by Mathieu Calmont, Area Business Manager EMEA (Europe – Middle East – Africa), France, and Kristin Sandersen Gretland, Technical Sales Manager, Borregaard, Scandinavia, Norway

 

In many countries, shrimp aquaculture production is depressed by disease, particularly caused by vibrio bacteria. Antibiotics, which have been used in large quantities, are in many cases ineffective, or result in increased virulence of pathogens. Formaldehyde products have been widely used, and resistance behavior has started to appear in shrimp farming.

Vibrio parahaemolyticus and Vibrio harveyi have been described as the main pathogens in shrimp species. These pathogens cause serious infections and decreased productivity both in hatcheries and ponds. In addition, this leads to reduced feed conversion and reduced growth rate in surviving individuals.

Most bacterial aquatic diseases are related to vibrio bacteria. These bacteria usually dominate the normal microflora of farmed and wild penaeoidean shrimp. Bacteria will colonise in the digestive tract, release toxins, and eventually destroy the digestive organ known as hepatopancreas. This is leading to high mortality - up to 90 percent.

A new product, SoftAcid Aqua Deca, has been developed and optimised to be used in all phases of shrimp production (from algae to growing phase). The product inhibits growth and biofilm formation of vibrio, not allowing vibrio species to colonise the digestive tracts of the shrimp. Shrimp feeding studies have shown positive results – with higher survival rate, increase in biomass and reduction in feed conversion cost.

Introduction to the work done

The goals for this project were to develop a unique product that can be used in several stages of the shrimp production, in addition to having a positive impact on reduction of biofilm- and bacteria formation. The work started with laboratory trials where the bactericidal effect towards different vibrio strains was studied. As a second step, the bacteriostatic effect was studied with respect to quorum sensing and biofilm formation.

Quorum sensing (QS) is a mechanism by which bacteria coordinate the expression of certain genes in response to the presence or absence of small signal molecules. It appears that several of the virulence factors of Vibrio harveyi are regulated by QS and extracellular toxin production is known to be one of the biggest causes of vibrio toxicity. Cultivated shrimp can be protected from vibriosis by disruption of quorum sensing.

Earlier findings have stated that pyrogallol units disrupts QS in Vibrio [1-2]. The lignosulphonic acid present in SoftAcid Aqua Deca has a polyphenolic structure, similar to the pyrogallol unit seen in other polyphenols.

Earlier experiences have shown positive effects towards biofilm removal when using SoftAcid. In the shrimp industry, it is believed that vibrio bacteria stick to the digestive tract of the shrimp by creating a biofilm. Laboratory trials with biofilm adhesion were performed in order to see the effect of SoftAcid Aqua Deca specifically on vibrio biofilms.

Based on initial studies, shrimp feed trials in Ecuador were set up.

Trials in laboratory: Minimal Inhibitory Concentration (MIC)

The antimicrobial effect of SoftAcid Aqua Deca was tested by using the MIC procedure. MIC was determined for three additives: SoftAcid Aqua Deca, a commercial buffered acid and a formaldehyde solution. The antibiotic Enrofloxacin was used as reference.

Three different strains of bacteria were tested: Vibrio parahaemolyticus (Vp), Vibrio harveyi (Vh) and Pseudomonas sp.(Ps). The results were presented as percentages, and the OD (optical density) of the positive control (+) was set to 100 percent.

The MIC values show that SoftAcid Aqua Deca inhibits bacterial growth of Vibrio parahaemolyticus, Vibrio Harveyi and Pseudomonas at 2000 ppm (> 98% reduction in optical density for all the strains tested). The commercial buffered acid showed limited effect on the strains when using the maximum test concentration 4000 ppm: a reduction in optical density of 31 percent, 0 percent and eight percent was found for the three strains tested

The formaldehyde solution inhibits growth by 80 percent reduction at 700ppm for all strains and has the same inhibition pattern with 80 percent reduction at the maximum concentration of 4000ppm. Resistance behavior is commonly seen with formaldehyde products.

QS: Quorum sensing

Quorum sensing in vibrio can be observed by following its luminescence. Laboratory trials have been performed with SoftAcid Aqua Deca studying the luminescence spectrum of Vibrio harveyi and Vibrio campbellii.

From the studies one can see that SoftAcid Deca disrupt the quorum sensing system. Cultivated shrimp can be protected from vibriosis by this kind of disruption of the quorum sensing system.

Biofilm

In the shrimp industry, it is believed that vibrio bacteria stick to the digestive tract of the shrimp by creating a biofilm. Earlier findings have shown positive effect towards biofilm removal when using SoftAcid.

Biofilm adhesion was studied in order to see the effect of SoftAcid Aqua Deca towards vibrio biofilms including Vibrio harveyi (Vh), Vibrio parahaemolyticus ATCC (Vp), Vibrio vulnificus (Vv), Vibrio campbelli (Vc) and Pseudomonas sp (Ps). A buffered organic acid was used as reference.

The results are presented in percentages, where the luminescence of the positive control (+) is set to 100 percent. No adhesion of biofilm in the treatment with SoftAcid Aqua Deca, whereas all the strains tested with buffered organic acid were able to attach a biofilm.

Plant trials

Several plant trials have been performed; one of these was a plant trial performed at a land-based shrimp farm in Ecuador, in order to study the effect of SoftAcid Aqua Deca. The farm had limited water renewal and the survival rate of the shrimp was normally at 30-40 percent.

Different parameters were studied for the growing and finishing phases of the shrimp: total biomass, shrimp density, conversion index, survival rate as well as feed cost. 

SoftAcid Aqua Deca - Positive effect on algae growth

Algae are the most important dietary source for the penaeoidean shrimp, specifically the larval stages. Algal species used to feed shrimp in hatcheries are generally chosen according to size and ability to grow in cultures. Nutritional composition is important to support good growth.

During the last phase of algae growth, before each type is added to the larvae tanks, the algae need to be preserved from degradation and oxidation. SoftAcid has shown positive effects in this application, both as a preservative and as a growth promotor.

Lignosulphonates are water soluble lignin. These are available as lignosulphonic acid salt. SoftAcid Aqua Deca is a combination of lignosulphonic acid and short chain fatty acids.

Humic and fulvic substances have shown a positive effect on microalgae growth. It is well known that lignin plays a key role in the formation of natural humic and fulvic substances – equal to what will be found in the algae tanks.

Today the most accepted view is that a major building block of humic/fulvic substances originate from polyphenols of lignin. One pathway of forming humic/fulvic substances is through a breakdown of plant biopolymers, including lignin, into their structural units [3].

Therefore, many of the functional groups found in humic/fulvic acids can also be found in lignin. In addition, the amount of equivalent fulvic acid in the lignosulphonic acid used in SoftAcid is 94 percent [4]. The positive effect of SoftAcid towards algae growth, could hence be attributed to the presence of fulvic acids.

SoftAcid Aqua Deca – Improvement of the sanitary conditions

Depending on the different stages/lifecycle of the shrimp, water treatment can be performed by using SoftAcid Aqua Deca. The product can be used during all phases of the shrimp development from protozoa to larvae phases.

  • Protozoa phase: 2-4ppm per day in the water (applied 2 times)
  • Mysis phase: 4-6ppm per day in the water (applied 2 times)
  • Post larvae phase (PL1 to PL9): 10-20ppm per day in the water (applied 2 times)
  • Post larvae phase (>PL10): 20ppm/6h (80ppm in total) Tank pH should not be below 7.0.

In addition, SoftAcid Aqua Deca can be used as a sanitizer for all the elements within the nursery to avoid pathogen colonization.

Sustainability

The world faces a number of important challenges related to climate and the environment, food production and resource management. Borregaard`s advanced bio refinery can help address some of these challenges. Borregaard uses renewable and natural raw materials and converts these into valuable products that can replace oil-based chemicals.

High raw material utilisation, lower greenhouse gas emissions and products to replace oil-based alternatives, provide sustainable and future-oriented solutions [5]. The SoftAcid products, and more specifically SoftAcid Aqua Deca, contribute to a more sustainable alternative for the shrimp industry.

Conclusion

SoftAcid Aqua Deca has been developed to work as a unique, versatile and sustainable product which:

  • Inhibits growth and biofilm formation of vibrio not allowing vibrios to colonise the digestive tracts of the shrimp
  • Shows positive results in shrimp feeding studies with higher survival rate, increase in biomass and reduction in feed conversion cost
  • Should be used in all phases of shrimp production (from algae to the growing phase)

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