in **Figure 1**.

### Fish Feeding Experiment

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### Trial Experiment

After selection of the protein extraction protocol, experiments to formulate a protein-rich algae meal for substitution in appropriate quantities in the standard fish diet, was carried out. Trial experiment for investigating the ability of the freshwater fish species, rohu (Labeo rohita), mrigal (Cirrhinus mrigala), and catla (Catla catla), to feed on S. obliquus whole biomass as their only feed resource was performed for a period of 30 days. Additionally, the ability of the dried microalgal biomass pellet to sink to the bottom and remain undispersed till eaten by the fishes, was also checked. It was observed that the microalgal biomass was being consumed by the fishes with significant improvement in growth without much wastage of the supplied feed. The water quality parameters measured, were found to be well maintained within the tolerable limits of the fishes during the trial period.

### Experimental Set-Up 1

After confirmation of the acceptability of S. obliquus biomass as a feed supplement by the freshwater fishes, the first experimental set-up for 90 days was conducted with fish fed with three different test diets at 2% body weight per day. The body weight of the fish in the test tanks on the 0 day (on the day of the start of the experiment) was 4.5 ± 0.72, 4.9 ± 0.65, and 4.7 ± 0.69 g in the tank fed with the standard diet (control), whole S. obliquus biomass diet, and standard + whole microalgal biomass (50:50) diet, respectively. The control diet consisted of a standard fish meal-based pelleted feed with 30% crude protein, 3.5% lipid, and 40% carbohydrate, with the rest of the composition being crude fiber, minerals, ash, and moisture. The S. obliquus biomass consisted of 53.2% protein, 12.5% lipid, and 22% carbohydrate.

### **Fish growth performance and nutrient utilization parameters**

The growth parameters for assessing the effect of different diets on the freshwater fishes was studied as per the methods described in Section "Analysis of Fish Growth Performance and Nutrient Utilization Parameters." Eight different growth performance and nutrient utilization parameters i.e., BWG, SGR, FCR, PER, ANLU, omega-3 fatty acid content, PPV and MGR were observed. The results of the growth study have been shown in **Figures 2A–H**. It was observed that, all the above parameters showed maximum improvement in the fishes fed with 50:50 ratio of standard + whole S. obliquus biomass diet. Under this diet condition, maximum BWG of 6.95 g was observed in the third fortnight, i.e., within 30–45 days of feeding. The maximum SGR when fed with this diet was 0.026 µ. Out of the three test diets, this diet was observed to have the lowest FCR value, indicating the cost-effectiveness of the formulated diet. Hence it was implied that the maximum portion of 50:50 ratio of standard + whole S. obliquus biomass diet was able to be assimilated in the body of the fishes. Similarly, the PER value of this diet was found to be the highest among all three diet conditions indicating higher contribution of the protein in the standard + whole S. obliquus biomass feed toward raising the weight of the fish. It was also observed that the proportion of the lipid assimilated into the body of the fish out of the total crude lipid fed, i.e., ANLU was maximum (45.7%) in the third fortnight of feeding the 50:50 ratio of standard + whole S. obliquus biomass diet. Hence the omega-3 fatty acid content of 11.2% was also found to be maximum during that period. Similar to these observations, the PPV, i.e., the amount of protein gained by the fish after being fed with the test diet was found to be maximum (30.6%) within 30–45 days. With all the growth performance and nutrient utilization parameters showing maximum value in the third fortnight of feeding with 50:50 ratio of standard + whole S. obliquus biomass diet, the MGR was consequently found to be maximum during the same time period. This finding was found to be well in agreement with Radhakrishnan et al. (2015) in which 50% inclusion of C. vulgaris in the control diet, increased the growth parameters in Macrobrachium rosenbergii. Higher feeding rate and better assimilation of the protein fraction of the microalgal diet in the prawn were reasons cited by the authors for such an observation. The other two diets, i.e., standard (control) diet and whole S. obliquus biomass diet showed comparatively poorer performance as assessed through the above mentioned parameters. The 100% inclusion level of whole S. obliquus biomass diet in this research study must have reduced the palatability of the feed thus showing comparatively lower growth (**Figure 2**). Contrary to this observation, 100% inclusion percentage of Spirulina platensis was found to improve the specific growth rate and PER of the freshwater fish, Labeo rohita as observed in the report of Nandeesha et al. (2001). Algal inclusion percentages of <50% have also been recommended by few researchers (Teimouri et al., 2013; Ghosh and Mitra, 2015; Basri et al., 2015) for improving growth and/or functional component accumulation in some fish species. The examples cited, thus justify the species-specific dependence of both, the microalga (to be included in the fish diet) and the fishes (to be fed with the microalga included diet) on each other for improvement in the fish growth and nutrient utilization parameters. Moreover, standardization of the inclusion percentage of protein-rich algal meal in the diets of the freshwater fishes is also suggested to be of utmost importance.

### **Water quality parameters**

The water quality parameters of the fish tanks were analyzed as per the protocols described in Section "Analysis of Water Quality Parameters." As the experiment was carried out from September to November, the temperature gradually saw a declining trend due to gradual decline in the atmospheric temperature in this geographical locale. The three parameters, i.e., temperature, pH and DO were not found to have any significant difference in the fish tanks administered with the three test diets. However, in case of turbidity, TAN, nitrate and nitrite content, the fish tanks provided with standard diet, showed higher values of the said parameters (data not shown) which could have been due to the accumulation of the residual feed and inadequate oxidation of the fecal matter (Ghosh and Mitra, 2015).

### Experimental Set-Up 2

As 50% inclusion of whole S. obliquus biomass showed the best results in the first set-up (**Figure 2**), the second set-up was designed to reconfirm the results of the first set-up, and in addition explore the effect of 50% inclusion of extracted microalgal protein in the standard diet. Another 90 days feeding experiment was conducted with fish fed with three different test diets, standard diet (control), standard + whole microalgal biomass (50:50) diet, and standard + extracted microalgal protein (50:50) diet at 2% body weight per day. The body weight for the fish in the test tanks on the 0 day (on the day of the start of the experiment) was 4.6 ± 0.83, 5.3 ± 0.79, and 5.8 ± 0.72 g,

respectively. The experimental set-up was similar to that of the first experiment.

**Fish growth performance and nutrient utilization parameters** The results of the experiment have been shown in **Figures 3A–H**. Out of the three test diets, the standard + whole S. obliquus biomass (50:50) diet in this experimental set-up also, showed the best growth performance. It was observed that, BWG of the fishes in these test tanks increased gradually with maximum BWG of 5.65 g in the third fortnight of feeding. The SGR of the fish fed on 50:50 ratio of control and whole S. obliquus biomass diet was also found to be maximum (0.02 µ), with a gradual decline in the subsequent time periods. An observation of the FCR, PER and PPV values further attested the best growth performance in the fish fed with this test diet. The percentage of ANLU and omega-3 fatty acid content, were also found to be maximum between 30 to 45 days of feeding with their respective values being, 35.7 and 10.5%. The diet formulation with standard + extracted microalgal protein (50:50) diet was however, not found to be very significant as compared to the other two test diets which could be due to the lower palatability of this diet which was clearly demonstrated in the poorer growth performance in the fishes administered with this diet, as compared to the control and 50% whole S. obliquus biomass included diet. Such an experiment with inclusion of only extracted plant or animal protein has not been carried out by any researcher, to the extent of our knowledge and hence is not available for reference.

### **Water quality parameters**

The temperature, as recorded daily was found to drift toward higher degrees gradually due to the change in season during the months of December–February, in this geographical locale. The three parameters, i.e., temperature, pH and DO were not found to have any significant difference in the fish tanks administered with the three test diets. However, in case of turbidity, TAN, nitrate and nitrite content, the fish tanks provided with standard + extracted microalgal protein (50:50) diet, showed a higher value of the said parameters due to higher accumulation of the uneaten feed. Comparatively, the fish tanks with the standard diet showed lower wastage of the administered feed, hence lower values of turbidity, TAN, nitrite and nitrate but the lowest values of the three measured parameters was observed in the tanks with the standard + whole S. obliquus biomass (50:50) diet. These four parameters were found to increase gradually in the test tanks with increased days of feeding (data not shown).

### Experimental Set-Up 3

As 50% inclusion of the extracted microalgal protein along with 50% standard diet, did not show any improvement in the growth parameters of the freshwater fish varieties in the second experimental set-up, the proportion of the standard diet was reduced to 25 and 25% of whole S. obliquus biomass was included along with 50% extracted algal protein in the third set-up, to increase the palatability of the test diet for consumption by the fishes. The third experimental set-up for 90 days was carried out in a similar way as was done in the other two experimental set-ups. The weight of the fishes before the start of the experiment were 5.6 ± 0.72, 5.9 ± 0.66, and 5.5 ± 0.71 g in the fish tanks administered with standard diet (control), standard + whole microalgal biomass (50:50) diet, and standard + whole microalgal biomass + extracted microalgal protein diet (25:25:50) diet, respectively.

**Fish growth performance and nutrient utilization parameters** The results of the experiment have been shown in **Figures 4A–H**. Out of the three test diets, the standard + whole microalgal biomass + extracted microalgal protein (25:25:50) diet showed the best growth performance. It was observed that, BWG of the fishes in this test tank increased gradually with maximum BWG (7.3 g) in the third fortnight of feeding. The SGR of the fish fed on the same diet was also found to be 0.028 µ. Higher incorporation of protein due to higher nutrient digestibility (Nandeesha et al., 1994; Mustafa and Nakagawa, 1995) could have led to this increased body weight gain and SGR in the selected test diet. Although the microalgal protein during extraction and precipitation could have been denatured, the amino acids to which it was broken down to were successfully incorporated in the body of the fishes (Sward, 2014). While this diet condition showed maximum gain in body weight, the BWG in case of standard + whole microalgal biomass (50:50) diet was found to be only marginally different. An observation of the FCR and PER values further confirmed the best growth performance in the fish fed with this test diet. The percentage of ANLU and omega-3 fatty acid content, were however, found to be maximum within 30–45 days of feeding with the standard + whole microalgal biomass (50:50) diet, their respective values being, 46.8 and 15.5%. But in case of PPV and MGR, maximum improvement was seen in the third fortnight of feeding the fishes with standard + whole microalgal biomass + extracted microalgal protein (25:25:50) diet. The maximum PPV was recorded to be 36.3% and maximum MGR was found to be 6.21 day−1 , thus, attesting the use of the microalgal protein as an aquafeed, during the formation of an algal refinery.

### **Water quality parameters**

The water quality parameters in the fish tanks were analyzed as per the protocols described in Section "Analysis of Water Quality Parameters." Temperature, as recorded daily was found to drift toward higher degrees gradually due to the change in change in atmospheric temperature during March – May. As observed in the other two experimental set-ups, these three parameters, i.e., temperature, pH and DO, were not found to have any significant difference in the fish tanks administered with the three different test diets. However, in case of turbidity, TAN, nitrate and nitrite content, the fish tanks provided with standard diet, again showed higher values of the said parameters. Comparatively, the fish tanks with the standard + whole microalgal + extracted microalgal protein (25:25:50) and standard + whole microalgal biomass (50:50) diets showed lower wastage of the administered feed, hence lower values of turbidity, TAN, nitrite and nitrate, varying marginally from each other (data not shown).

Fish feeds account for a significant portion of the costs in an aquaculture enterprise. Hence, to minimize the feed costs while simultaneously looking into environmental sustainability,

accurate information about the nutritional requirement of the fishes is imperative so as to adopt balanced diet formulations and feeding practices for their optimal growth and development. Fishes mostly require diets composed of proteins, lipids and carbohydrates for good growth and maintenance, however, amongst them, proteins are required in maximum proportions, comprising about 65–70% of the fish muscle. Proteins consumed by the fishes not just provide the essential and non-essential

amino acids for their muscle formation and enzymatic function, but also provide energy for their maintenance. Insufficient amounts of protein in the fish diets lead to a decline or diminution of their growth parameters probably due to restricted supply of proteins to more vital organs and tissues. While, excessive amounts of protein in the fish diets result in accelerated energy requirements, higher costs, more nitrogenous waste excretions and decreased fish growth. Moreover, as proteins

are the most expensive item in the commercially available fish feeds, it is indeed wise to include optimum dietary protein levels in their diets, thus avoiding biological and economical loss without compromising with their growth and maintenance. Furthermore, as fish growth also depends on the effectiveness of the withholding time of the dietary protein, optimum protein proportions for each species including their cultivation phase needs should also be determined for maximum profitability.

### Proposition of an Integrated Sequential Processing Approach for an Algal Refinery

### Sequential Production of Biodiesel and Various Co-products

### **Selection of the sequence for maximum extraction of the microalgal components**

The extraction of the components from S. obliquus biomass in this part of the study was done under the optimized culture condition as detailed in Section "Proposition for an Algal Refinery." During a sequential extraction process, the biggest problem posed is by the interference of the solvents and the methodologies used for extraction of one component on the yield of the other microalgal components. Hence proper placement of the lipids and other components one after the other, without much effect on their extracted yields is essential. For this purpose four different sequences were tried. A display of the variation in the yields of the different components when arranged in different sequential positions has been given in **Table 1**.

Before elaborating on the data given in **Table 1**, a mention of the yields of the different components (each component extracted individually from the whole S. obliquus biomass without any sequential processing) obtained under the optimized condition from 100 g of dry S. obliquus biomass is important. The β-carotene, lipid, carbohydrate and protein yield, extracted individually was 0.06, 56.2, 30.2, and 10.2 g, respectively.

From the Table above, it was observed that when sequence 1 was followed, the yields of β-carotene, lipid and carbohydrate were insignificantly affected, but the protein yield was reduced

TABLE 1 | Variation in the yields of the different microalgal components when arranged in different sequential positions.

drastically to 5.4 g from 1 Kg wet (≈100 g dry) S. obliquus biomass. This observation indicated that neither did the use of acetone for β-carotene extraction affect the lipid yield from the microalgal biomass as the amount of lipid obtained after β-carotene extraction was nearly the same as that obtained directly from the test microalga, nor did the solvents acetone, chloroform and methanol affect the carbohydrate yield, extracted sequentially after β-carotene and lipid, but the positioning of protein extraction at the end of the sequential process affected the protein yield severely. The reduced yield of protein could possibly have been due to leakage during acid hydrolysis of the biomass for carbohydrate extraction. The loss in biomass during the sequential extraction procedure was however, found to be negligible. In case of sequence 2, a reduction in both, the lipid and protein yield to 32.2 and 2.4 g, respectively was observed when the lipid and protein extractions were preceded by carbohydrate extraction from the dry microalgal biomass due to loss of lipids and proteins during acid hydrolysis of the dry microalgal biomass. β-carotene extraction on the top of the sequence was not found to be responsible for this observation which can also be confirmed from the results of the first

sequential extraction process. The results of the third sequential extraction process showed that if β-carotene and lipid extraction are followed by protein extraction instead of carbohydrate extraction then the yield of the extracted carbohydrate was reduced marginally to 26.7 g but the yield of the protein obtained was still significantly lower than that obtained directly from the whole S. obliquus biomass under the optimized condition which was due to the interference of the solvents used during lipid extraction in the extraction of proteins using ultra-pure water (Wessel and Flugge, 1984), although after being placed in the third position of the sequential extraction process the protein yield showed an increase to 6.4 g.

However, implementation of the fourth sequential extraction process resulted in maximum yields of the microalgal components which were unaffected by any form of loss due to use of solvents or methods of cell hydrolysis. The chosen sequence extracted β-carotene followed by protein, lipid and carbohydrate. β-carotene and protein extraction were done from wet S. obliquus biomass, but the data presented, has been given in terms of dry cell weight for a better representation of the microalgal biomass composition.

### **Microalgal refinery**

The design of the proposed algal refinery has been shown in **Figure 5**. The harvested wet biomass of S. obliquus was treated with acetone for β-carotene extraction followed by protein extraction using ultrapure water and alkali hydrolysis of the microalgal cells. The S. obliquus biomass was then dried at 60◦C followed by lipid extraction using a binary solvent system (chloroform-methanol). The de-fatted biomass was then subjected to acid hydrolysis for the breakdown of the complex polysaccharides to simple monomers for bioethanol production through fermentation by Saccharomyces cerevisae. 1 Kg wet (≈100 g dry) S. obliquus biomass under the optimized condition yielded 0.06 g of β-carotene, 10 g of protein, 38 g (43 mL) of biodiesel, 2 g of omega-3 fatty acid, 3 g (2.4 mL)

independent determinations.

of glycerol and 18 g (23 mL) of bioethanol. As shown in **Figure 5**, during conversion of carbohydrates to ethanol, it was observed that ∼60% of the total carbohydrate contained in the microalgal biomass under the optimized condition was converted to bioethanol. Although theoretically, 50% conversion of the carbohydrates in the microalgal cells is possible, conversions of carbohydrates to bioethanol by >50% may reflect fermentation of additional carbohydrates beside glucose and mannose, which was not accounted for in the theoretical calculations (Laurens et al., 2015). Hence 70% of the test microalgal biomass was successfully used for production of biodiesel and other value-added products by using an algal refinery approach.

The approach of algal refinery as a method to reduce the cost of the algal biodiesel by co-production of various value-added chemicals, antioxidants, fertilizers etc. is the most up-coming strategy suggested for the economic viability of the microalgal biodiesel. This concept is a relatively new and novel idea where abundant literature is not available although fragmented data on some feedstocks are present. A report by Dong et al. (2016) on the valorization of Scenedesmus acutus by simultaneous

production of bioethanol and biodiesel in a combined algal processing method has shown to utilize nearly 66% of the microalgal biomass for energy generation thus bringing down the cost of the total energy produced by 9% from \$10.86/GGE (Gallons of Gasoline equivalent) to \$9.91/GGE. In another report by Marinho et al. (2016), the macroalga Saccharina latissima in a biorefinery approach has been shown to produce succinic acid after which the leftover solid residue has been tested for their total phenolic compounds and macronutrients (Ca, K, Na, Mg, P, N, and Fe) content. These phenolic compounds and macronutrients contained in the leftover residue have been proposed to be used as antioxidants and fertilizers, respectively, thus, demonstrating the potential of ∼70% of the S. latissima biomass to be converted to value-added products. In the present study, a successful conversion of 70% of the Scenedesmus obliquus biomass into industrially important products such as β-carotene, protein for aqua-feed, biodiesel, omega-3 fatty acid, glycerol and bioethanol through defined sequential processing steps has been demonstrated unlike the report by Marinho et al. (2016) in which conversion of the phenolic compounds and macronutrients to antioxidants and fertilizers has not been carried out. Additionally, the production of multiple high-value products as shown in the refinery design further confirms the comparative richness and relevance of the present research study in providing an economically sustainable model in future during application of the refinery design in a pilot scale.

Cost-intensive production process of microalgal biodiesel is a major obstacle in its commercialization. Hence efforts should be made to reduce the costs associated with microalgal production and conversion of the algal intermediates to biodiesel. But merely improving the biomass productivity or augmenting the quantity of lipids in microalgae is not expected to bring enough cost reductions. For further progress, complete utilization of all algal components is essential. Previous research reports have shown substantial reductions in microalgal biofuel production costs by applying combined algal processing techniques through simultaneous production of proteins, carbohydrates and other valuable cellular compounds. But, the production of higher value co-products differs depending on the algal biomass composition. Therefore for making maximum profit from algal biodiesel production, decisions should be made judiciously regarding the algal strains possessing maximum potential for producing different highvalue products; methodologies used for sequential extraction of different cellular components so as to incur minimal losses in terms of loss of compounds; the potential for nutrients and solvents recycling; and degradation or denaturation of the cellular compounds during the extraction process. Furthermore, colocation of the refinery units with algal farms can reduce the transportation and pre-processing costs. Algal refineries are expected to lead to high quality job creation and energy independence by bridging the gap between high-value smallmarket products and low-value large-market products. Hence extensive research on these refinery designs is expected to be of immense benefit.

# CONCLUSION

An algal refinery is a concept that is being followed nowadays as a strategy to reduce the high costs incurred during microalgal biodiesel production. Hence, a study with a sequential processing design for production of biodiesel and other industrially important products such as β-carotene, protein, biodiesel, omega-3 fatty acid, glycerol and bioethanol, might appear conceptually incomplete without an assessment of the economics of the entire process to ascertain its viability and sustainability. But the experiments having been carried out in the laboratory under controlled culture condition, calculation of the costs incurred in the entire process will not be realistic. Therefore, scaling up of the present study to pilot scale under outdoor condition should be carried out in future to valuate and validate the economic sustainability of the designed algal refinery. Furthermore, as the present study demonstrates conversion of 70% of the S. obliquus biomass into industrially important products, efforts to convert the remaining 30% of the microalgal biomass into value-added products through improved methodologies to valorize the whole algal biomass with minimal loss would further enrich the proposed strategy in future.

# DATA AVAILABILITY

All datasets generated for this study are included in the manuscript/the supplementary files.

# ETHICS STATEMENT

The animal study was reviewed and approved by Institutional Animal Ethics Committee, IIT Kharagpur.

# AUTHOR CONTRIBUTIONS

RP wrote the article, conducted all the three fish feeding experiments including the experiments for the final algal refinery design, analyzed and interpreted the results of all the experiments. NS performed the first two set-ups of the experiments during which he analyzed four growth parameters and seven water quality parameters. SB also contributed to this work through his help during data analysis. NM and PR supervised the design of the experiments. NM checked the manuscript. All authors have read and approved the final manuscript.

# FUNDING

Financial support from the Indian Council of Agricultural Research, New Delhi, India, is highly acknowledged (grant number NFBSFARA/AE-2007/2010-2011).

### REFERENCES

fmicb-10-02114 September 14, 2019 Time: 12:26 # 14

Gracilaria cornea as dietary ingredients in European sea bass (Dicentrarchus labrax) juveniles. Aquaculture 252, 85–91. doi: 10.1016/j.aquaculture.2005. 11.052

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Patnaik, Singh, Bagchi, Rao and Mallick. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Biodegradation of Deoxynivalenol by a Novel Microbial Consortium

*Yanxia Wang1,2 , Gang Wang2 , Yijun Dai1 \*, Yu Wang1,2 , Yin-Won Lee2,3 , Jianrong Shi2,4\* and Jianhong Xu2,4\**

*1 Jiangsu Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing, China, 2 Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base, Ministry of Science and Technology, Key Laboratory for Agro-Product Safety Risk Evaluation (Nanjing), Ministry of Agriculture and Rural Affairs, Collaborative Innovation Center for Modern Grain Circulation and Safety, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing, China, 3 Department of Agricultural Biotechnology, Seoul National University, Seoul, South Korea, 4 School of Food and Biological Engineering, Jiangsu University, Zhenjiang, China*

### *Edited by:*

*Carlo Giuseppe Rizzello, University of Bari Aldo Moro, Italy*

### *Reviewed by:*

*Yun Chen, Zhejiang University, China Aibo Wu, Shanghai Institutes for Biological Sciences (CAS), China*

### *\*Correspondence:*

*Yijun Dai<EMAIL_ADDRESS>Jianrong Shi<EMAIL_ADDRESS>Jianhong Xu<EMAIL_ADDRESS>
### *Specialty section:*

*This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology*

*Received: 30 June 2019 Accepted: 09 December 2019 Published: 08 January 2020*

### *Citation:*

*Wang Y, Wang G, Dai Y, Wang Y, Lee Y-W, Shi J and Xu J (2020) Biodegradation of Deoxynivalenol by a Novel Microbial Consortium. Front. Microbiol. 10:2964. doi: 10.3389/fmicb.2019.02964*

Deoxynivalenol (DON), a common mycotoxin of type B trichothecene, is produced mainly by several *Fusarium* species. DON causes great losses in farming and poses severe safety risks to human and animal health. Thus, DON contamination in cereals and DON toxicity are of worldwide concern. In this study, we screened the bacterial consortium C20, which efficiently degraded almost 70 μg ml−1 DON within 5 days. The bacterial consortium also had the ability to degrade 15-acetyl-DON, 3-acetyl-DON, and T-2 toxin. The bacterial consortium C20 was able to degrade DON under a wide range of pH and temperature conditions. The optimal temperature and pH for DON degradation were 30°C and pH 8.0, respectively. The bacterial consortium C20 comprised of different bacterial genera, and several strains were found to significantly increase when cultured in Mineral Medium with 100 μg ml−1 DON based on the analysis of the sequences of the hypervariable V3-V4 region of the 16S rRNA gene. 3-keto-DON was confirmed as a degradation product of DON by liquid chromatography/time-of-flight/mass spectrometry (LC-TOF-MS) and nuclear magnetic resonance (NMR) analyses. The results indicated that the bacterial consortium C20 is a potential candidate for the biodegradation of DON in a safe and environmentally friendly manner.

### Keywords: deoxynivalenol, bacterial consortium, biodegradation, 3-keto-DON, mycotoxin

# INTRODUCTION

Mycotoxins have adverse effects on grain quality, resulting in economic losses, besides the threat to human and animal health. The main groups of mycotoxins in world-wide concern are aflatoxin, ochratoxin, patulin, fumonisin, trichothecene, deoxynivalenol, and zearalenone due to their toxicity and their contamination of cereals (James and Zikankuba, 2018). Among these mycotoxins, trichothecenes are mainly produced by a range of fungi, including *Fusarium, Myrothecium*, *Trichoderma*, and *Stachybotrys* (Pessu et al., 2011). Trichothecenes not only cause growth impairment and immune dysfunction at low doses but also contribute to diarrhea, emesis, leukocytosis, hemorrhage, endotoxemia, and death at higher doses (Ueno, 1983). Deoxynivalenol (DON), also known as vomitoxin, produced by *Fusarium graminearum* and *Fusarium culmorum*, is one of the most important type B-trichothecene mycotoxins (Sobrova et al., 2010). It causes acute toxicity, vomiting, anorexia, growth retardation, reproductive and teratogenic effects, skin disorders, diarrhea, carcinogenesis, and even immune dysregulation (Pestka, 2010; Sobrova et al., 2010). DON is a frequent contaminant of grain cereals, such as wheat, maize, and barley, which can be also detected in the foods of many countries (Thuvander et al., 2001; Rasmussen et al., 2003; Leblanc et al., 2005; Schollenberger et al., 2005; Castillo et al., 2008; Sugiyama et al., 2009; Alkadri et al., 2014; Stanciu et al., 2016; Shi et al., 2018). Since DON is commonly detected in grains and is chemically stable, it represents a permanent health risk to humans and domestic animals. Therefore, the development of efficient decontamination strategies is essential to DON in grains and agricultural products.

Several strategies have been developed to control DON contamination. Firstly, cultural practices may help to combat plant diseases and fungal growth, such as the multi-field rotation of crops, the selection of seeds that are resistant to *Fusarium* head blight, and the early harvest of crops (Awad et al., 2010). The suitable storage of grains after harvest can also help to avoid increased DON contamination (Shapira et al., 2004). Physical methods, such as damaged grain removal, sieving, dehulling, radiation, and adsorbent use, were successful in detoxifying DON in cereal crops (Leibetseder, 2006). A wide variety of chemical methods have also been found to be effective against DON, including heating, alkaline hydrolysis, and oxidation (Young et al., 1986; He et al., 2010).

Compared with physical and chemical detoxification, microbial biotransformation attracted considerable interests due to high degradation efficiency, the metrics of mild reaction condition, sustainable and eco-friendly process (Awad et al., 2010). Several studies have demonstrated that DON could be metabolized to lower toxicity compounds performed by several microorganisms. For instance, DON was found to be converted into 3-keto-DON under aerobic conditions by the *Agrobacterium-Rhizobium* strain E3–39, isolated from soil samples (Shima et al., 1997). Zhou et al. revealed that two metabolites of DON during degradation by *Barpee* were identified as stereoisomers of DON and 3-keto-DON (Zhou et al., 2008). In addition, microbial consortia have shown the potential to degrade DON. One mixed culture was able to convert DON into two chromatographically separable metabolites (Andrea et al., 2004). Chicken intestinal microbes were also capable of degrading DON by de-epoxidation (Young et al., 2007).

Compared with single bacterium and anaerobic microbes, bacterial consortia with ability of DON degradation under aerobic conditions are rarely reported. Moreover, the DON degradation conditions of these anaerobic bacteria were still limited. In the present study, we isolated a novel bacterial consortium C20 from the wheat field in Jiangsu Province, China, which can stably and efficiently degrade DON into 3-keto-DON under a wide range of pH and temperature with aerobic condition. Further, the performance of biodegradation of other trichothecene mycotoxins was also evaluated.

# MATERIALS AND METHODS

### Samples

A total of 174 samples from different locations, including soil, rice, corn, wheat, rice panicles and fresh corn leaves, were collected during 2016–2017 in Jiangsu Province, China (**Table 1**). Notably, soil samples were randomly collected from the upper layers (0–30 cm) of different crop fields, such as rice, soybean, corn, and winter wheat fields.

# Culture Media and Mycotoxins

Minimal medium (MM) was used as a culture medium for the enrichment and isolation of DON-degrading strains. The composition of MM (pH 7.0) was described by Wang et al. (2019b). Ingredients were purchased from Sigma-Aldrich (Oakville, Ontario, Canada), Guanghua Sci-Tech Co., Led. (Shantou, Guangdong, China), XILONG SCIENTIFIC (Shantou, Guangdong, China), and Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Toxin standards for DON, HT-2, T-2, 3-acetyldeoxynivalenol (3-ADON), and 15-acetyldeoxynivalenol (15-ADON) were purchased from Romer Labs (Erber Campus, Getzersdorf, Austria).

### Selection of Deoxynivaleno-Degrading Microbes

Five-gram aliquots of each sample from the 174 field samples were smashed by a pulverizer (JYL-c020E, Joyoung, China), dispersed in 50 ml distilled water, and cultured with shaking at 180 rpm for 30 min. About 500 μl of the suspension was immediately transferred to 4.5 ml MM broth media with DON (10 μg ml−1) as the sole carbon source. Tebuconazole and Carbendazim (1 or 10 μg ml−1) were added in the cultures to inhibit fungal growth, respectively. The cultures were grown at 30°C, 180 rpm. To exclude physical adsorption, samples autoclaved at 121°C for 20 min were set as negative controls. The extent of DON biotransformation was determined by highperformance liquid chromatography (HPLC). Then, the cultures with the ability of DON degradation were further inoculated on the MM broth containing 10 μg ml−1 DON at intervals of

TABLE 1 | Information for samples collected during 2016–2017 in Jiangsu province.

7 days. To investigate DON degradation activity, the cultures were used to degrade 100 μg ml−1 DON. MM with DON (100 μg ml−1) was set as negative control. The bacterial growth was measured by optical density. The changes in DON concentration and OD 600 values were monitored every day.

### Sample Preparation and High-Performance Liquid Chromatography Analyses

DON was extracted from fermentation broth with an equal volume of ethyl acetate for three times. After dried under an N2 stream, it was re-dissolved in methanol and quantified by HPLC (Waters 2695, Milford, USA) equipped with a PDA (photodiode array) detector at a wavelength of 218 nm and an Atlantis T3 column (octadecylsilyl, ODS; 4.6 mm × 150 mm, 3 μm; Waters), as described previously (Wang et al., 2019b). The mobile phase was methanol/water (20/40, v/v) with a flow rate of 0.6 ml min−1 at 35°C. The identification of mycotoxin was achieved by comparing the retention times while using DON as a standard. DON and degradation products were quantified by measuring peak areas and comparing with MS calibration curves. All data are presented as mean ± standard deviation of three replicates. Degradation percentage of DON was calculated as follows:

$$D\_r = \left(1 - C\_t \, / \, C\_{ck} \right) \times 100\%$$

where *D*r represents the degradation percentage, and *Ct* and *Cck* are the DON concentrations in the experimental treatment and the control groups, respectively (Wang et al., 2019b).

### Analysis of Bacterial Population Diversity

To analyze the bacterial population diversity and its change at high concentrations of DON, the enriched microbial consortium C20 was isolated from environmental samples and was added to MM to degrade DON, with DON concentrations of 10 and 100 μg ml−1. The bacteria were incubated in media with different concentrations of DON at 30°C for 72 h. Genomic DNA of the enriched microbial cultures was extracted and purified with Gentra Puregene Yeast/Bacteria Kit (Qiagen Inc., Mississauga, Ontario, Canada). The hypervariable V3-V4 region of the 16S rRNA gene was amplified by PCR with primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC) (Ahad et al., 2017) and sequenced by Sangon Biotech Co. (Shanghai, China). Further, the 16S rRNA (V3-V4 region) gene sequences were compared with the ribosomal database project (RDP)1 . All treatments were conducted in triplicates.

### Factors Influencing Deoxynivaleno Degradation

To screen for optimal degrading conditions of DON by C20, the degradation rates of DON were determined at the different conditions including temperature, pH, and bacterial concentrations. An aliquot (200 μl) of each microbial culture was added to 1.8 ml of MM supplemented with 10 μg ml−1 DON. The effect of incubation temperature (20, 25, 30, 35, and 40°C), medium pH (4.0, 5.0, 6.0, 7.0, 8.0, and 9.0), and inoculation size (1, 3, 5, 10, 15, and 20%) on biodegradation of DON were investigated. All treatments were performed by three independent experiments with three replicates. The optimal degradation conditions of temperature, pH, and inoculation amount were determined by the degradation rates of DON.

### Type A- and B-Trichothecene Mycotoxins Degradation

To examine the ability of C20 to transform type A- and B-trichothecene mycotoxins, which included two type A-trichothecene mycotoxins (HT-2 toxin and T-2 toxin) and three type B-trichothecene mycotoxins (DON, 3-ADON, and 15-ADON), 10 μg ml−1 of each mycotoxin was added to MM in 2 ml microcentrifuge tubes. Each tube was inoculated with 200 μl of C20, which was cultured in MM with 10 μg ml−1 DON for 72 h. Three replicate cultures were evaluated for each trichothecene mycotoxin, and the negative controls lacked C20. All cultures were incubated at 30°C and 180 rpm in a shaking incubator for 72 h, extracted three times with ethyl acetate, as described, re-dissolved in methanol and analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS).

The LC-MS/MS system was equipped with a Shimadzu 20 AD XR (Kyoto, Japan) coupled with an AB Sciex QTRAP 3500 mass spectrometer (Foster City, CA, USA) and an XDB-C18 analytical column (2.1 mm × 150 mm, 3.5-μm bead diameter; Agilent) maintained at 30°C. Nitrogen was used as the drying gas. The capillary voltage was 4 kV, the nebulizer pressure was 30 psi, and the drying gas temperature was 300°C. The gradient elution conditions were methanol/water (5/95, v/v) to methanol/water (90/10, v/v) in 10 min and were held at 90% methanol for 3 min, the flow rate was set at 0.2 ml min−1 (Wang et al., 2019b). Analyst 1.6.1 software was used for data acquisition and processing.

### Identification of Deoxynivaleno Degradation Products

To analyze the transformation of DON, C20 was added to broth containing 10 μg ml−1 DON and incubated at 30°C on a rotary shaker (180 rpm) for 72 h. Culture broth containing C20 alone was used as a control. Samples were prepared as described above and detected by HPLC. The mobile phase was methanol/water (50:50, v/v) with the flow rate of 0.8 ml min−1.

To investigate the structure of DON degradation products, C20 was inoculated into MM medium with 100 μg ml−1 DON and cultured for 72 h at 30°C with shaking at 180 rpm. The DON degradation products were extracted three times with ethyl acetate. The organic phase was pooled, concentrated and applied to a Waters 1525 Prep-HPLC system (Milford, MA, USA) equipped with a UV detector. Each 20 μl extract was injected onto an XBridge™ Prep C18 column (19 mm × 100 mm, 5 μm, Waters) with methanol/water (50:50, v/v) at a flow rate of 2 ml min−1. DON and its degradation products were detected at a wavelength of 218 nm at 35°C. The resulting degradation products were dried under an N2 stream and re-dissolved in CDCl3 for nuclear magnetic resonance (NMR) analysis.

1 http://rdp.cme.msu.edu/misc/resources.jsp

The 1 H NMR spectra of the biodegradation products were measured by a Bruker DRX-600 spectrometer operated at 600 MHz. The chemical shift (δ) was recorded in parts per million (ppm) relative to the solvent signals [δ(H) 7.26], and the coupling constant (*J*) was measured in Hz.

### Data Analysis

Data were organized using Microsoft Excel 2010. All values are expressed as the mean of three replicates ± standard deviation. The mean differences among treatments were considered significantly at *p* < 0.05 and were evaluated with Tukey's-honestly significant difference (HSD) test (Ahad et al., 2017).

### RESULTS

### Isolation of Deoxynivaleno-Degrading Bacteria

To screen for the microbes that degraded DON, DON was used as the sole carbon source for the enrichment and isolation of the mixed culture. The mixed microbial culture C20, isolated from a soil sample from Suqian, degraded 74.29 μg ml−1 DON after incubation at 30°C for 10 days. DON was degraded rapidly within 5 days of incubation and was continuously degraded after incubation for 10 days. The biomass increased continuously with the amount of DON decreased (**Figure 1**). Furthermore, no difference was found between the samples with and without fungal inhibitors. And the DON concentration in the autoclaved sample was not reduced (**Supplementary Figure S5**). Importantly, the enriched bacterial consortium C20 could efficiently degrade DON, even after more than 100 subcultures in broth (**Supplementary Figure S3**).

### Bacterial Diversity Analysis of the Microbial Consortium C20

To examine mainly bacteria involved in DON degradation, the microbial diversity dynamics of C20 with different

concentrations of DON was defined through analyzing the sequences of the 16S rRNA genes, as described in methods. The results showed that the enriched bacterial consortium C20 mainly consisted of the genera *Hyphomicrobium* (69.54%), followed by *Acidovorax* (12.09%), unknown microbes (7.04%), *Prosthecomicrobium* (2.71%), *Pseudomonas* (2.68%), and *Methylophilus* (2.31%) in MM with 10 μg ml−1 DON (**Figure 2A**). In contrast, the culture in MM with 100 μg ml−1 DON contained a heterogeneous community composed of *Methylophilus* (35.48%), followed by *Hyphomicrobium* (27.71%), *Ancylobacter* (14.73%), *Pseudomonas* (4.87%), *Prosthecomicrobium* (4.6%), unknown microbes (2.39%), *Taonella* (2.33%), *Bosea* (2.33%), and others (**Figure 2B**). Noticeably, as the DON concentration increased to 100 μg ml−1, there was a marked increase in *Methylophilus*, *Ancylobacter*, *Pseudomonas, Prosthecomicrobium, Taonella*, *Bosea*, *Terrimonasin*, and *Devosia* in C20.

### Factors Influencing Deoxynivaleno Degradation

The effects of the temperature, pH, and inoculum on DON degradation by C20 were investigated (**Figure 3**). DON degradation activity was examined at various temperatures from 20 to 40°C in the enriched cultures (**Figure 3A**). The highest degradation rate occurred at 30°C and decreased only slightly at 25 and 35°C. Importantly, approximately 50% of DON degradation was observed even at 40°C, indicating that the consortium C20 can effectively degrade DON over a wide range of temperatures (25–35°C). Moreover, the effects of pH on DON degradation was detected under six different pH levels conditions. The degradation rate of DON increased markedly from pH 4.0 to 8.0. The maximum DON degradation rate was observed at pH 8.0, and the degradation for 10 μg ml−1 DON at pH 9.0 was reduced to 70.08% (**Figure 3B**). The inoculum size significantly affected the degradation of DON. The degradation rates of 10 μg ml−1 DON after 72 h of incubation increased from 30 to 85% when the inoculum size was raised from 1 to 10% (**Figure 3C**).

### Degradation of Type A- and B-Trichothecene Mycotoxins by Consortium C20

To investigate the degradation of other trichothecene mycotoxins by C20, the consortium C20 was incubated with T-2, HT-2, 3-ADON, and 15-ADON for 72 h. The results demonstrated that the consortium C20 degraded 58% of 15-ADON, 28% of 3-ADON, and 21% of T-2. However, the degradation of HT-2 was not observed (**Figure 4**). 15-ADON and 3-ADON are the main acetylated derivatives of DON, and the degradation of 15-ADON and 3-ADON indicates that C20 possesses the ability to degrade acetylated DON derivatives (Payros et al., 2016).

### Identification of Deoxynivaleno Degradation Products

The metabolic products of DON were identified by using HPLC, LC-TOF-MS, and NMR. HPLC analysis showed that DON

was transformed to another metabolite during degradation by the enriched bacterial consortium C20 (Compound P, **Figure 5**). LC-TOF-MS results (**Supplementary Figure S1**) showed that the molecular ion of the metabolite was at *m/z* 295.1151 ([M + H]+ , C15H19O6 + ; calcd. 295.1182), which indicated a di-dehydrogenation of the DON molecule. The 1 H-NMR spectrum for compound P (**Supplementary Figure S2**) was 6.55 (dd, J = 6.0, 1.5), 4.91 (d, J = 1.3), 4.57 (d, J = 6.0), 3.91 (d, J = 11.6), 3.81 (d, J = 1.5), 3.75 (d, J = 11.6), 3.52 (s), 3.36 (d, J = 4.2), 3.23 (d, J = 4.2), 3.13 (d, J = 19.5), 2.29 (d, J = 19.5), 1.90 (3H, s), and 1.34 (3H, s). The chemical formula and the 1 H-NMR signals of compound P were identified as 3-keto-DON (Hassan et al., 2017; Wang et al., 2019b).

# DISCUSSION

DON is one of the most commonly detected contaminants of crops in worldwide, such as maize, wheat, and rice (Lee and Ryu, 2015). The widespread occurrence of this mycotoxin,

coupled with the detriments to human health and animal productivity resulting from its consumption, has promoted intensive efforts to identify detoxification measures, including microbial biotransformation (Pestka, 2010; Sobrova et al., 2010; Kris et al., 2014). Although several single microbial strains for the degradation of DON were isolated, there are still many limitations to application for agricultural fields because of the diversity and complexity of crop cultivation and storage environments (Brenner et al., 2008). Fortunately, microbial consortia provide a promising application for mycotoxin decontamination owing to their special advantages in the degradation of complex compounds. Microbial consortia combine multiple metabolic capacities of different species, improving the ability and efficiency of complex substrates biotransformation process, as well as the tolerance of complex environments (Shong et al., 2012). To date, microbial consortia has been widely used for a variety of important processes, such as the treatment of wastewater (Daims et al., 2006), the biosynthesis of compounds for medicine and industry (Shong et al., 2012), the biodegradation of bispyribac-sodium and phenanthrene (Janbandhu and Fulekar, 2011; Ahmad et al., 2018), and the production of hydrogen (Zhang et al., 2017), due to their high adaptability and broad substrate spectra (Kleerebezem and Van Loosdrecht, 2007). However, few attempts have been made to degrade DON by a microbial consortium. In this study, the highly enriched, stable and effective microbial consortium C20 was found to be able to degrade DON, resulting in a significant reduction in DON (**Figure 1**). In addition, tebuconazole and

carbendazim have no effects on the degradation of DON by the enriched culture, indicating that the degradation of DON is owing to bacteria. Furthermore, there was no change in DON concentration in the autoclaved sample, suggesting that the soil sample cannot detoxify DON by physical adsorption (**Supplementary Figure S5**).

The characterization of DON degradation in this study showed the activity of the microbial culture at a wide range of pH (6.0–9.0) and temperatures (25–35°C) compared with previously studied microbial cultures (Guan et al., 2009; Wang et al., 2019b). The microbial consortium C20 might degrade DON in complex and diverse environments. The optimal pH (8.0) and temperature (30°C) of the microbial consortium C20 for DON degradation are similar to that of most arable plants. It has been suggested that microbial consortia have potential applications for the detoxification of DON in field crops (Karlovsky, 1999; Islam et al., 2012).

The functional and stability of microbial consortium are usually modulated through interactions among microorganisms. These interactions can be indirect, such as exchanging different metabolites and information signals (Wintermute and Silver, 2010). For example, *Bacillus cereus* A1 and *Brevundimonas naejangsanensis* B1 mutually cooperate to enhance hydrogen production and starch utilization. In this consortium, strain A1 produced lactate as carbon source for strain B1 to cell growth and hydrogen production. In return, formate produced by strain B1 as an electron shuttle for strain A1 to generate hydrogen (Wang et al., 2019a). In direct interactions, microorganisms exchange electrons or deliver macromolecules such as DNA and proteins by direct interspecies contact. Rotaru et al. reported that besides H2 or formate as the electron donor for interspecies electron transfer, direct electrical connections was also found in the production of methane between *Geobacter metallireducens* and *Methanosaeta harundinacea* (Rotaru et al., 2014).

As the sole carbon, DON provided more substrate to the microbial consortium C20 in MM with 100 μg ml−1 DON than in the original culture. The increasement of DON might enhance the proportion of DON-degraders during the consecutive enrichment, resulting in a change in the community composition (Ahad et al., 2017). Strains belonging to *Methylophilus*, *Ancylobacter*, *Pseudomonas, Prosthecomicrobium, Bosea*, *Taonella* and *Devosia* were significantly increased (*p* < 0.05, one-way ANOVA) in C20 after enrichment with 100 μg ml−1 DON. These results suggest that the changes in these bacterial populations might be related to the microbial degradation of DON. It was demonstrated that many bacteria isolated for the degradation of DON were identified as *Devosia* strains (Sato et al., 2012; Onyango et al., 2014; Yin et al., 2016; Zhao et al., 2016; Hassan et al., 2017). Moreover, DON was transformed by these bacteria to 3-epi-DON, 3-keto-DON, or other products. Therefore, the *Devosia* strains in C20 have great potential for DON degradation.

The 3-OH group in DON, as well as the epoxide group on the trichothecene backbone, is responsible for its toxicity. When DON was epimerized or degraded, its interaction with ribosome will be changed, leading to an absence of MAP kinase activation and toxicity reduction. It was reported that the intestinal toxicity of DON would be decreased when it was converted into depoxy-DON or 3-epi-DON by microbial transformation (Pierron et al., 2016). In this study, 3-keto-DON was found to the main degradation product of DON analyzed by HPLC, LC-TOF-MS and NMR. It is all known that the oxidation of DON to 3-keto-DON plays a vital role in the detoxification of DON-contaminated grains (Shima et al., 1997). In addition, the 3-keto-DON showed a reduction of more than 90% in immunosuppressive toxicity compared with that of DON (He et al., 2015).

A DON degrading bacterial strain was isolated from the consortium C20, but the strain did not maintain its DON degradation activity after a few generations of culture in broth medium (**Supplementary Figure S4**). It is still challenging to isolated DON-degrader through selective enrichment procedures because the degradation reaction involves the removal of an oxygen molecule (Islam et al., 2012; Yoshizawa et al., 2014), which does not supply any nutrients for bacteria (Horvath, 1972; Fedorak and Grbić Galić, 1991) and prevents the utilization of DON as the sole carbon or nitrogen source for the selective isolation of degrader from the consortium C20 (Ahad et al., 2017).

The enriched culture C20 had the ability to degrade 15-ADON, 3-ADON and T-2, as well as DON in this study. Previous studies demonstrated that 15-ADON is higher toxicity than DON or 3-ADON (Pinton et al., 2012; Kadota et al., 2013). Moreover, both DON and 15-ADON are frequently found in wheat and other cereals (Han et al., 2014; Dong et al., 2016). Thus, the microbial consortium C20 has great potential to degrade DON and 15-ADON in agricultural products.

In conclusion, the enriched bacterial consortium C20, which can efficiently degrade DON and its derivatives 15-ADON and 3-ADON through the combined strengths of individual organisms, was isolated from environmental samples in this study. C20 is comprised of different bacterial genera, with *Methylophilus*, *Ancylobacter,* and *Devosia* significantly increasing in cultures with high concentrations of DON. Importantly, about 70 μg ml−1 DON could be degraded by consortium C20 within 5 days under a wide range of pH and temperature conditions. And the final degradation product was identified as 3-keto-DON with less toxicity. These results suggest that the C20 microbial consortium provides a new potential method for DON degradation. Extending DON degradation capabilities from single bacteria to microbial consortia represents a new frontier in DON microbial biotransformation. Although stable community behavior remains a great challenge in this area, it still has great potential to be used to degrade DON in food/feedstuffs and to improve food/feed quality and safety.

### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/**Supplementary Material**.

# AUTHOR CONTRIBUTIONS

YaW performed experiments and wrote the paper. GW, YuW, and Y-WL designed and performed experiments. JX, JS, and YD designed experiments, supervised the students, and wrote the paper. All authors have approved the final version of the manuscript.

# FUNDING

This work was supported by the National Natural Science Foundation of China (31872914, U1604234), Jiangsu Provincial Natural Science Foundation (BK20160595), Jiangsu Agriculture Science and Technology Innovation Fund (CX(17)1003), The Special Fund for Risk Assessment of China (GJFP2019002), and International Science and Technology Cooperation Program of China (2016YFE0112900).

### REFERENCES

# SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2019.02964/ full#supplementary-material

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2020 Wang, Wang, Dai, Wang, Lee, Shi and Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Assessment of a Potential Role of Dickeya dadantii DSM 18020 as a Pectinase Producer for Utilization in Poultry Diets Based on in silico Analyses

### Dana K. Dittoe1 , Ravi D. Barabote2 , Michael J. Rothrock3 and Steven C. Ricke1 \*

1 Department of Food Science and Center for Food Safety, University of Arkansas, Fayetteville, AR, United States, 2 Department of Biological Sciences, University of Arkansas, Fayetteville, AR, United States, 3 Egg Safety and Quality Research Unit, U.S. National Poultry Research Center, United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Athens, GA, United States

### Edited by:

Andrea Gianotti, University of Bologna, Italy

### Reviewed by:

Sébastien Bontemps-Gallo, Institut Pasteur de Lille, France Byron Brehm-Stecher, Iowa State University, United States Richard Ducatelle, Ghent University, Belgium

> \*Correspondence: Steven C. Ricke<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

Received: 31 May 2019 Accepted: 30 March 2020 Published: 23 April 2020

### Citation:

Dittoe DK, Barabote RD, Rothrock MJ and Ricke SC (2020) Assessment of a Potential Role of Dickeya dadantii DSM 18020 as a Pectinase Producer for Utilization in Poultry Diets Based on in silico Analyses. Front. Microbiol. 11:751. doi: 10.3389/fmicb.2020.00751 Currently, the poultry industry has been faced with consumer pressure to utilize only vegetable feedstuffs in poultry diets, eliminate antibiotics from poultry production, and rear poultry in free range systems. To maintain current production standards, the industry must determine ways to enhance nutrient uptake and utilization further. One possible solution is the supplementation of pectinase, an enzyme that degrades pectin within the cell walls of plants, in poultry diets. Therefore, the objective of the current study was to determine the potential role of a pectinase producer, Dickeya dadantii DSM 18020, as a commercially utilized pectinase producer in poultry diets against other known pectinase producers, in silico. In the current study, whole genomes of Dickeya dadantii DSM 18020 (Dd18020), D. dadantii 3937 (Dd3937), D. solani IPO 2222 (Ds2222), Bacillus halodurans C-125 (BhC125), and B. subtilis subsp. subtilis str. 168 (Bs168) were compared using bioinformatic approaches to compare the chromosomal genome size, GC content, protein coding genes (CDS), total genes, average protein length (a.a.) and determine the predicted metabolic pathways, predicted pectin degrading enzymes, and pectin-degradation pathways across pectinase producers. Due to insufficient information surrounding the genome of Dd18020 (lack of annotation), the genome of Dd3937, a 99% identical genome to Dd18020, was utilized to compare pectinaseassociated enzymes and pathways. The results from the current study demonstrated that Dd3937 possessed the most significant proportion of pathways presented and the highest number of pathways related to degradation, assimilation, and utilization of pectin. Also, Dd18020 exhibited a high number of pectinase-related enzymes. Both Dd3937 and Dd2222 shared the pectin degradation I pathway via the EC <IP_ADDRESS>, EC <IP_ADDRESS>, and EC 4.2.2.- enzymes, but did not share this pathway with either Bacillus species. In conclusion, Dd18020 demonstrated the genetic potential to produce multiple pectinase enzymes that could be beneficial to the degradation of pectin in poultry diets. However, for Dd18020 to become a commercially viable enzyme producer for the poultry industry, further research quantifying the pectinase production in vitro and determining the stability of the produced pectinases during feed manufacturing are necessary.

Keywords: free range, pectinase, Dickeya dadantii, all-vegetable diets, in silico

# INTRODUCTION

fmicb-11-00751 April 21, 2020 Time: 14:29 # 2

Dickeya dadantii, previously classified as Erwinia chrysanthemi by Burkholder (1957), is one the many bacteria responsible for bacterial soft rot disease (Dickeya spp., Erwinia spp., Pectobacterium spp., etc.) that occurs in a wide range of crops (Samson et al., 2005; Toth et al., 2011). The facultative anaerobic, Gram-negative bacilli typically grow at an optimal temperature of 39◦C, but can grow between the ranges of 25–40◦C (Samson et al., 2005). The typical transmission of D. dadantii to plants is through direct contact with contaminated soil (Grenier et al., 2006). Contamination of soil can occur through several vectors such as water (irrigation, runoff, etc.), various insects, and specific agricultural techniques such as plowing (Grenier et al., 2006). Due to the pectinolytic nature of D. dadantii, it causes an infection in crops that induces soft rot that can be characterized by the rapid disorganization of the parenchymatous tissues (Hugouvieux-Cotte-Pattat et al., 1996). Although the colonization of plants by soft rot Erwinia is primarily driven by the production of pectic enzymes, the process is multifactorial requiring cellulases, iron assimilation, an Hrp type III secretion system, exopolysaccharides, motility, and proteins involved in resistance against plant defense mechanisms (Barabote et al., 2003; Toth et al., 2003; Ravirala et al., 2007). Due to the capability of D. dadantii to produce pectinases, it has the potential to be advantageous to multiple industries.

Pectinases consist of a group of enzymes that hydrolyze pectin present in plant cell walls and exist in higher order plants and microorganisms (Whitaker, 1991). In plants, pectinases are found to enhance cell wall extension (Jayani et al., 2005) and promote softening of specific plant tissues during maturation and later storage (Aguilar and Huirton, 1990; Sakai, 1992). Therefore, pectinases are used to degrade plant materials in multiple food processing techniques such as reducing the time it takes to extract fruit juice from fruit puree (Junwei et al., 2000) and enhancing the clarity in wine (Kobayashi et al., 2001). Currently, pectinase is applied in several processes such as textile processing and bio-scouring of cotton fibers, the degumming and retting of plant bast fibers, wastewater treatment, coffee and tea fermentation, paper and pulp industry, purification of plant viruses, and in animal feed (Sharma et al., 2013). Across industries, pectinases are mass manufactured by utilizing pectinase producing bacteria and fungi. Of such bacteria and fungi, Bacillus and Aspergillus are the most characterized pectinase producers utilized across manufacturing of agricultural and food products (Garg et al., 2016).

Of the many uses of pectinases, the poultry industry would greatly benefit from the addition of pectinases in poultry diets. Recently, consumer pressure has motivated the poultry industry to introduce no antibiotic ever (NAE), all -vegetable fed broilers (Weil, 2017). In addition, there has also been an increased demand for free range poultry and forage feeding. In efforts to utilize locally available feed sources, sectors of the poultry industry have initiated forage feeding, pasture farming, or supplementary feeding regimens (Buchanan et al., 2007). With the move to all -vegetable diets and forage feeding, broiler diets may contain an increased concentration of cereals containing non-starch polysaccharides (NSPs) in their endosperm (Broz and Beardsworth, 2002). Poultry are unable to produce many of the enzymes necessary to degrade NSP's and broiler performance is decreased due to the impairment of digestive enzymes, decreased nutrient absorption, and increase in viscous excreta (Broz and Beardsworth, 2002).

The supplementation of diets with pectin has specifically resulted in the decrease of growth rate, decrease in feed efficiency, increase in feed intake, increase in sticky droppings, and overgrowth of Clostridium species (Wagner and Thomas, 1977, 1978; Ricke et al., 1982). More recently, research has demonstrated that pectinases have the ability to degrade pectin more readily in poultry diets alone and in combination with other NSP-hydrolyzing enzymes. In addition, the supplementation of pectinase, cellulase, or hemicellulase alone in poultry diets has proven to be less effective than their combination on improving poultry performance (Tahir et al., 2005, 2006).

Currently, the major sources of NSP-containing plant material within poultry diets is corn and soybean meal (Pierson et al., 1980; Chesson, 2001). However, due to the increasing demand for corn and soybean meal from the swine industry and other sectors, the poultry industry has explored alternative sources of carbohydrates. Such sources are wheat, barley, and lupins (Australia) which contain high amounts of NSPs compared to those contained in corn and soybean meal, specifically pectin (Bailoni et al., 2005; Leeson, 2008; Kim et al., 2011).

Therefore, there is a need for a wide variety of enzymes, especially those that break down plant material that poultry are unable to utilize on their own due to the vast differences in plant composition. For example, of the pectins contained within the cell wall of plants vary in amount and in the physiochemical properties such as structural differences among the types of plants and fruits (Kamnev et al., 1998). Pectin structure consists of an α(1-4)-linked polygalacturonans with varying chain lengths, intermittent rhamnosyl residues, and other potential structural variations (Selvendran, 1985; Piknik, 1990; King, 1993). Further, there are currently no products on the market that solely target the complex structures of pectins, especially those contained in lupins (Kim et al., 2011). Pectin contain galactose residues of the polygalacturonans that are methyl-esterfied at C-6, which protects the substrate from the access of pectinases (Ali et al., 2005). Further, the in vitro supplementation of pectin methyl esterase and polygalacturonase in combination with ground dehulled lupins (Lupinus angustifolius in 70 mL acid buffer) reduced the length of pectin chains by 65% and molecular weight of pectin by 56% (Ali et al., 2005). As such, it is imperative to identify bacterial enzyme sources capable of producing a wide variety of pectinases for potential commercial development.

Pectinases have been provided in poultry diets in addition to other enzymes to enhance nutrient digestibility of broilers and other poultry species. Previous research has demonstrated that the use of pectinases in an enzyme cocktail has the capability to reduce the viscosity of the feed, release nutrients through the hydrolysis of non-biodegradable fibers or those blocked by these fibers and reduce the total amount of excreted feces (Jayani et al., 2005). Therefore, the overall objectives of the current study were to determine if the genome of Dickeya dadantii DSM 18020 (NZ\_CP023467.1) encodes pectinases, if, in turn, those pectinases are comparable to those currently utilized in the poultry industry, and if D. dadantii would make a suitable alternative source of novel pectinases to those bacteria currently used commercially to produce pectinases.

# MATERIALS AND METHODS

### Genomic Data Retrieval

fmicb-11-00751 April 21, 2020 Time: 14:29 # 3

In the current study, genomic data was accessed and collected from the National Center for Biotechnology Information (NCBI) on December 7, 2018 (O'Leary et al., 2016). The genomic data for Dickeya dadantii DSM 18020 (NZ\_CP023467.1), D. dadantii 3937 (NC\_014500.1; Glasner et al., 2011), D. solani IPO 2222 (NZ\_CP015137.1; Pédron et al., 2014), Bacillus subtilis subsp. subtilis str. 168 (NC\_000964.3; Kunst et al., 1997; Barbe et al., 2009; Belda et al., 2013; Borriss et al., 2018) and B. halodurans C-125 (NC\_002570.2; Takami et al., 2000), was utilized in the current study to compare novel pectinase homologs (Dickeya spp.) to known industrialized pectinase producers (Bacillus spp.) and their associated metabolic pathways.

# Genomic Relation Across Pectinase Producers

Genomic data that was retrieved from NCBI was utilized to compare the genomic characteristics of Dickeya dadantii DSM 18020 (Dd18020), D. dadantii 3937 (Dd3937), D. solani IPO 2222 (Ds2222), Bacillus subtilis subsp. subtilis str. 168 (Bs168), and B. halodurans C-125 (BhC125). Genomic characteristics that were compared among bacterium were chromosomal genome size (Mb), Guanidine to Cytosine content (GC Content; %), protein-coding genes (CDS), total genes, and average protein length (a.a).

Dd3937, a wild-type strain originally isolated from Saintpaulia ionantha, is most commonly used as a model organism for soft rot pathogenesis since the 1980's (Glasner et al., 2011). As Dd3937 is a known model organism, the authors wanted to explore the candidacy of a more recently discovered strain, DSM 18020. Dd18020 was recently isolated from Pelargonium capitum in Comoros and deposited to the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell

Culture (Braunschweig, Germany). The authors were particularly motivated to choose Dd18020 as the candidate pectinase producer in the current study as there are no current Nagoya Protocol restrictions to its use, it is obtainable through multiple collections of plant-associated bacteria, and has a complete genome assembly available on NCBI. Therefore, Dd18020 can be annotated (genome), purchased, and applied in research or industrial use consistently and without restrictions.

However, due to the absence of Dd18020 in several databases, Dd18020 and Dd3937 were compared against one another by comparing syntany and homogeneity. Syntany was explored using the progressive MAUVE whole genome alignment (Darling et al., 2004). Homogeneity was investigated using Web Blast on NCBI (Altschul et al., 1990). As other research has utilized reference genomes to identify close proximity to the target bacteria or organism (Tyson et al., 2004; Iverson et al., 2012; Wrighton et al., 2012; Albertsen et al., 2013; Sharon et al., 2013), the comparison of Dd18020 and Dd3937 was utilized to validate if Dd3937 was an appropriate reference genome for Dd18020. Therefore, further genomic comparisons were done in the absence of Dd18020, with Dd3937 in its stead. In addition, a rooted phylogenetic tree was constructed from the output obtained from progressiveMAUVE whole genome alignment of Dd18020, Dd3937, Ds2222, Bs168, and BhC125 (Darling et al., 2010).

# Predicted Pathways Across Pectinase Producers

In the current study, genomes of Dd3937, Ds2222, Bs168, and BhC125 were compared in MetaCyc (MetaCyc 22.5; Caspi et al., 2014) to evaluate the protein and pathway similarities between genomes of the four pectinase producers. Numerical comparisons were performed in Microsoft Excel for Office 365 MSO (16.0.11601.20174; Microsoft, Redmond, WA, United States) to highlight the numerical difference in pathways between the four pectinase producers utilized in the current study.

# Predicted Pectin-Related Enzymes Across Pectinase Producers

The list of proteins and enzymes encoded in the genomes of Dd18020, Dd3937, Ds2222, Bs168, and BhC125 was retrieved from NCBI (O'Leary et al., 2016) and utilized to determine the pectin-related proteins and enzymes encoded. Proteins encoded in the genome of the pectinase producers were aligned in MegaX (Kumar et al., 2018) using ClustalW (Thompson et al., 1994) where Maximum Likelihood was utilized to produce a rooted phylogenetic tree. In addition, Metacyc was subsequently utilized to compare pectinase related enzymes and genes across the four pectinase producers: Dd3937, Ds2222, Bs168, and BhC125. The top eleven related proteins were chosen for comparison in the current study.

# Pectinase Pathway

As Dd18020 is less studied, Dd3937 was chosen to elucidate the pectin-related pathways. MetaCyc 22.5 was utilized to determine the pathways of Dd3937. Dd3937 was subsequently compared to the other pectinase producers utilized in the current study: Ds2222, Bs168, and BhC125.

### TABLE 3 | Analysis of metabolic pathways in the genomes of four pectinase producing bacteria.

aGreen shading represents the highest incidence of pathways between the four pectinase producing bacteria, with white representing the lowest incidence of pathways between the four pectinase producing bacteria.

# RESULTS

In relation to the genomic make-up of the pectinase producers, all D. dadantii species, Dd18020 and Dd3937, and Ds2222 were more similar in their genome size (5.00, 4.92, and 4.92 Mb, respectively), GC content (56.40, 56.30, and 56.20%, respectively) and average protein-coding length (326, 323, 339 a.a., respectively) than the size, GC content, and average protein-coding length of the two Bacillus species (4.22 and 4.20 Mb, 43.50 and 43.70%, and 290 and 298 a.a., respectively, **Table 1**). All bacteria in the current study possessed similar levels of proteins and genes, with BhC125 having the fewest number of proteins and genes (3,950 CDS and 4,134 total genes). In addition, both bacilli possessed smaller average protein lengths than the Dickeya species. The differences in size, GC content, average proteincoding length, proteins, and genes may be attributed to Dickeya and Bacillus species belonging to different phyla

(Proteobacteria and Firmicutes, respectively) (**Figure 1**, Galperin, 2015; Lockwood et al., 2019).

Due to the paucity of literature and available genomic research concerning Dd18020, comparison of Dd18020 and Dd3937 genomic information was performed in Web BLAST (**Table 2**). The results of the global alignment in Web BLAST revealed that the two genomes were a 99% match and therefore the genome of Dd3937 was sufficient to compare to other pectinase producers in lieu of Dd18020. The E-value, maximum score, and maximum identity of Dd18020 and Dd3937 genomes were 0.0, 1.661e + 05, and 99%, respectively, indicating that the local coverage was high. The total metrics were also relatively high, with a total score and query coverage of 9.161e + 06 and 91%, respectively. In addition, synteny was explored between Dd18020 and Dd3937 (**Figure 2**). The Dd18020 genome is highly syntenic with the Dd3937 genome, exhibiting only two disruptions. One region of about 250 kb is present at different locations in both chromosomes (location 4.6–4.85 Gb in Dd18020 and 0–242 kb in Dd3937). A second region in Dd18020 chromosome is split into two regions. The first region of Dd18020 is at position 0 to 228 kb and the second region at position 4.85–4.92 Gb.

The metabolic pathways of the four pectinase producers were compared in MetaCyc 22.5, with Dd3937 representing Dd18020. The pathways did differentiate in several ways (**Table 3**). In all, Dd3937 and Bs168 exhibited the highest representation of pathways, however, Dd3937 had the highest proportion of total pathways presented. In relation to degradation, assimilation, and utilization, Dd3937 possessed the highest number of related pathways. Carbohydrate biosynthesis was present in the highest number (14) in Dd3937 when compared to the three other pectinase producers. Ds2222 had the second highest number of carbohydrate biosynthesis pathways (12) than both Bacillus species (8 and 6); however, when carbohydrate degradation pathways were examined, Dd3937 had the lowest representation of pathways (17), with Ds2222 and Bs168 possessing more carbohydrate degradation pathways (20 and 20, respectively).

Dd18020 was screened for pectinase-related genes by exploring the genome in NCBI (**Table 4**). The genome of Dd18020 contained 13 identified extracellular pectinaserelated proteins: pectin acetylesterase, pectinesterase A, MULTISPECIES: pectin degradation protein KdgF, and several pectate lyases. The pectin degrading enzymes and proteins

encoded in the genome of the five pectinase producers in the current study demonstrated no distinct pattern in genetic divergence (**Figure 3**). For example, Dd18020, Dd3937, Ds2222,

TABLE 5 | Comparison of key pectinase enzymes between four pectinase producing bacteria.

and Bs168 possess the genetic potential to produce pectin lyase which evolutionarily is the most recent with Dd18020, Dd3937, and Ds2222 being a polytomy. Overall, the evolutionary

divergence of pectin degrading enzymes is primarily due to the emergence of new proteins and enzymes, rather than the differences between pectinase producing bacteria. Pectin degrading enzymes encoded by Dadantii species were less divergent between one another than those encoded by the Bacilli.

Due to the paucity of genomic research related to Dd18020, Dd3937 was compared to the three other pectinase-producing bacteria in MetaCyc 22.5 (**Table 5**). The top eleven pectinaserelated enzymes that were shared among pectinase producers are also present in Dd18020 (**Tables 4**, **5**), however, Ds2222

did not appear to share similar enzymes to either Dd3937 or both Bacillus species. It appears that D. dadantii possesses more proteins dedicated to degradation, utilization, and assimilation (105) than Ds2222, Bs168, and BhC125 (97, 95, and 94 proteins, respectively). However, in vitro modeling should be done to quantify the pectinase production of D. dadantii compared to other pectinase-producing bacteria.

In addition to comparing the pectinase-related proteins produced by Dd18020 and the four other bacteria utilized in the current study, the metabolic pathways related to pectin degradation were compared simultaneously for shared pectinase related pathways in MetaCyc 22.5. Of the shared pathways, only the two Dickeya species shared similar pathways, EC <IP_ADDRESS>, EC <IP_ADDRESS>, and EC 4.2.2.- (**Figure 4A**). The presence of shared pathways between the two Dickeya species was relatively surprising, as the genome-encoded pectinaserelated enzyme comparison did not reveal similarities in the proteins encoded by the two Dickeya species. In fact, Ds2222 did not possess similarly coded proteins to that of the other three bacteria (**Table 5**). However, the metabolic pathway related to pectin degradation I was shared between Dd3937and Ds2222.

Pectinases have been divided into three separate groups dependent on the cleavage site. These three groups of pectinases are comprised of (1) hydrolases consisting of polygalacturonase, PG (EC <IP_ADDRESS>); (2) lyase/trans-eliminases comprising pectin lyase, PNL (EC <IP_ADDRESS>), and pectate lyase, PL (EC <IP_ADDRESS>); and (3) pectinesterase, PE (EC <IP_ADDRESS>) (Yadav et al., 2009; Visser et al., 2004). In the current study, similar pathways (**Figures 4A–D**) of all categories were observed in Dd18020, Dd3937, and Ds2222.

In the pectin degradation pathway, both Dd3937and Ds2222 possessed the same enzyme, pectinesterase A (EC <IP_ADDRESS>), encoded for by the DDA3937\_RS15630 and DSOIPO2222\_RS15080 genes (data not shown). In the same pathway, the enzymes and genes of similar relation from Dd3937and Ds2222 were several exo-polyα-D-galacturonosidases (EC <IP_ADDRESS>) encoded for by the DDA3937\_RS20090, DDA3937\_RS20095, DDA3937\_RS20100, and DDA3937\_RS05830 genes of Dd3937and the DSOIPO2222\_RS19410, DSOIPO2222\_RS19420, and DSOIPO2222\_RS19425 genes of Ds2222. Lastly, in the metabolic pathway EC 4.2.2.-, the enzymes, and genes of similar relation from Dd3937and Ds2222 were oligogalacturonate lyase encoded in the genes DDA3937\_RS11450 and DSOIPO2222\_RS11150, respectively. Neither Bacillus species utilized in the current study shared any similar enzymes or genes in the pectin degradation I pathway to the Dickeya species.

The pectin degradation pathway I shared by the Dickeya species degrades pectin to D galactopyranuronate + 4-deoxy-Lthreo-hex-4-enopyranuronate (**Figure 4A**). Pectin is degraded to pectate via pectinesterase A in the metabolic reaction catalyzed by EC <IP_ADDRESS>, with methanol byproduct (**Figure 4B**). Pectate is degraded to α-D, α-D-digalacturonate via several exo-polyα-D-galacturonosidases in the metabolic reaction catalyzed by EC <IP_ADDRESS> with a pectate byproduct (**Figure 4C**). The α-D, α-D-digalacturonate is degraded to D galactopyranuronate + 4 deoxy-L-threo-hex-4-enopyranuronate in the metabolic pathway catalyzed by EC 4.2.2.- in Dd3937 via oligogalacturonate lyase (**Figure 4D**).

# DISCUSSION

As pectinase is a vital enzyme utilized in the degradation of different fruit and vegetable products, it has value to the food industry (Sharma et al., 2013). Likewise, based on consumer preference, poultry industry diets are increasingly becoming vegetable-based void of animal protein (Weil, 2017). To make all-vegetable diets commercially viable, the poultry industry will need additional sources of feed grade pectinases with different hydrolytic capabilities to break down vegetable products with widely different compositional levels and subsequently improve broiler performance. Therefore, the utilization of a more flexible set of supplementary enzymes will become increasingly crucial to poultry nutritionists in enhancing the nutrient uptake in all-vegetable diets.

However, as D. dadantii has been reported to be a pathogen to plants and insects, another potential strategy for use of D. dadantii DSM 18020 pectinase genes would be to incorporate them into a current commercial poultry probiotic. Therefore, the genes of pectin degrading enzymes of Dd18020 could be inserted into a Bacillus, a known probiotic currently utilized in poultry diets. A Bacillus spp. would be a suitable candidate as they have the potential to withstand feed manufacturing conditions due to spore forming capabilities and can be genetically modified for industrial use (Ricke and Saengkerdsub, 2015). Though it is feasible, the insertion of Gram-negative genes (D. dadantii) into a Gram-positive organisms (Bacillus) is not easily accomplished as it requires proper plasmid origin recognition, promoter recognition, and codon usage between the organisms. Another limitation to consider is that certain Bacillus species such as B. subtilis produce proteases that have been known to hinder the production of heterologous proteins (Puohiniemi et al., 1992). As such, the meticulous insertion of Gramnegative genes into Bacillus spp. has been possible through plasmid insertion (Luchansky et al., 1988; Ohta et al., 2005) and more recently through CRISPR-Cas9 systems (Toymentseva and Altenbuchner, 2019).

In the current study, data demonstrated that D. dadantii DSM 18020 possessed several pectinase-related enzymes such as polygalacturonases (PG), pectin lyase (PL), polygalacturonate lyase (PGL), and pectinesterase or pectin methylesterase (PE) (Sharma et al., 2013). The optimal pH for most endo PG, PL, PGL, and PEs are 2.5–6.0, 4.0–7.0, 6.0–11.0, and 4.0–7.0, respectively (Sharma et al., 2013). The optimal temperatures range from 30 to 50◦C (PGs) and 40–60◦C (PEs) (Sharma et al., 2013). The internal body temperature of poultry is 41.5◦C (Dawson and Whittow, 2000) and the pH of the avian gastrointestinal tract (GIT) varies from highly acidic (below 4) in the proventriculus and gizzard and neutral in the lower intestines (above 7; Sturkie, 1999).

Therefore, these enzymes should function as the avian GIT is within the optimal parameters for enzyme utilization.

In fact, the in vitro evaluation of PG and pectin methylesterase (PME) demonstrated an improvement in the digestion of pectin and a reduction in the water holding capacity of lupins in comparison to PG or PME alone (Ali et al., 2005). Therefore, Ali et al. (2005) concluded that the inclusion of legumes such as lupin could be increased by utilizing the combination of the two pectinases in poultry diets. Furthermore, the supplementation of pectinase with either cellulase or hemicellulase in corn-soybean meal broiler diets from days 15 to 27 has demonstrated the improvement in the ileal digestibility of crude protein (CP) and organic matter and increased apparent metabolizable energy content of the diet (Tahir et al., 2006). Other researchers have also found the supplementation of a multi-enzyme preparation with pectinase improved CP digestibility (Marsman et al., 1997; Kocher et al., 2002; Saleh et al., 2005). The improvement in CP digestibility may in part be due to the degradation and subsequent digestion of pectic polysaccharides (Slominski and Campbell, 1990; Simbaya et al., 1996). Due to 10% of the protein being entrapped in the cell wall matrix of soybeans, the degradation of the cell wall via the utilization of pectinase-related enzymes may release the contained proteins (Chesson, 2001). Also, half of the NSP's contained within the cell matrix of the soybean meal are pectic polysaccharides (Chesson, 2001). Their subsequent depolymerization may improve CP digestibility, which has been noted previously (Tahir et al., 2006).

### CONCLUSION

The current study validates the genetic potential of D. dadantii DSM 18020 as an enzymatic pectinase producer to utilize commercially within the poultry feed industry. The results demonstrated that several pectin-related enzymes are encoded in the genome of D. dadantii, and D. dadantii possesses the most significant number of pectin degradation-related pathways. Also, the study demonstrates the genomic capability for metabolic breakdown of pectin via the pectin degradation pathway belonging to D. dadantii; however, further studies are necessary to evaluate the capabilities of D. dadantii DSM 18020 as a source of commercially viable pectinase for large scale production.

### REFERENCES

For example, more in-depth knowledge of pectin degradation by D. dadantii is necessary to quantify the pectinase production of D. dadantii to design scale up production. Based on the current in silico research study, D. dadantii does appear to produce pectinases that can further enhance the utilization of poultry diets and the continual supplementation via production by poultry probiotics fed to poultry may enhance nutrient absorption and subsequent performance. In relation to poultry, further analyses are necessary to determine if the supplementation of D. dadantii pectin-related enzymes in poultry diets is feasible. Some caution should be exercised as diet manufacturing involves steam and high temperature; this could potentially denature these key enzymes, thus reducing the effectiveness of pectin-related enzymes. Therefore, further research evaluating the survival of pectinases in poultry diets is necessary.

# DATA AVAILABILITY STATEMENT

The datasets generated for this study can be found in the NCBI, Dickeya dadantii DSM 18020 (NZ\_CP023467.1), D. dadantii 3937 (NC\_014500.1), D. solani IPO 2222 (NZ\_CP015137.1), Bacillus subtilis subsp. subtilis str. 168 (NC\_000964.3) and B. halodurans C-125 (NC\_002570.2).

### AUTHOR CONTRIBUTIONS

DD wrote the manuscript with the assistance from SR, RB, and MR. All authors significantly contributed to the work of the current manuscript.

### ACKNOWLEDGMENTS

DD would like to acknowledge the Graduate College at the University of Arkansas for its support of the research and the stipend provided through the Distinguished Academy Fellowship and the continued support from the Center for Advanced Surface Engineering, under the National Science Foundation Grant No. OIA-1457888 and the Arkansas EPSCoR Program, ASSET III. We would like to acknowledge Elena Olson for her assistance on the current manuscript.

supplementation on the performance, carcass composition and meat quality of grower/finisher pigs. Anim. Prod. Sci. 51, 37–43. doi: 10.1071/AN10087

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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